WO2016194993A1 - Nucleic acid medicine that targets cancer-type slco1b3 - Google Patents

Nucleic acid medicine that targets cancer-type slco1b3 Download PDF

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WO2016194993A1
WO2016194993A1 PCT/JP2016/066332 JP2016066332W WO2016194993A1 WO 2016194993 A1 WO2016194993 A1 WO 2016194993A1 JP 2016066332 W JP2016066332 W JP 2016066332W WO 2016194993 A1 WO2016194993 A1 WO 2016194993A1
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slco1b3
nucleic acid
cancer
seq
cells
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PCT/JP2016/066332
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Japanese (ja)
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和丈 辻川
裕子 上田
雅美 佐藤
中村　好宏
古川　龍彦
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国立大学法人鹿児島大学
国立大学法人大阪大学
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Priority to JP2017522232A priority Critical patent/JP6823320B2/en
Publication of WO2016194993A1 publication Critical patent/WO2016194993A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

Definitions

  • the present invention relates to a nucleic acid for use in suppressing the expression of cancer-type SLCO1B3 or a pharmaceutical composition containing the nucleic acid.
  • EML4 Echinoderm microtubule-associated protein-like 4
  • ALK anaplastic lymphoma kinase
  • solute carrier organic anion transporter family member 1B3 is a 12-transmembrane transporter expressed on the cell membrane and is known to show a wide range of substrate recognition properties. ing. SLCO1B3 is normally expressed specifically in the liver, but it has been reported that expression is also observed in other organs with canceration.
  • Patent Document 1 describes that an alternative splicing variant of OATP1B3 (also known as SLCO1B3) is specifically expressed in cancer cells.
  • OATP1B3 also known as SLCO1B3
  • SLCO1B3 mutants having exon-like sequences are expressed in colon cancer and pancreatic cancer cells instead of exon1 and exon2 of wild-type SLCO1B3.
  • Non-patent Document 1 Show that the mutant is proteasomally degraded through post-translational modification, and that the mutant has less uptake of cholecystokin-8, one of the substrates of SLCO1B3, compared to the wild type. It is reported that. Compared to the wild type, this mutant has less localization to the cell membrane, and most of them are localized in the cell (Non-patent Document 1).
  • the SLCO1B3 variant having a transcription initiation site between exon2 and exon3 of wild-type SLCO1B3 was reported to be specifically expressed in cancer cells, and was shown to be able to function as a cancer biomarker. However, it is not clear at all whether these variants contribute to the onset and / or progression of cancer.
  • a cancer-specific variant of the SLCO1B3 gene encodes a novel human organic anion transporting polypeptide 1B3 (OATP1B3) localized mainly in the cytoplasm of colon and pancreatic cancer cells. 16.January 2013
  • the object of the present invention is to identify a molecule that is expressed in cancer cells and not expressed in normal cells or very low in expression, and that contributes to the onset and / or progression of cancer. It is to provide a substance that inhibits, and to provide a pharmaceutical composition for preventing or treating cancer using the same. Furthermore, the present invention also provides a method for screening a substance having an effect of preventing and / or treating cancer, a method for testing the malignancy or risk of malignancy of cancer in a test animal, and a method for preventing or treating cancer.
  • the present inventors have conducted a comprehensive exon expression analysis using exon specimens after lung cancer, and exon (exon1 *) specific to the cancer part compared to the non-cancer part.
  • SLCO1B3 a cancer type SLCO1B3 (Ct-SLCO1B3), which is SLCO1B3 using exon1 *, is a molecule that promotes proliferation, migration, and invasion in lung cancer cells.
  • Ct-SLCO1B3 increases the expression of snail and slug, suppresses the expression of E-cadherin and occludin, and induces the expression of matrix metalloproteinase 9 (MMP9). Therefore, the present inventors searched for a target Ct-SLCO1B3 region that can effectively suppress the expression and function of Ct-SLCO1B3, and found a nucleic acid that can effectively suppress the expression and function of Ct-SLCO1B3. . Based on these findings, the present inventors have demonstrated that suppression of Ct-SLCO1B3 expression can suppress the proliferation, migration, and invasion of cancer cells, thereby completing the present invention.
  • MMP9 matrix metalloproteinase 9
  • the present invention is as follows. 1) a double-stranded nucleic acid consisting of a sense strand and an antisense strand, and comprising a double-stranded region of at least 11 base pairs, wherein the antisense strand has at least 17 nucleotides and at most 30 Target Ct-SLCO1B3 (cancer-type solute carrier organic anion transporter family member 1B3) that is completely complementary to the nucleotide sequence of the DNA described in any of (I) to (II) below in the oligonucleotide chain of nucleotide length Double-stranded nucleic acid that reduces the expression of Ct-SLCO1B3 that is complementary to the RNA sequence: (I) DNA comprising the sequence of SEQ ID NO: 1, (II) A nucleic acid having substantially the same sequence as the DNA described in (I).
  • a single-stranded nucleic acid comprising only the antisense strand of the double-stranded nucleic acid according to any one of 1) to 6).
  • a pharmaceutical composition comprising a nucleic acid that suppresses the expression of Ct-SLCO1B3 as an active ingredient.
  • a pharmaceutical composition comprising the nucleic acid according to any one of 1) to 7) as an active ingredient.
  • the pharmaceutical composition according to 8) or 9 which is used for treatment or prevention of cancer.
  • the pharmaceutical composition according to 10) wherein the cancer is non-small cell lung cancer, liver cancer, pancreatic cancer, or esophageal cancer that expresses Ct-SLCO1B3.
  • a screening method for a substance having a preventive and / or therapeutic action for cancer comprising the following steps (1) to (3): (1) a step of contacting a test substance with a cell expressing Ct-SLCO1B3, (2) a step of measuring the expression level or function of Ct-SLCO1B3 in the cell, and (3) a decrease in the expression level or function of Ct-SLCO1B3 as compared to the case of measurement in the absence of the test substance.
  • a step of selecting a compound as a candidate for a substance having a preventive and / or therapeutic action for cancer comprising the following steps (1) to (3): (1) a step of contacting a test substance with a cell expressing Ct-SLCO1B3, (2) a step of measuring the expression level or function of Ct-SLCO1B3 in the cell, and (3) a decrease in the expression level or function of Ct-SLCO1B3 as compared to the case of measurement in the absence of the test substance.
  • a method for testing malignancy or risk of malignant cancer characterized by measuring the expression level of Ct-SLCO1B3 in a sample collected from a test animal, including the steps (1) and (2): (1) a step of measuring the expression level of Ct-SLCO1B3 in a sample collected from a test animal, (2) A step of determining that a test animal having an increased expression level has a high cancer malignancy or a high risk of becoming malignant in the future, as compared with a case of measuring in a sample derived from a normal animal.
  • Ct-SLCO1B3 It is possible to suppress the expression of Ct-SLCO1B3 by administering the nucleic acid of the present invention or the pharmaceutical composition containing the nucleic acid. Accordingly, it is possible to treat or prevent diseases associated with the expression of Ct-SLCO1B3, particularly cancer.
  • each SLCO1B3 in the established Ct-SLCO1B3 and Lt-SLCO1B3 high expression cell lines It is a figure which shows the anchorage independent proliferation of Ct-SLCO1B3 and Lt-SLCO1B3 high expression NCI-H23 cell line. It is a figure which shows the migration ability of CCI-SLCO1B3 and Lt-SLCO1B3 high expression NCI-H23 cell line. It is a figure which shows preparation of the C549-SLCO1B3 expression suppression A549 cell using a CRISPR / Cas9 system. It is a figure which shows the anchorage independent proliferation suppression of Ct-SLCO1B3 expression suppression A549 cell.
  • Solute carrier organic anion transporter family member 1B3 Solute carrier organic anion transporter family member 1B3
  • SLCO1B3 is also referred to as organic anion-transporting polypeptide 1B3 (OATP1B3; organic anion-transporting polypeptide 1B3) or LST-2 (Liver specific organic anion transporter-2).
  • the SLCO1B3 protein is a protein encoded by the SLCO1B3 gene, and its amino acid sequence and nucleotide sequence are known.
  • the amino acid sequence of human SLCO1B3 protein is registered as GenBank Accession No. NP — 062818 (SEQ ID NO: 3).
  • the nucleotide sequence encoding human SLCO1B3 protein is registered as GenBank Accession No. NM_019844 (SEQ ID NO: 4).
  • SLCO1B3 is a mammalian molecule.
  • mammals include humans and mammals other than humans.
  • mammals other than humans include rodents such as mice, rats, hamsters, and guinea pigs, and laboratory animals such as rabbits, pigs, cows, and goats.
  • Primates such as domestic animals such as horses and sheep, pets such as dogs and cats, monkeys, orangutans and chimpanzees.
  • SLCO1B3 is preferably a human, chimpanzee or orangutan molecule.
  • human-derived SLCO1B3 is preferred for the prevention or treatment of human diseases.
  • human liver-type SLCO1B3 (Lt-SLCO1B3) protein means human SLCO1B3 gene exon1, exon2, exon3, exon4, exon5, exon6, exon7, exon8, exon9, exon10, exon11, exon12, exon13, exon14 and A protein consisting of an amino acid sequence encoded by DNA consisting of exon15.
  • An example of the amino acid sequence of liver-type Lt-SLCO1B3 protein is SEQ ID NO: 3.
  • the liver-type SLCO1B3 protein refers to a protein comprising the same or substantially the same amino acid sequence as the protein consisting of the amino acid sequence of SEQ ID NO: 3.
  • human cancer-type SLCO1B3 (Ct-SLCO1B3) protein is a protein encoded by RNA of SLCO1B3 transcribed from the transcription start site between exon2 and exon3 of human Lt-SLCO1B3 gene, A protein comprising an amino acid sequence identical or substantially identical to the amino acid sequence encoded by No. 1.
  • Examples of human Ct-SLCO1B3 protein include a protein consisting of the amino acid sequence of SEQ ID NO: 5.
  • Ct-SLCO1B3 protein refers to a protein comprising the same or substantially the same amino acid sequence as the protein consisting of the amino acid sequence of SEQ ID NO: 5.
  • proteins having substantially the same amino acid sequence include amino acid sequences of about 80% or more, preferably about 90% or more, more preferably about 95% or more, more preferably about 97% or more, particularly A protein having an identity of preferably about 98% or more, most preferably about 99% or more is mentioned.
  • the protein having substantially the same amino acid sequence is preferably 1 to 10, preferably 1 to 8, more preferably 1 to 5, and more preferably 1 to 3 in the amino acid sequence.
  • proteins having substantially the same amino acid sequence have one or more common functions.
  • the functions of Ct-SLCO1B3 include (1) promotion of cell anchorage-independent growth, (2) promotion of cell migration ability, (3) promotion of cell invasion ability, (4) increase in snail expression level, ( 5) Increase in slug expression level, (6) Decrease in E-cadherin expression level, (7) Decrease in occludin expression level, (8) Increase in MMP9 expression level.
  • the amino acid sequence of SEQ ID NO: 5 A protein having substantially the same amino acid sequence as the human Ct-SLCO1B3 protein comprising any one of the above functions (1) to (8). Whether or not a protein has the functions (1) to (8) can be verified according to the method described in the examples or the method described in the screening method of the present invention.
  • exon1 * of the human Ct-SLCO1B3 gene refers to a region between the transcription start point existing between exon2 and exon3 of the Lt-SLCO1B3 gene and 5 ′ of the exon3 region of the Lt-SLCO1B3 gene and the transcription start point. Refers to the region up to the splicing donor site.
  • SEQ ID NO: 1 can be mentioned as the base sequence of exon1 * of the human Ct-SLCO1B3 gene.
  • RNA sequence that is completely complementary to the base sequence of the Ct-SLCO1B3 gene, which is the target of the nucleic acid of the present invention (herein, simply referred to as “target Ct-SLCO1B3 RNA sequence” or “ The “target Ct-SLCO1B3 RNA sequence of the nucleic acid of the present invention” is also described below.
  • the nucleic acid of the present invention targets RNA having a base sequence that is completely complementary to the base sequences of the following DNAs (I) to (II): (I) DNA comprising the sequence of SEQ ID NO: 1, (II) A nucleic acid having substantially the same sequence as the DNA described in (I).
  • a DNA sequence is used synonymously with a DNA base sequence
  • an RNA sequence is used synonymously with an RNA base sequence
  • nucleic acids having substantially the same base sequence include a base sequence of about 80% or more, preferably about 90% or more, more preferably about 95% or more, more preferably about 97% or more, particularly Preferably, the nucleic acid has about 98% or more, most preferably about 99% or more identity.
  • the nucleic acids having substantially the same nucleotide sequence are preferably 1 to 10, preferably 1 to 8, more preferably 1 to 5, even more preferably 1 to 3, particularly preferably nucleotide sequences. Examples include a nucleic acid having a base sequence in which deletion, substitution and / or addition mutation has occurred in one or two nucleotides, most preferably one nucleotide.
  • the target Ct-SLCO1B3 RNA sequence of the nucleic acid of the present invention is preferably a sequence included in SEQ ID NO: 2, more preferably any one of SEQ ID NOS: 6 to 13 described below, and particularly preferably SEQ ID NOS: 6, 7 , 8 or 9.
  • nucleic acid containing a base sequence complementary to the target Ct-SLCO1B3 RNA sequence is called an antisense strand nucleic acid
  • a nucleic acid containing a base sequence complementary to the base sequence of the antisense strand nucleic acid is the sense strand.
  • nucleic acid of the present invention is used to include an antisense strand nucleic acid, a sense strand nucleic acid, and a double-stranded nucleic acid paired with a sense strand and an antisense strand nucleic acid. It is done.
  • the nucleic acid of the present invention may be any molecule as long as it is a molecule obtained by polymerizing nucleotides or molecules having functions equivalent to the nucleotides, for example, RNA that is a polymer of ribonucleotides, or a polymer of deoxyribonucleotides.
  • RNA that is a polymer of ribonucleotides
  • deoxyribonucleotides examples thereof include DNA, chimeric nucleic acids composed of RNA and DNA, and nucleotide polymers in which at least one nucleotide of these nucleic acids is substituted with a molecule having a function equivalent to that of the nucleotide.
  • derivatives containing at least one molecule having a function equivalent to nucleotide in these nucleic acids are also included in the nucleic acid of the present invention.
  • Uracil (U) can be uniquely read as thymine (T).
  • nucleotide derivatives examples include nucleotide derivatives.
  • the nucleotide derivative may be any molecule as long as it is a nucleotide-modified molecule.
  • affinity with a complementary strand nucleic acid is increased.
  • a molecule in which ribonucleotides or deoxyribonucleotides are modified is preferably used.
  • nucleotide examples include a sugar moiety-modified nucleotide, a phosphodiester bond-modified nucleotide, a base-modified nucleotide, and a nucleotide in which at least one of the sugar moiety, phosphodiester bond and base is modified.
  • any or all of the chemical structure of the sugar of the nucleotide may be modified or substituted with any substituent, or may be substituted with any atom.
  • '-Modified nucleotides are preferably used.
  • Examples of 2′-modified nucleotides include those in which the 2′-OH group of ribose is H, OR, R, R′OR, SH, SR, NH 2 , NHR, NR 2 , N 3 , CN, F, Cl, Br, and Substituted with a substituent selected from the group consisting of I (R is alkyl or aryl, preferably alkyl having 1 to 6 carbon atoms and R ′ is alkylene, preferably alkylene having 1 to 6 carbon atoms)
  • a nucleotide preferably a nucleotide in which the 2′-OH group is substituted with H, F or a methoxy group, more preferably a nucleotide in which the 2′-OH group is substituted with F or a methoxy group.
  • 2'-OH group is 2- (methoxy) ethoxy group, 3-aminopropoxy group, 2-[(N, N-dimethylamino) oxy] ethoxy group, 3- (N, N-dimethylamino) propoxy group, 2- A substituent selected from the group consisting of [2- (N, N-dimethylamino) ethoxy] ethoxy group, 2- (methylamino) -2-oxoethoxy group, 2- (N-methylcarbamoyl) ethoxy group and 2-cyanoethoxy group Examples thereof include substituted nucleotides.
  • sugar-modified nucleotide examples include a bridged structure type artificial nucleic acid (BNA) having two cyclic structures by introducing a crosslinked structure into the sugar part, specifically, an oxygen at the 2 ′ position.
  • BNA bridged structure type artificial nucleic acid
  • LNA Locked ⁇ Nucleic Acid
  • ENA ethylene bridged nucleic acid
  • PNA peptide nucleic acids
  • OPNA oxypeptide nucleic acids
  • Peptide ribonucleic acid PRNA
  • any or all of the chemical structure of the nucleotide phosphodiester bond modified or substituted with any substituent or any atom may be used.
  • a nucleotide in which a phosphodiester bond is replaced with a phosphorothioate bond a nucleotide in which a phosphodiester bond is replaced with a phosphorodithioate bond, a nucleotide in which a phosphodiester bond is replaced with an alkylphosphonate bond, a phosphate
  • examples thereof include nucleotides in which a diester bond is substituted with a phosphoramidate bond.
  • the base-modified nucleotide may be any nucleotide as long as it is part or all of the nucleotide base chemical structure modified or substituted with an arbitrary substituent, or substituted with an arbitrary atom.
  • oxygen atoms are substituted with sulfur atoms
  • hydrogen atoms are substituted with alkyl groups having 1 to 6 carbon atoms, halogens, etc.
  • methyl groups are hydrogen, hydroxymethyl, alkyl groups with 2 to 6 carbon atoms, etc.
  • the amino group is substituted with an alkyl group having 1 to 6 carbon atoms, an alkanoyl group having 1 to 6 carbon atoms, an oxo group, a hydroxy group, or the like.
  • nucleotide derivative examples include a peptide, protein, sugar, lipid, phospholipid, phenazine, folate, phenanthridine, anthraquinone, acridine, a nucleotide derivative in which at least one of nucleotide or sugar moiety, phosphodiester bond or base is modified.
  • examples include those obtained by adding another chemical substance such as fluorescein, rhodamine, coumarin, and dye, directly or via a linker.
  • Specific examples include 5'-polyamine-added nucleotide derivatives, cholesterol-added nucleotide derivatives, and steroid-added nucleotide derivatives.
  • the nucleotide derivative may form a crosslinked structure such as an alkylene structure, a peptide structure, a nucleotide structure, an ether structure, an ester structure, and a structure obtained by combining at least one of these with other nucleotides or nucleotide derivatives in the nucleic acid.
  • the nucleic acid of the present invention includes those in which some or all atoms in the nucleic acid molecule are substituted with atoms (isotopes) having different mass numbers.
  • “complementary” means a relationship that allows base pairing between two bases, for example, a moderate hydrogen such as a relationship between adenine and thymine or uracil, and a relationship between guanine and cytosine. It means a double-stranded structure as a whole double-stranded region through a bond.
  • the antisense strand complementary to the target Ct-SLCO1B3 RNA sequence may contain one or more base substitutions in the base sequence that is completely complementary to the partial base sequence of the RNA.
  • the antisense strand contains 1 to 8, preferably 1 to 6, 1 to 4, 1 to 3, particularly 2 or 1 mismatch bases to the target sequence of the target gene. You may have.
  • the antisense strand when it is 21 bases long, it may have 6, 5, 4, 3, 2 or 1 mismatch bases with respect to the target sequence of the target gene.
  • the position of the mismatch may be the 5 ′ end or 3 ′ end of each sequence.
  • “complementary” includes a case where one nucleotide sequence is a sequence in which one or a plurality of bases are added and / or deleted in a nucleotide sequence that is completely complementary to the other nucleotide sequence.
  • the target Ct-SLCO1B3 RNA and the antisense strand nucleic acid of the present invention include one or two of the antisense strand and / or the target Ct-SLCO1B3 RNA due to the addition and / or deletion of a base in the antisense strand. You may have a bulge base.
  • “completely complementary” of two nucleotide sequences refers to having 0 mismatch bases between the nucleotide sequences, that is, complementary in all bases.
  • the nucleic acid of the present invention is a nucleic acid containing a base sequence complementary to a part of the base sequence of the target Ct-SLCO1B3 RNA and / or a nucleic acid containing a base sequence complementary to the base sequence of the nucleic acid. , May be composed of any nucleotide or derivative thereof.
  • a nucleic acid containing a base sequence complementary to a target Ct-SLCO1B3 RNA sequence and a nucleic acid containing a base sequence complementary to the base sequence of the nucleic acid are double-stranded.
  • the length of the sequence capable of forming a duplex is usually 11 to 35 bases, preferably 15 to 30 bases, more preferably 17 to 25 bases, -23 bases are more preferred, and 19-23 bases are particularly preferred.
  • nucleic acid containing a base sequence complementary to the target Ct-SLCO1B3 RNA sequence is used.
  • these nucleic acids 1 to 3 bases, preferably 1 to 2 bases, more preferably May be one in which one base is deleted, substituted or added.
  • the nucleic acid that suppresses the expression of Ct-SLCO1B3 is a single-stranded nucleic acid that includes a base sequence complementary to the target Ct-SLCO1B3 RNA sequence and suppresses the expression of Ct-SLCO1B3, or the target Ct -Double-stranded nucleic acid consisting of a nucleic acid containing a base sequence complementary to the SLCO1B3 RNA sequence and a nucleic acid containing a base sequence complementary to the base sequence of the nucleic acid, and suppressing the expression of Ct-SLCO1B3 Are preferably used.
  • a double-stranded nucleic acid refers to a nucleic acid having a double-stranded region in which two nucleotide chains are paired.
  • a double-stranded region refers to a portion where nucleotides constituting a double-stranded nucleic acid or a derivative thereof constitute a base pair to form a double strand.
  • the double-stranded region is usually 11 to 27 base pairs, preferably 15 to 25 base pairs, more preferably 15 to 23 base pairs, still more preferably 17 to 21 base pairs, and particularly preferably 17 to 19 base pairs.
  • the single-stranded nucleic acid constituting the double-stranded nucleic acid usually consists of 11 to 30 bases, preferably 15 to 29 bases, more preferably 15 to 27 bases, and 15 to 25 bases. More preferably, it consists of 17 to 23 bases, particularly preferably 19 to 21 bases.
  • the double-stranded nucleic acid of the present invention has an additional nucleotide or nucleotide derivative that does not form a duplex on the 3 ′ side or 5 ′ side following the double-stranded region, this is referred to as an overhang.
  • the nucleotide constituting the overhang may be ribonucleotide, deoxyribonucleotide or a derivative thereof.
  • the double-stranded nucleic acid having a protruding portion one having a protruding portion consisting of 1 to 6 bases, usually 1 to 3 bases at the 3 ′ end or 5 ′ end of at least one strand is used.
  • Those having protrusions are preferably used, and examples thereof include those having protrusions made of dTdT or UU.
  • a double-stranded nucleic acid having a protruding portion on both the antisense strand and the sense strand can be provided on the antisense strand only, the sense strand only, and both the antisense strand and the sense strand. Is preferably used.
  • the antisense strand is sufficiently complementary to the target Ct-SLCO1B3 RNA in an oligonucleotide strand consisting of at least 17 nucleotides and at most 30 nucleotides, including the double-stranded region and the subsequent overhang. is there.
  • the double-stranded nucleic acid of the present invention has, for example, a nucleic acid molecule (WO2005 / 089287) that generates the above-mentioned double-stranded nucleic acid by the action of a ribonuclease such as Dicer, or a 3′-end or 5′-end overhang.
  • a double-stranded nucleic acid that forms a blunt end a double-stranded nucleic acid in which only the sense strand protrudes (US2012 / 0040459), and the like can also be used.
  • the antisense strand of the double-stranded nucleic acid of the present invention comprises a sequence selected from the group consisting of SEQ ID NOs: 14-21, more preferably a sequence selected from the group consisting of SEQ ID NOs: 14-17. Including.
  • the sense strand of the double-stranded nucleic acid of the present invention comprises a sequence selected from the group consisting of SEQ ID NOs: 22-29, more preferably a sequence selected from the group consisting of SEQ ID NOs: 22-25 including.
  • the double-stranded nucleic acid of the present invention comprises SEQ ID NO: 14 / SEQ ID NO: 22, SEQ ID NO: 15 / SEQ ID NO: 23, SEQ ID NO: 16 / SEQ ID NO: 24, SEQ ID NO: 17 / SEQ ID NO: 25 described in Table 1.
  • a pair of antisense strands / sense strands selected from the group consisting of: SEQ ID NO: 18 / SEQ ID NO: 26, SEQ ID NO: 19 / SEQ ID NO: 27, SEQ ID NO: 20 / SEQ ID NO: 28, and SEQ ID NO: 21 / SEQ ID NO: 29 Contains an array.
  • the double-stranded nucleic acid of the present invention comprises SEQ ID NO: 14 / SEQ ID NO: 22, SEQ ID NO: 15 / SEQ ID NO: 23, SEQ ID NO: 16 / SEQ ID NO: 24, and SEQ ID NO: 17 / SEQ ID NO: 25 described in Table 1.
  • a pair of antisense / sense strand sequences selected from the group consisting of:
  • double-stranded nucleic acid of the present invention a nucleic acid having the same sequence as the base sequence of the target gene or its complementary strand may be used, but the 5 ′ end or 3 ′ of at least one strand of the nucleic acid may be used.
  • a double-stranded nucleic acid comprising a nucleic acid from which one to four bases have been deleted and a nucleic acid containing a base sequence complementary to the base sequence of the nucleic acid may be used.
  • the double-stranded nucleic acid of the present invention is a double-stranded RNA (dsRNA) in which RNAs form a double strand, a double-stranded DNA (dsDNA) in which DNAs form a double strand, or a double-stranded RNA and DNA. It may be a hybrid nucleic acid that forms a strand. Alternatively, one or both of the double strands may be a chimeric nucleic acid of DNA and RNA. Double-stranded RNA (dsRNA) is preferred.
  • the second nucleotide from the 5 ′ end of the antisense strand of the present invention is preferably complementary to the second deoxyribonucleotide from the 3 ′ end of the target Ct-SLCO1B3 RNA sequence, and 2 nucleotides from the 5 ′ end of the antisense strand.
  • the seventh nucleotide is completely complementary to the second to seventh deoxyribonucleotides from the 3 ′ end of the target Ct-SLCO1B31RNA sequence, and the second to eleventh nucleotides from the 5 ′ end of the antisense strand Is more preferably completely complementary to deoxyribonucleotides 2 to 11 from the 3 ′ end of the target Ct-SLCO1B3 RNA sequence.
  • the 11th nucleotide from the 5 ′ end of the antisense strand in the nucleic acid of the present invention is preferably complementary to the 11th deoxyribonucleotide from the 3 ′ end of the target Ct-SLCO1B3 RNA sequence, More preferably, the 9th to 13th nucleotides from the end are completely complementary to the 9th to 13th deoxyribonucleotides from the 3 'end of the target Ct-SLCO1B3BRNA sequence, and 7 to 7 from the 5' end of the antisense strand. More preferably, the 15th nucleotide is completely complementary to the 7th to 15th deoxyribonucleotides from the 3 ′ end of the target Ct-SLCO1B3 RNA sequence.
  • the method for producing the nucleic acid of the present invention is not particularly limited, and examples thereof include a method using known chemical synthesis or an enzymatic transcription method.
  • methods using known chemical synthesis include phosphoramidite method, phosphorothioate method, phosphotriester method, CEM method [Nucleic® Acid® Research, 35, 3287 (2007)].
  • ABI3900 high-throughput nucleic acid synthesis Can be synthesized by a machine (Applied Biosystems). After the synthesis is completed, elimination from the solid phase, deprotection of the protecting group, purification of the target product, and the like are performed. It is desirable to obtain a nucleic acid having a purity of 90% or more, preferably 95% or more by purification.
  • the synthesized and purified sense strand and antisense strand are in an appropriate ratio, for example, 0.1 to 10 equivalents, preferably 0.5 to 2 equivalents of sense strand to 1 equivalent of antisense strand.
  • 0.9 to 1.1 equivalents, more preferably equimolar amounts are mixed and then annealed, or the mixed product may be used directly without the step of annealing. Annealing may be performed under any conditions that can form a double-stranded nucleic acid.
  • the sense strand and the antisense strand are mixed in approximately equimolar amounts, and then heated at about 94 ° C. for about 5 minutes.
  • Examples of the enzymatic transcription method for producing the nucleic acid of the present invention include a method by transcription using a phage RNA polymerase, for example, T7, T3, or SP6 RNA polymerase, using a plasmid or DNA having the target base sequence as a template. .
  • the nucleic acid of the present invention can be introduced into cells using a transfection carrier, preferably a cationic carrier such as a cationic liposome. It can also be directly introduced into cells by the calcium phosphate method, electroporation method or microinjection method.
  • a transfection carrier preferably a cationic carrier such as a cationic liposome. It can also be directly introduced into cells by the calcium phosphate method, electroporation method or microinjection method.
  • the 5 ′ end, 3 ′ end and / or the inside of the sequence may be modified with one or more ligands or fluorophores, and a nucleic acid modified with a ligand or fluorophore is also called a conjugate nucleic acid.
  • a nucleic acid modified with a ligand or fluorophore is also called a conjugate nucleic acid.
  • the 5 'end, 3' end and / or the inside of the sequence can be modified by reacting a modifying agent capable of reacting on the solid phase.
  • a conjugated nucleic acid can be obtained by previously synthesizing and purifying a nucleic acid into which a functional group such as an amino group, a mercapto group, an azide group, or a triple bond has been introduced, and allowing a modifying agent to act on them.
  • the ligand may be a molecule having affinity for a biomolecule.
  • lipids such as cholesterol, fatty acid, tocopherol, and retinoid, N-acetylgalactosamine (GalNAc), galactose (Gal), mannose (Man), etc.
  • Saccharides full antibodies, antibodies such as Fab and VHH, low density lipoprotein (LDL), proteins such as human serum albumin, peptides such as RGD, NGR, R9, and CPP, small molecules such as folic acid, synthetic polyamino acids, etc.
  • LDL low density lipoprotein
  • fluorophores include the Cy3 series, Alexa series, and black hole quenchers.
  • a vector that can be introduced into cells and expressed can be used.
  • the nucleic acid or the like can be expressed by inserting the sequence encoding the nucleic acid of the present invention downstream of the promoter in the expression vector, constructing the expression vector, and introducing it into a cell.
  • Expression vectors include pCDNA6.2-GW / miR (Invitrogen), pSilencer® 4.1-CMV (Ambion), pSINsi-hH1 DNA (Takara Bio), pSINsi-hU6 DNA (Takara Bio), pENTR / U6 (Invitrogen) etc. can be mentioned.
  • virus vectors include retrovirus vectors, lentivirus vectors, adenovirus vectors, adeno-associated virus vectors, and the like.
  • the double-stranded nucleic acid of the present invention can suppress the expression of Ct-SLCO1B3 mRNA after being introduced into cells at a concentration of several pM to several nM and then cultured for 24 hours or more, for example 48 hours.
  • the evaluation of the Ct-SLCO1B3 mRNA expression inhibitory activity of the double-stranded nucleic acid of the present invention was carried out by transfecting the nucleic acid or the like into a human cell line using a cationic liposome, etc. This can be done by quantifying the expression level of Ct-SLCO1B3 mRNA in the cell line.
  • the nucleic acid having a Ct-SLCO1B3 expression-suppressing activity is a nucleic acid containing a base sequence complementary to a part of the base sequence of Ct-SLCO1B3 mRNA, and Ct-SLCO1B3 A single-stranded nucleic acid that suppresses the expression of SLCO1B3.
  • the single-stranded nucleic acid constituting the nucleic acid usually consists of 8 to 30 bases, preferably 12 to 30 bases, more preferably 12 to 20 bases.
  • the partial base sequence of Ct-SLCO1B3 mRNA is preferably a part of a continuous base sequence included in an RNA sequence completely complementary to SEQ ID NO: 1, and more preferably a sequence included in SEQ ID NO: 2.
  • the single-stranded nucleic acid of the present invention can suppress the expression of Ct-SLCO1B3 mRNA after being introduced into cells at a concentration of several pM to several nM and then cultured for 24 hours or more, for example 48 hours.
  • the evaluation of the Ct-SLCO1B3 mRNA expression inhibitory activity of the single-stranded nucleic acid of the present invention was carried out by transfecting the nucleic acid or the like into a human cell line using a cationic liposome or the like, culturing for a certain time, This can be done by quantifying the expression level of Ct-SLCO1B3 mRNA in a human cell line.
  • composition Containing Nucleic Acid that Suppresses Ct-SLCO1B3 Expression comprises a nucleic acid that suppresses Ct-SLCO1B3 expression.
  • a nucleic acid that suppresses the expression of Ct-SLCO1B3, which is contained as an active ingredient in the pharmaceutical composition of the present invention, has a growth inhibitory action, an invasion metastasis inhibitory action, etc. on various cancer cells characterized by the expression of Ct-SLCO1B3. It becomes possible to suppress malignant transformation of cancer. Therefore, it can be used for treatment, prevention, progression prevention, and malignant inhibition of cancer characterized by the expression of Ct-SLCO1B3.
  • the pharmaceutical composition of the present invention can be used in mammals (eg, humans, chimpanzees, gorillas, monkeys, cats, pigs, horses, cows, mice, rats, guinea pigs, dogs, rabbits, etc., preferably humans, chimpanzees).
  • mammals eg, humans, chimpanzees, gorillas, monkeys, cats, pigs, horses, cows, mice, rats, guinea pigs, dogs, rabbits, etc., preferably humans, chimpanzees.
  • gorillas, particularly preferably humans are useful as therapeutic or prophylactic agents for cancer characterized by the expression of Ct-SLCO1B3.
  • the nucleic acid that suppresses the expression of Ct-SLCO1B3, which is contained in the pharmaceutical composition of the present invention, is an increase in the expression of snail and slug due to the high expression of Ct-SLCO1B3, and the expression suppression of E-cadherin and occludin And has the effect of inhibiting the induction of MMP9 expression. Therefore, a substance that inhibits the expression of Ct-SLCO1B3 has an action of suppressing malignant cancer characterized by the expression of Ct-SLCO1B3. It is known that increased expression of snail and slug promotes epithelial-mesenchymal transition.
  • Whether or not a cell expresses Ct-SLCO1B3 can be verified by a method known per se.
  • the expression level of Ct-SLCO1B3 is determined based on the nucleic acid (DNA) that can hybridize under stringent conditions with a nucleic acid that can hybridize with DNA encoding Ct-SLCO1B3 under a stringent condition or with a complementary base sequence.
  • DNA nucleic acid
  • the expression level of Ct-SLCO1B3 can also be measured at the protein level by detecting Ct-SLCO1B3 protein using an antibody against Ct-SLCO1B3.
  • total RNA was extracted from cells suspected of expressing Ct-SLCO1B3, cDNA was synthesized by reverse transcription reaction, and PCR was performed using primers of SEQ ID NO: 30 and SEQ ID NO: 31. Also good.
  • the stringent conditions include, for example, the conditions described in Current Protocols in Molecular Biology, John Wiley & Sons, 6.3.1-6.3.6, 1999, for example, 6 ⁇ SSC (sodium chloride / sodium citrate) / 45 ° C. Hybridization, followed by one or more washings at 0.2 ⁇ SSC / 0.1% SDS / 50 to 65 ° C., etc., but those skilled in the art will appropriately select the hybridization conditions that give the same stringency. can do.
  • the target cancer of the pharmaceutical composition of the present invention is not particularly limited as long as cancer cells express Ct-SLCO1B3.
  • solid cancer transitional cell cancer, colon cancer, colorectal cancer, colon cancer, lung cancer ( Small cell carcinoma), lung cancer (non-small cell lung cancer), renal cancer (renal cell carcinoma), renal pelvic and ureteral cancer, biliary tract cancer, liver cancer (hepatocellular carcinoma), brain tumor, glioma (glioma), glioblastoma, multi Type glioblastoma, pancreatic cancer, head and neck cancer (squamous cell carcinoma), multiple myeloma, bone soft tissue tumor, prostate cancer, penile cancer, testicular cancer, ovarian cancer, gastrointestinal stromal tumor (GIST), gastric cancer , Female genital cancer, cervical cancer, breast cancer, melanoma, lymphoma (non-Hodgkin), lymphoma (Hodgkin), lymphoma (diffuse large cell type), leukemia (
  • the pharmaceutical composition of the present invention and the nucleic acid of the present invention can be suitably used for the prevention or treatment of cancer exemplified above.
  • prevention of cancer refers to suppressing or delaying the occurrence of cancer by administering to an animal (patient) who is at risk of suffering from cancer, or to an animal (patient) after cancer treatment. It is also used as a term encompassing prevention of cancer recurrence by administration.
  • treatment of cancer refers to a tumor that suppresses or delays the progression of cancer or suppresses or delays the growth of a tumor by administration to an animal (patient) suffering from cancer. Is used as a term encompassing reducing or eliminating the like.
  • the nucleic acid that suppresses the expression of Ct-SLCO1B3 contained as an active ingredient in the pharmaceutical composition of the present invention is particularly limited as long as it acts on the transcribed Ct-SLCO1B3 RNA and specifically inhibits the expression. It is not something.
  • Ct-SLCO1B3 may be a double-stranded RNA or a single-stranded RNA.
  • the nucleic acid that suppresses the expression of Ct-SLCO1B3 contained as an active ingredient in the pharmaceutical composition of the present invention may be a ribozyme nucleic acid.
  • the nucleic acid contained as an active ingredient in the pharmaceutical composition of the present invention can target any region of RNA encoding Ct-SLCO1B3 including a region other than exon1 * of Ct-SLCO1B3 as long as it has a desired effect.
  • the pharmaceutical composition of the present invention expresses an SLCO1B3 variant other than Ct-SLCO1B3, such as liver-type SLCO1B3. It is preferably administered to non-tissue.
  • the pharmaceutical composition of the present invention contains the nucleic acid of the present invention as an active ingredient.
  • the method for producing a nucleic acid contained as an active ingredient in the pharmaceutical composition of the present invention is produced according to the above-described method for producing a nucleic acid of the present invention.
  • the nucleic acid that suppresses the expression of Ct-SLCO1B3 contained as an active ingredient in the pharmaceutical composition of the present invention may contain a nucleic acid that suppresses the expression of multiple types of Ct-SLCO1B3 as long as it has a desired effect.
  • the content of the nucleic acid that suppresses the expression of Ct-SLCO1B3, which is contained as an active ingredient in the pharmaceutical composition of the present invention, is not particularly limited as long as it has a desired effect. About 99.9% by weight, preferably about 0.1 to about 50% by weight.
  • the pharmaceutical composition of the present invention can be administered alone, or can be used in appropriate combination with other anticancer agents and / or radiation therapy.
  • anticancer agents include antimetabolites (eg, methotrexate, 5-fluorouracil, etc.), alkylating agents (eg, cyclophosphamide, ifosfamide, etc.), platinum anticancer agents (eg, cisplatin, carboplatin, etc.), topoisomerase, etc.
  • Inhibitors eg, etoposide, etc.
  • anticancer antibiotics eg, mitomycin, adriamycin, etc.
  • plant-derived anticancer agents eg, vincristine, vindesine, taxol, etc.
  • tyrosine kinase inhibitors eg, gefitinib, imatinib, etc.
  • humanized antibodies eg, Herceptin
  • the nucleic acids of the invention may be administered per se or as a suitable pharmaceutical composition.
  • the pharmaceutical composition used for administration may contain the nucleic acid of the present invention and a pharmacologically acceptable carrier, diluent or excipient.
  • a pharmaceutical composition is provided as a dosage form suitable for oral or parenteral administration.
  • the nucleic acid of the present invention can be formulated and administered according to a method known per se. That is, as long as it has a desired effect, the nucleic acid of the present invention is inserted alone or in a functional manner into an appropriate expression vector for mammalian cells such as a retrovirus vector, adenovirus vector, adenovirus associated virus vector, etc.
  • the nucleic acid can be administered as it is or together with an auxiliary agent for promoting intake by a catheter such as a gene gun or a hydrogel catheter. Alternatively, it can be aerosolized and locally administered into the trachea as an inhalant, but is not limited thereto.
  • the nucleic acid of the invention is formulated in a form suitable for local administration to a tumor (and / or surrounding tumor) of a subject animal.
  • the nucleic acid of the present invention may be formulated as a gel such as atelocollagen gel, cream, liposome, exosome or the like.
  • injections are dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, infusions, and the like. May be included.
  • Such an injection can be prepared according to a known method.
  • a method for preparing an injection it can be prepared, for example, by dissolving, suspending or emulsifying the nucleic acid of the present invention in a sterile aqueous liquid or oily liquid usually used for injection.
  • an aqueous solution for injection for example, an isotonic solution containing physiological saline, glucose and other adjuvants, and the like are used, and suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct-of-hydrogenated-castor-oil)) and the like may be used in combination.
  • alcohol eg, ethanol
  • polyalcohol eg, Propylene glycol, polyethylene glycol
  • nonionic surfactants eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct-of-hydrogenated-castor-oil)
  • oily liquid for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solub
  • the prepared injection solution is preferably filled in a suitable ampoule.
  • Suppositories used for rectal administration may be prepared by mixing the nucleic acid with a normal suppository base.
  • examples of compositions for parenteral administration include, but are not limited to, inhalants such as gels and aerosols, creams, ointments, lotions and the like in addition to the above compositions.
  • compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), and syrups. Agents, emulsions, suspensions and the like.
  • Such a composition is produced by a known method and may contain a carrier, a diluent or an excipient usually used in the pharmaceutical field.
  • carriers and excipients for tablets include lactose, starch, sucrose, and magnesium stearate, but are not limited thereto.
  • the above parenteral or oral pharmaceutical composition is conveniently prepared in a dosage unit form suitable for the dosage of the active ingredient.
  • dosage form of such a dosage unit include, but are not limited to, tablets, pills, capsules, injections (ampoules), and suppositories.
  • the pharmaceutical composition of the present invention is further formulated into a formulation (injection) with the above-mentioned nucleic acid alone or with a carrier such as a liposome for the purpose of improving pharmacokinetics, prolonging the half-life, and improving cellular uptake efficiency, It may be administered subcutaneously or the like.
  • the pharmaceutical composition of the present invention can further contain a carrier effective for transferring nucleic acid into cells.
  • a carrier effective for transferring the nucleic acid into the cell include a cationic carrier.
  • the cationic carrier include cationic liposomes and cationic polymers.
  • a carrier utilizing a viral envelope may be used as an effective carrier for transferring nucleic acids into cells.
  • JetSI Qbiogene
  • Jet-PEI polyethyleneimine; Qbiogene
  • As a carrier using a viral envelope GenomeOne (HVJ-E liposome; Ishihara Sangyo Co., Ltd.) and the like are preferably used.
  • the composition containing the nucleic acid of the present invention and the above carrier can be prepared by methods known to those skilled in the art. For example, it can be prepared by mixing a carrier dispersion having an appropriate concentration and a nucleic acid solution.
  • a cationic carrier since the nucleic acid is usually negatively charged in an aqueous solution, it can be easily prepared by mixing in an aqueous solution by a conventional method.
  • the aqueous solvent used for preparing the composition include electrolyte solutions such as water for injection, distilled water for injection, and physiological saline, and sugar solutions such as glucose solution and maltose solution.
  • those skilled in the art can appropriately select conditions such as pH and temperature when preparing the composition.
  • the composition can be made into a uniform composition by carrying out a dispersion treatment using an ultrasonic dispersing device or a high-pressure emulsifying device.
  • the optimum method and conditions for the preparation of the composition containing the carrier and the nucleic acid depend on the carrier to be used, so that those skilled in the art can select the optimum method for the carrier to be used without being bound by the above method.
  • compositions composed of composite particles containing nucleic acids and lead particles and a lipid membrane covering the composite particles is also preferably used.
  • the lead particles include lipid aggregates, liposomes, emulsion particles, polymers, metal colloids, fine particle preparations, and the like, and cationic liposomes are preferably used.
  • the lead particles in the present invention may be composed of a complex obtained by combining two or more lipid aggregates, liposomes, emulsion particles, polymers, metal colloids, fine particle formulations, etc., and lipid aggregates, liposomes, emulsion particles, A complex formed by combining a polymer, a metal colloid, a fine particle preparation, and the like with another compound (eg, sugar, lipid, inorganic compound, etc.) may be used as a constituent component.
  • the lipid membrane that coats the composite particles include non-cationic lipids, lipids that prevent aggregation of particles, and cationic lipids.
  • composition can be prepared according to the method described in, for example, International Publication No. 2006/080118 Pamphlet.
  • the nucleic acid of the present invention is preferably contained, for example, usually 5 to 500 mg per dosage unit dosage form, particularly 5 to 100 mg for injections and 10 to 250 mg for other dosage forms.
  • the administration method of the nucleic acid of the present invention or the pharmaceutical composition of the present invention is not particularly limited as long as the desired effect is obtained.
  • a desired site for example, a tumor site and / or a tumor periphery
  • Topical administration As an example of the local administration method, a method known per se such as injection can be appropriately used. Since the nucleic acid of the present invention targets Ct-SLCO1B3 expressed specifically in cancer tissues and is expected to act only in cancer tissues even when administered systemically, it may be administered systemically.
  • nucleic acid of the present invention or the pharmaceutical composition of the present invention is administered systemically, in order to achieve stable and highly efficient delivery to a desired site (for example, tumor site and / or surrounding tumor)
  • a desired site for example, tumor site and / or surrounding tumor
  • Known drug delivery techniques can also be used as appropriate.
  • the dosage of the above-mentioned pharmaceutical containing the nucleic acid of the present invention varies depending on the administration subject, target disease, symptom, administration route, etc., but for example, when used for the treatment / prevention of cancer, the nucleic acid of the present invention. Is usually about 0.01 to 20 mg / kg body weight, preferably about 0.1 to 10 mg / kg body weight, more preferably about 0.1 to 5 mg / kg body weight, about 1 to 5 times a day, preferably 1 day a day. It is convenient to administer about 3 times by intravenous injection. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If symptoms are particularly severe, the dose may be increased according to the symptoms.
  • compositions may appropriately contain other active ingredients as long as an undesirable interaction is not caused by blending with the nucleic acid of the present invention.
  • Ct-SLCO1B3 Screening of drug candidate compounds for disease Expression of Ct-SLCO1B3 promotes anchorage-independent growth of cells and enhances cell migration ability and invasion ability. Therefore, a compound that suppresses the expression and / or function of Ct-SLCO1B3 is used as a prophylactic and / or therapeutic agent for cancer that suppresses anchorage-independent growth, increased invasive ability and / or enhanced migration ability of cells. be able to. That is, a cell producing Ct-SLCO1B3 can be used as a tool for screening a substance having a preventive and / or therapeutic action for cancer by using the expression level and / or function of Ct-SLCO1B3 as an index.
  • Ct-SLCO1B3 increases the expression of snail and slug involved in epithelial-mesenchymal transition.
  • Ct-SLCO1B3 suppresses the expression of E-cadherin and occludin, which are known to be down-regulated in cancer, and induces the expression of MMP9 related to invasion. Therefore, cells that produce Ct-SLCO1B3 are screened for substances that have an activity to change the expression level of a molecule selected from the group consisting of snail, slug, E-cadherin, occludin and MMP9 due to the expression of Ct-SLCO1B3. Can be used as a tool for.
  • the present invention provides a method for screening a substance having a preventive and / or therapeutic action for cancer (also referred to as the screening method of the present invention), comprising the following steps (1) to (3): (1) a step of contacting a test substance with a cell expressing Ct-SLCO1B3, (2) a step of measuring the expression level or function of Ct-SLCO1B3 in the cell, and (3) a compound that decreases the expression level or function compared to the case of measurement in the absence of the test substance. Selecting as a candidate for a substance having a preventive and / or therapeutic action.
  • the screening method involves culturing cells capable of producing Ct-SLCO1B3 in the presence or absence of a test substance. And comparing the expression level and / or function of Ct-SLCO1B3 under both conditions.
  • Cells having the ability to produce Ct-SLCO1B3 used in the above screening method include cells derived from humans or other mammals that naturally express Ct-SLCO1B3 or biological samples containing the same (for example, excised cancer Part, blood, tissue, organ, etc.). In the case of blood, tissues, organs, etc. derived from non-human animals, they may be isolated from the living body and cultured, or the test substance is administered to the living body itself, and these biological samples are isolated after a certain period of time. May be. As a human-derived cultured cell line expressing Ct-SLCO1B3, A549 cells which are human alveolar basal epithelial adenocarcinoma cells are preferably used.
  • Examples of cells having the ability to produce Ct-SLCO1B3 include various transformants prepared by known and commonly used genetic engineering techniques.
  • animal cells such as NCI-H2 cells, HEK293 cells, COS7 cells, and CHO cells are preferably used. Specifically, it hybridizes under stringent conditions with a base sequence represented by DNA encoding Ct-SLCO1B3 (SEQ ID NO: 32) or a base sequence complementary to the base sequence, and SEQ ID NO: 5
  • a DNA comprising a nucleotide sequence that encodes a polypeptide having the same function as the protein consisting of the amino acid sequence represented by is ligated downstream of a promoter in an appropriate expression vector and introduced into a host animal cell. be able to.
  • a method for preparing a gene encoding Ct-SLCO1B3 is described below.
  • the gene encoding Ct-SLCO1B3 can be obtained by conventional genetic engineering methods (eg, Sambrook J., Frisch E.F., Maniatis T., Molecular Cloning 2nd edition), Cold Spring Harbor Laboratory ( The method can be obtained according to the method described in Cold Spring Harbor Laboratory press). That is, DNA encoding Ct-SLCO1B3 is derived from cells / tissues that produce Ct-SLCO1B3 as described above by synthesizing an appropriate oligonucleotide as a probe or primer based on the base sequence represented by SEQ ID NO: 32, for example.
  • the cDNA or cDNA library can be cloned using a hybridization method or a PCR method.
  • Hybridization can be performed, for example, according to the method described in Molecular Cloning 2nd edition (above). When a commercially available library is used, hybridization can be performed according to the method described in the instruction manual attached to the library.
  • the cloned DNA can be used as it is or after digestion with a restriction enzyme or addition of a linker, if desired.
  • the DNA may have ATG as a translation initiation codon on the 5 ′ end side and TAA, TGA or TAG as a translation termination codon on the 3 ′ end side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.
  • Ct-SLCO1B3 protein
  • Ct-SLCO1B3 protein
  • Ct-SLCO1B3 can be obtained from the obtained culture.
  • the plasmid include a promoter that can replicate autonomously in a host cell, can replicate autonomously, can be easily isolated and purified from the host cell, and can function in the host cell.
  • Preferred examples include those in which a gene encoding Ct-SLCO1B3 is introduced into an expression vector having a detectable marker.
  • animal cell expression plasmids eg, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo
  • bacteriophages such as ⁇ phage
  • animal virus vectors such as retrovirus, vaccinia virus, adenovirus, etc. It can also be used.
  • the promoter may be any promoter as long as it is appropriate for the host used for gene expression.
  • SR ⁇ promoter for example, SR ⁇ promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter, ⁇ -actin A gene promoter, aP2 gene promoter, etc. are used.
  • CMV promoter, SR ⁇ promoter and the like are preferable.
  • an expression vector containing an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), etc. is used as desired. Can do.
  • selectable markers examples include a dihydrofolate reductase gene (hereinafter abbreviated as dhfr, methotrexate (MTX) resistance), an ampicillin resistance gene (hereinafter abbreviated as ampr), a neomycin resistance gene (hereinafter abbreviated as amplicon). , G418 resistance, which may be abbreviated as neor).
  • dhfr dihydrofolate reductase gene
  • ampr ampicillin resistance gene
  • amplicon a neomycin resistance gene
  • G418 resistance which may be abbreviated as neor
  • the target gene can also be selected using a medium that does not contain thymidine.
  • the plasmid obtained as described above can be introduced into the host cell by an ordinary genetic engineering method.
  • the transformant can be cultured by a method known per se. Transformation can be performed by calcium phosphate coprecipitation method, PEG method, electroporation method, microinjection method, lipofection method and the like.
  • the transformed cells obtained as described above, the mammalian cells having the ability to produce native Ct-SLCO1B3, or the tissues / organs containing the cells are, for example, the minimum essential medium containing about 5 to 20% fetal calf serum. (MEM), Dulbecco's modified Eagle's medium (DMEM), RPMI ⁇ ⁇ 1640 medium, 199 medium, and the like.
  • the pH of the medium is preferably about 6-8. Cultivation is usually carried out at about 30-40 ° C, with aeration and agitation as necessary.
  • test substance examples include proteins, peptides, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. These substances may be novel or may be known ones.
  • a control cell that does not contact the test substance can also be used as a comparative control.
  • “do not contact the test substance” means that the same amount of solvent (blank) as the test substance is added instead of the test substance, or the expression level of Ct-SLCO1B3 or Ct-SLCO1B3 gene or Ct- This includes cases where a negative control substance that does not affect the function of SLCO1B3 is added.
  • the contact of the test substance with the cells may be performed by, for example, the above-mentioned medium or various buffers (for example, HEPES buffer, phosphate buffer, phosphate buffered saline, Tris-HCl buffer, borate buffer, acetic acid).
  • the test substance can be added to a buffer solution or the like, and the cells can be incubated for a certain time.
  • concentration of the test substance to be added varies depending on the type of compound (solubility, toxicity, etc.), but is appropriately selected within the range of about 0.1 nM to about 100 ⁇ M, for example. Examples of the incubation time include about 10 minutes to about 24 hours.
  • the state of the animal individual is not particularly limited.
  • a model mouse transplanted with cancer cells for example, A549 cells in Balb / It may be a model animal such as a mouse produced by subcutaneous implantation in the back of a c slc-nu / nu mouse.
  • the breeding conditions of the animals to be used it is preferable that the animals are raised in an environment of SPF grade or higher.
  • the administration route is not particularly limited, for example, intravenous administration, intraarterial administration, subcutaneous administration, intradermal administration, Examples include intraperitoneal administration, oral administration, intratracheal administration, and rectal administration.
  • the dose is not particularly limited. For example, a dose of about 0.5 to 20 mg / kg can be administered 1 to 5 times a day, preferably 1 to 3 times a day for 1 to 14 days.
  • the screening method of the present invention is characterized in that the expression of the protein (gene) in a cell capable of producing Ct-SLCO1B3 is compared in the presence and absence of a test substance, or a cancer treatment or A method for screening a substance having prophylactic activity is provided.
  • the cells used in this method, the type of test substance, the mode of contact between the test substance and cells, etc. are the same as described above.
  • the comparison of gene expression levels can be performed using a method known per se.
  • the present invention provides: (A) Cells having the ability to produce Ct-SLCO1B3 are cultured in the presence or absence of a test substance, and the amount of mRNA encoding the protein under both conditions is determined using the nucleic acid for detection of the present invention.
  • a method for screening a substance having a therapeutic or prophylactic activity for cancer and (b) a cell having the ability to produce Ct-SLCO1B3 in the presence or absence of the test substance.
  • a method for screening a substance having a therapeutic or prophylactic activity for cancer comprising culturing and measuring and comparing the amount of the protein under both conditions using a detection antibody.
  • screening for a substance that changes the expression level of Ct-SLCO1B3 can be performed as follows.
  • Normal or disease for example, a mouse produced by subcutaneous implantation of A549 cells into Balb / c slc-nu / nu mice
  • Model non-human mammals for example, mice, rats, rabbits, sheep, pigs, After the test substance is administered to cows, cats, dogs, monkeys, etc., after a certain period of time (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours)
  • a specific organ for example, lung, etc.
  • a tissue or cell isolated from the organ is obtained.
  • the mRNA of Ct-SLCO1B3 can be quantified by extracting mRNA from cells or the like by a usual method, or can be quantified by Northern blot analysis known per se.
  • the protein amount of Ct-SLCO1B3 can be quantified by a method known per se.
  • the amount of Ct-SLCO1B3 protein can be quantified using Western blot analysis or various immunoassay methods described in detail below, but is not limited thereto.
  • Ct-SLCO1B3 gene-expressing cells for example, transformants into which Ct-SLCO1B3 has been introduced
  • the test substance is added to the medium or buffer when culturing according to conventional methods
  • Ct-SLCO1B3 contained in the cell or the mRNA encoding the same is added to the above (i ) Can be quantified and analyzed in the same manner.
  • Ct-SLCO1B3 gene mRNA
  • a known method such as Northern blotting or RT-PCR using RNA prepared from the cells or a complementary polynucleotide transcribed therefrom. it can.
  • RNA prepared from the cells or a complementary polynucleotide transcribed therefrom.
  • the expression of the Ct-SLCO1B3 gene in RNA can be detected. Presence / absence and its expression level can be detected and measured.
  • Such a probe or primer is based on the base sequence of the Ct-SLCO1B3 gene, for example, primer 3 (http://www.genome.wi.mit.edu/cgi-bin/primer/primer3.cgi) or a vector It can be designed using NTI (Infomax).
  • the primer or probe When using Northern blotting, the primer or probe is labeled with a radioisotope (32P, 33P, etc .: RI) or a fluorescent substance and hybridized with cell-derived RNA transferred to a nylon membrane or the like according to a conventional method. After soy, the formed duplex of the primer or probe (DNA or RNA) and RNA is used as a signal from the primer or probe label (RI or fluorescent material) as a radiation detector (BAS- 1800II (manufactured by Fuji Film) or a method of detecting and measuring with a fluorescence detector can be exemplified.
  • a radioisotope 32P, 33P, etc .: RI
  • RI a fluorescent substance
  • the probe is labeled according to the protocol, hybridized with cell-derived RNA, and then the signal derived from the probe label is expressed in multibioin.
  • a method of detecting and measuring with a major STORM860 can also be used.
  • cDNA is prepared from cell-derived RNA according to a conventional method, and based on this, the target Ct-SLCO1B3 gene region can be amplified using this as a template, based on the sequence of the Ct-SLCO1B3 gene.
  • An example is a method in which a pair of prepared primers are hybridized with this, followed by PCR according to a conventional method, and detection of the resulting amplified double-stranded DNA.
  • the detection of the amplified double-stranded DNA was performed by a method for detecting the labeled double-stranded DNA produced by performing the PCR using a primer previously labeled with RI or a fluorescent substance.
  • a method can be used in which double-stranded DNA is transferred to a nylon membrane or the like according to a conventional method, and the labeled primer is used as a probe to hybridize with this to detect it.
  • the produced labeled double-stranded DNA product can be measured with an Agilent 2100 Bioanalyzer (manufactured by Yokogawa Analytical Systems).
  • Agilent 2100 Bioanalyzer manufactured by Yokogawa Analytical Systems.
  • RT-PCR reaction solution according to the protocol using SYBR Green RT-PCR Reagents (Applied Biosystems) and react with ABI PRIME 7900 Sequence Detection System (Applied Biosystems) to detect the reaction product. You can also.
  • Ct-SLCO1B3 gene in cells added with test substance is about 70% or less, preferably about 50% or less, more preferably about 30% or less compared to the expression level in control cells not containing test substance Furthermore, if it is more preferably about 20% or less, particularly preferably about 10% or less, the test substance can be selected as a Ct-SLCO1B3 gene expression inhibitor.
  • Ct-SLCO1B3 in the sample solution is quantified by competitively reacting the detection antibody with the sample solution and labeled Ct-SLCO1B3 and detecting the labeled protein bound to the antibody. How to do, (Ii) After reacting the sample solution with the detection antibody insolubilized on the carrier and another labeled detection antibody simultaneously or successively, the amount (activity) of the labeling agent on the insolubilized carrier is determined.
  • a method of quantifying Ct-SLCO1B3 in the sample solution by measuring it may be mentioned.
  • Detection and quantification of the protein expression level of Ct-SLCO1B3 can be quantified according to a known method such as Western blotting using an antibody recognizing Ct-SLCO1B3.
  • Western blotting uses an antibody that recognizes Ct-SLCO1B3 as the primary antibody, followed by a primary antibody labeled with a radioisotope such as 125 I, a fluorescent substance, or an enzyme such as horseradish peroxidase (HRP) as the secondary antibody. It can be carried out by labeling with an antibody that binds to, and measuring the signal derived from these labeling substances with a radiation measuring instrument (BAI-1800II: manufactured by Fuji Film Co., Ltd.), a fluorescence detector, or the like.
  • a radiation measuring instrument BAI-1800II: manufactured by Fuji Film Co., Ltd.
  • detection is performed according to the protocol using ECL Plus Western Blotting Detection System (Amersham Pharmacia Biotech), and multi-biomeasuring STORM860 (Amersham Pharmacia Biotech) ).
  • the form of the above-mentioned detection antibody is not particularly limited, and may be a polyclonal antibody having Ct-SLCO1B3 as an immunizing antigen or a monoclonal antibody thereof, and further an amino acid sequence constituting Ct-SLCO1B3 Among them, an antibody having an antigen binding property to a polypeptide consisting of at least continuous, usually 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids can also be used.
  • Methods for producing these antibodies are already well known, and antibodies can be produced according to these conventional methods (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.12- 11.13).
  • the detection antibody may be an antibody that recognizes both Ct-SLCO1B3 and Lt-SLCO1B3.
  • an antibody that recognizes both Ct-SLCO1B3 and Lt-SLCO1B3 in order to distinguish between Ct-SLCO1B3 and Lt-SLCO1B3, a process for distinguishing both of them according to biochemical and physical properties is included. May be.
  • Ct-SLCO1B3 and Lt-SLCO1B3 can be distinguished from the difference in protein size by combining the sample solution with SDS-PAGE electrophoresis or chromatography.
  • the two antibodies recognize different portions of Ct-SLCO1B3.
  • a labeling agent used in a measurement method using a labeling substance for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or the like is used.
  • the radioisotope for example, [ 125 I], [ 131 I], [ 3 H], [ 14 C] and the like are used.
  • the enzyme is preferably stable and has a large specific activity.
  • ⁇ -galactosidase ⁇ -glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used.
  • fluorescent substance for example, fluorescamine, fluorescein isothiocyanate and the like are used.
  • luminescent substance for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used.
  • a biotin- (strept) avidin system can be used for binding of an antibody or antigen to a labeling agent.
  • the Ct-SLCO1B3 quantification method using the detection antibody is not particularly limited, and the amount of the antibody, antigen or antibody-antigen complex corresponding to the amount of antigen in the sample solution is chemically or physically measured. Any measurement method may be used as long as it is a measurement method that is detected from the above and calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, competition method, immunometric method and sandwich method are preferably used. In view of sensitivity and specificity, for example, the sandwich method described later is preferably used.
  • insolubilization of the antigen or antibody physical adsorption may be used, or a chemical bond usually used for insolubilizing / immobilizing proteins or enzymes may be used.
  • the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or glass.
  • the sample solution is reacted with the insolubilized detection antibody (primary reaction), and further reacted with another labeled detection antibody (secondary reaction), and then the amount of labeling agent on the insolubilized carrier or By measuring the activity, Ct-SLCO1B3 in the sample solution can be quantified.
  • the primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at different times.
  • the labeling agent and the insolubilizing method can be the same as those described above.
  • the antibody used for the immobilized antibody or labeled antibody is not necessarily one type, and a mixture of two or more types of antibodies is used for the purpose of improving measurement sensitivity. May be.
  • the detection antibody can also be used in measurement systems other than the sandwich method, such as a competitive method, an immunometric method, or nephrometry.
  • a competitive method Ct-SLCO1B3 in the sample solution and labeled Ct-SLCO1B3 are reacted with the antibody competitively, then the unreacted labeled antigen (F) and the labeled antigen bound to the antibody (B) Is separated (B / F separation), and the amount of labeling of either B or F is measured to quantify Ct-SLCO1B3 in the sample solution.
  • a soluble antibody is used as an antibody
  • B / F separation is performed using polyethylene glycol or a secondary antibody against the antibody (primary antibody)
  • a solid phase is used as the primary antibody.
  • Either an antibody is used (direct method), or a primary antibody is soluble, and a solid phase antibody is used as a secondary antibody (indirect method).
  • Ct-SLCO1B3 in the sample solution and Ct-SLCO1B3 immobilized on the solid phase are allowed to compete with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated or the sample solution is separated.
  • Ct-SLCO1B3 and an excess amount of labeled antibody are reacted, and then solid-phased Ct-SLCO1B3 is added to bind the unreacted labeled antibody to the solid phase. To separate. Next, the amount of label in any phase is measured to quantify the amount of antigen in the sample solution.
  • nephrometry the amount of insoluble precipitate produced as a result of the antigen-antibody reaction in a gel or solution is measured.
  • Laser nephrometry using laser scattering is preferably used even when the amount of Ct-SLCO1B3 in the sample solution is small and only a small amount of precipitate is obtained.
  • a measurement system for Ct-SLCO1B3 may be constructed by adding ordinary technical considerations to those skilled in the art to the usual conditions and operation methods in each method. For details of these general technical means, it is possible to refer to reviews, books and the like. For example, Hiroshi Irie “Radioimmunoassay” (Kodansha, published in 1974), Hiroshi Irie “Continue Radioimmunoassay” (published in Kodansha, 1974), “Enzyme Immunoassay” edited by Eiji Ishikawa et al.
  • the expression level (mRNA level or protein level) of Ct-SLCO1B3 in the presence of the test substance is about 20% or more, preferably about 20% or more compared to that in the absence of the test substance.
  • inhibition is 30% or more, more preferably about 50% or more
  • the test substance can be selected as a Ct-SLCO1B3 expression-suppressing substance, and thus a candidate for a substance having cancer therapeutic or prophylactic activity.
  • the screening method of the present invention can also be performed using as an index whether or not a test substance suppresses the function of Ct-SLCO1B3.
  • a test substance suppresses the function of Ct-SLCO1B3.
  • Ct-SLCO1B3 function selected from the group consisting of (1) to (8) above Of these, one or more functions may be inhibited, and preferably two or more, three or more, four or more, five or more, six or more, seven or more, or all eight functions are inhibited.
  • the anchorage-independent growth of cells expressing Ct-SLCO1B3 in the presence of the test substance is about 10% or more, preferably about 10% or more compared to the anchorage-independent growth of cells in the absence of the test substance.
  • the test substance is inhibited by 20% or more, more preferably about 30% or more, and still more preferably about 50% or more, the test substance is treated with a Ct-SLCO1B3 function inhibitory substance, and thus has a therapeutic and / or prophylactic activity for cancer.
  • the cells expressing Ct-SLCO1B3 the following 1) or 2) is preferably used. 1) Cells in which Ct-SLCO1B3 is expressed in cells that do not express Ct-SLCO1B3, and cells whose anchorage-independent growth of cells is enhanced compared to cells that do not express Ct-SLCO1B3 2) Cells that are Ct-SLCO1B3-expressing cells, and when Ct-SLCO1B3 expression is suppressed, cells that inhibit the anchorage-independent growth of cells compared to the parent Ct-SLCO1B3-expressing cells Independent growth means the ability to grow in the absence of an extracellular matrix such as collagen.
  • the anchorage-independent growth of cells can be evaluated according to the method described in Example 9, for example.
  • In vivo cell-independent growth of cells is measured, for example, by measuring the size of tumors formed by A549 cells in mice transplanted subcutaneously in the back to Balb / c slc-nu / nu mice. You can also.
  • the migration ability of a cell expressing Ct-SLCO1B3 in the presence of a test substance is about 10% or more, preferably about 20% or more, more preferably compared to the migration ability of a cell in the absence of the test substance.
  • the following 1) or 2) is preferably used.
  • a part of the cells is peeled off at the tip of the pipette tip, and after a certain time (for example, 6 hours, 12 hours, 24 hours, etc.) ) To measure the cell migration area.
  • the invasion ability of cells expressing Ct-SLCO1B3 in the presence of the test substance is about 10% or more, preferably about 20% or more, more preferably compared to the invasion ability of the cells in the absence of the test substance.
  • the following 1) or 2) is preferably used.
  • the invasive ability of cells means the ability to move out of the tissue.
  • the invasive ability of cells can be assayed by a method known per se.
  • an in vitro invasive ability assay of cancer cells can be performed using a transwell chamber such as CIM-Plate16. Before the seeding of cells, the upper part of the membrane of the upper chamber is covered with Matrigel in advance, and after seeding and culturing the cells, the number of cells infiltrating the lower chamber can be measured.
  • the invasive ability of cells can be evaluated according to the method described in Example 11, for example.
  • the snail expression level of cells expressing Ct-SLCO1B3 in the presence of the test substance is about 70% or less, preferably about 50% or less, compared to the invasion capacity of the cells in the absence of the test substance.
  • the test substance is reduced to about 30% or less, more preferably about 20% or less, and particularly preferably about 10% or less, the test substance is treated as a Ct-SLCO1B3 function inhibitor, and thus cancer treatment and / or It can be selected as a candidate for a substance having prophylactic activity.
  • the following 1) or 2) is preferably used.
  • the expression level of snail can be compared according to the method described in Example 13. Therefore, more specifically, the present invention (A) Cells having the ability to produce Ct-SLCO1B3 are cultured in the presence or absence of a test substance, and the amount of mRNA encoding the snail protein under both conditions is determined using the nucleic acid for mRNA detection.
  • a method for screening a substance having cancer therapeutic and / or prophylactic activity comprising measuring and comparing the amount of snail protein under both conditions using an antibody against the snail protein.
  • the amount of slug expression of cells expressing Ct-SLCO1B3 in the presence of the test substance is about 70% or less, preferably about 50% or less, compared to the invasive ability of the cells in the absence of the test substance.
  • the test substance is reduced to about 30% or less, more preferably about 20% or less, and particularly preferably about 10% or less, the test substance is treated as a Ct-SLCO1B3 function inhibitor, and thus cancer treatment and / or It can be selected as a candidate for a substance having prophylactic activity.
  • the following 1) or 2) is preferably used.
  • the expression level of slug can be compared according to the method described in Example 13. Therefore, more specifically, the present invention (A) Cells capable of producing Ct-SLCO1B3 are cultured in the presence or absence of a test substance, and the amount of mRNA encoding slug protein under both conditions is determined using the nucleic acid for mRNA detection.
  • a method for screening a substance having a therapeutic and / or prophylactic activity for cancer characterized by measuring and comparing, and (b) a cell capable of producing Ct-SLCO1B3 in the presence or absence of a test substance
  • a method for screening a substance having a therapeutic and / or prophylactic activity for cancer characterized by measuring and comparing the amount of slug protein under both conditions using an antibody against the slug protein.
  • E-cadherin expression level Specifically, in cells expressing Ct-SLCO1B3, it is measured whether the E-cadherin expression level in the cells increases by adding a test substance.
  • the E-cadherin expression level of cells expressing Ct-SLCO1B3 in the presence of the test substance is about 10% or more, preferably about 20% or more, compared to the invasive ability of the cells in the absence of the test substance More preferably about 30% or more, and even more preferably about 50% or more, the test substance is a Ct-SLCO1B3 function inhibitory substance, and therefore a substance having a therapeutic and / or prophylactic activity for cancer. Can be selected as a candidate.
  • the following 1) or 2) is preferably used.
  • the amount of E-cadherin expressed is about 10% or more, preferably about
  • the method for comparing the expression levels of cellular genes that rise to 20% or more, more preferably about 30% or more, and even more preferably about 50% or more is as described above.
  • the expression level of E-cadherin can be compared according to the method described in Example 12.
  • the present invention (A) Cells having the ability to produce Ct-SLCO1B3 are cultured in the presence or absence of a test substance, and the amount of mRNA encoding E-cadherin protein under both conditions is determined using the nucleic acid for mRNA detection.
  • a method for screening a substance having a therapeutic and / or preventive activity for cancer, characterized by using and measuring, and (b) a cell having the ability to produce Ct-SLCO1B3 in the presence or absence of the test substance Screening for a substance having therapeutic and / or prophylactic activity for cancer, characterized by measuring and comparing the amount of E-cadherin protein in both conditions using an antibody against E-cadherin protein Provide a method.
  • the expression level of occludin in cells expressing Ct-SLCO1B3 in the presence of the test substance is about 10% or more, preferably about 20% or more compared to the invasion ability of the cells in the absence of the test substance.
  • the test substance is regarded as a Ct-SLCO1B3 function inhibitory substance, and therefore a candidate for a substance having cancer therapeutic and / or prophylactic activity. You can choose.
  • the following 1) or 2) is preferably used.
  • a cell in which Ct-SLCO1B3 is expressed in a Ct-SLCO1B3 non-expressing cell, and the occludin expression level is about 70% or less, preferably about 50% or less compared to the parent strain Ct-SLCO1B3 non-expressing cell More preferably about 30% or less, still more preferably about 20% or less, particularly preferably about 10% or less.
  • the expression level of occludin is about 10% or more, preferably about 20%, compared to the parent Ct-SLCO1B3-expressing cells
  • the method for comparing the expression levels of cellular genes that rise to about 30% or more, more preferably about 50% or more is more as described above.
  • the expression level of occludin can be compared according to the method described in Example 12.
  • the present invention (A) Cells having the ability to produce Ct-SLCO1B3 are cultured in the presence or absence of a test substance, and the amount of mRNA encoding the occludin protein under both conditions is determined using the nucleic acid for mRNA detection.
  • a method for screening a substance having a therapeutic and / or prophylactic activity for cancer characterized by measuring and comparing the amount of occludin protein under both conditions using an antibody against occludin protein.
  • the expression level of MMP9 in cells expressing Ct-SLCO1B3 in the presence of the test substance is about 70% or less, preferably about 50% or less, compared to the invasion ability of the cells in the absence of the test substance.
  • the test substance is reduced to about 30% or less, more preferably about 20% or less, and particularly preferably about 10% or less, the test substance is treated as a Ct-SLCO1B3 function inhibitor, and thus cancer treatment and / or It can be selected as a candidate for a substance having prophylactic activity.
  • the following 1) or 2) is preferably used.
  • the expression level of MMP9 can be compared according to the method described in Example 13. Therefore, more specifically, the present invention (A) Cells having the ability to produce Ct-SLCO1B3 are cultured in the presence or absence of a test substance, and the amount of mRNA encoding MMP9 protein under both conditions is determined using the nucleic acid for mRNA detection.
  • a method for screening a substance having a therapeutic and / or prophylactic activity for cancer characterized by measuring and comparing, and (b) a cell capable of producing Ct-SLCO1B3 in the presence or absence of a test substance
  • a method for screening a substance having a therapeutic and / or prophylactic activity for cancer which comprises measuring and comparing the amount of MMP9 protein under both conditions using an antibody against MMP9 protein.
  • the test substance has the above function in a control cell not expressing the Ct-SLCO1B3 gene.
  • the mechanism of action of the candidate substance having cancer therapeutic or prophylactic activity obtained in the above screening method is based on suppression of Ct-SLCO1B3 or Ct-SLCO1B3 gene expression or Ct-SLCO1B3 function I can confirm.
  • a substance that suppresses the expression or function of Ct-SLCO1B3 obtained by using any one of the screening methods of the present invention is useful as a medicament for the prevention and / or treatment of cancer.
  • the compound obtained using the screening method of the present invention is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated in the same manner as the low molecular weight compound that suppresses the expression or function of Ct-SLCO1B3, and the same administration Administered orally or parenterally to humans or mammals (eg, mice, rats, rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, chimpanzees, etc.) by route and dose be able to.
  • mammals eg, mice, rats, rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, chimpanzees, etc.
  • Method for testing malignancy or risk of malignancy of cancer tests the malignancy or risk of malignancy of cancer characterized by measuring the expression level of Ct-SLCO1B3 in a sample collected from a test animal (Hereinafter also referred to as the test method of the present invention).
  • the method comprises the following steps: (A) a step of measuring the expression level of Ct-SLCO1B3 in a sample collected from the test animal, and (b) a test animal having a high expression level compared to the case of measuring in a sample derived from a normal animal, The process of determining that the malignancy of the cancer is high or that the risk of malignancy in the future is high, A method for testing the malignancy or risk of malignancy of cancer, characterized by measuring the expression level of Ct-SLCO1B3 in a sample collected from a test animal containing.
  • test animals include humans and other mammals, and preferred examples include humans, mice, rats, rabbits, dogs, monkeys, and the like that are widely used as experimental animals.
  • Samples to be measured include biopsy samples (eg, cells or a part of the tissue extracted from animal tissues suspected of having cancer (lung, liver, pancreas, esophagus, etc.), blood, plasma, serum, Examples include cerebrospinal fluid, lymph fluid, saliva, mucous membrane, urine, tears, semen, and joint fluid.
  • the sample to be measured is preferably a sample collected from a tissue suspected of having cancer.
  • Cancer malignancy refers to any cancer malignancy that can be associated with Ct-SLCO1B3 by those skilled in the art, and may be associated with, for example, the likelihood and / or the speed of metastasis, proliferation, invasion.
  • the expression level of Ct-SLCO1B3 in the sample can be measured by the amount of mRNA of Ct-SLCO1B3 or the amount of Ct-SLCO1B3 protein, and these are used in the above screening method using the expression level of the gene or the protein as an index. It can be measured by the same method as described.
  • the expression level of Ct-SLCO1B3 gene or the amount of Ct-SLCO1B3 protein in the sample collected from the test animal is equal to the expression level of Ct-SLCO1B3 gene or Ct-SLCO1B3 protein in the sample collected from the normal animal. If the amount is significantly higher than the amount, the subject animal can be determined to have developed cancer or have a higher risk of developing in the future.
  • the expression level in a normal animal is identified in advance, for example, the average value + 2SD (average value + twice the standard deviation) is defined as a cut-off value, and the Ct-SLCO1B3 gene in a sample collected from the test animal
  • the test animal can also be determined to have a high cancer malignancy or a high risk of becoming malignant in the future.
  • the present invention provides a method for preventing or treating cancer (hereinafter also referred to as the method for preventing or treating the present invention) using the nucleic acid of the present invention.
  • the definitions and embodiments of each term relating to the prevention or treatment method of the present invention are the same as those described above.
  • the preventive or therapeutic method of the present invention includes a step of administering a prophylactically or therapeutically effective amount of the nucleic acid of the present invention or the pharmaceutical composition of the present invention to an animal in need of prevention or treatment.
  • animals that require prevention or treatment include animals that have cancer or are highly likely to have cancer, preferably humans who have cancer or are highly likely to have cancer. It is.
  • the prevention or treatment method of the present invention can be applied to animals determined to be “high cancer malignancy or high risk of malignancy in the future” by the test method of the present invention.
  • Examples of preferable administration methods of the nucleic acid of the present invention or the pharmaceutical composition of the present invention include tissues suspected of having cancer, tissues suffering from cancer, risk of cancer having high malignancy or becoming malignant in the future. Local administration to high tissues is mentioned.
  • the test method step (a) of the present invention the nucleic acid of the present invention or the pharmaceutical composition of the present invention can be locally administered to a tissue (eg, lung, liver, pancreas, esophagus, etc.) from which the sample collected from the test animal is derived.
  • a tissue eg, lung, liver, pancreas, esophagus, etc.
  • Example 1 Identification of lung cancer cases with high expression of SLCO1B3 by analysis using lung cancer clinical specimens
  • QIAZOL QIAZOL
  • TOMY Micro smash MS-100
  • QIAGEN miRNeasy Mini kit
  • QIA Qube QIA Qube
  • An ultra-trace spectrophotometer Nano Drop was used for RNA concentration measurement.
  • 1 ⁇ L of RNA was added to an Experion RNA StdSens Analysis chip (BIO-RAD) and analyzed by a fully automatic chip electrophoresis system Experion Automated Electrophoresis station (BIO-RAD).
  • RQI represents RNA integrity as a number between 1 (very high degradation) and 10 (not degraded).
  • RNA with RQI greater than 7 was used.
  • Example 2 Identification of lung cancer cases with high expression of SLCO1B3 by analysis using lung cancer cell lines
  • QIAZOL QIAZOL
  • Example 1- (2) Concentration measurement and purity confirmation were performed in the same manner as Example 1- (2) and exon array in the same manner as in Example 1- (3). result: The results are shown in FIG. In non-small cell lung cancer cell lines, the presence of SLCO1B3 high-expressing cells was confirmed as in the case of lung cancer clinical specimens. Furthermore, the signal intensity of exon2 of SLCO1B3 was found to be low even in cells in which these high expression levels were observed.
  • Example 3 Expression of SLCO1B3 in normal human tissues Method: A solution obtained by diluting Human Multiple Tissue cDNA Panel (CLONTECH) 10 times with Milli-Q water was used as a template. A PCR reaction solution was prepared with the composition shown below, and the reaction was performed under the PCR conditions shown below. GAPDH was subjected to PCR reaction with AmpliTaq Gold (Applied biosystems), and SLCO1B3 full length was subjected to PCR reaction with KOD-plus- (TOYOBO).
  • SLCO1B3 fulllength F ATGGACCAACATCAACATTTGAATAAAAC (SEQ ID NO: 33)
  • SLCO1B3 fulllength R TTAGTTGGCAGCAGCATTGTCTTG (SEQ ID NO: 34)
  • GAPDH F CCATCACCATCTTCCAGGAG (SEQ ID NO: 35)
  • GAPDH R AATGAGCCCCAGCCTTCTCC (SEQ ID NO: 36) result: The results are shown in Figure 3. In normal tissues examined, SLCO1B3 was detected only in the liver.
  • Example 5 Immunohistochemical staining of SLCO1B3 in normal liver tissue and non-small cell lung cancer tissue
  • the expression of SLCO1B3 in normal liver tissue and non-small cell lung cancer tissue was examined by immunohistochemical analysis using anti-SLCO1B3 antibody.
  • the antibody used is Anti-SLCO1B3 antibody (Sigma-Aldrich, HPA004943). result: The results are shown in FIG. In the liver, SLCO1B3 was expressed in the cell membrane as is known. On the other hand, in non-small cell lung cancer tissues, membrane localization was not observed, but it was localized in the cytoplasm.
  • Example 6 Quantitative analysis of Ct-SLCO1B3 expression in non-small cell lung cancer tissues
  • Method Using 500 ng of total RNA extracted from clinical samples, cDNA was synthesized with PrimeScript RT reagent Kit (TaKaRa), diluted 10-fold with Milli-Q water, and used for real-time PCR.
  • Primer and SsoAdvanced SYBR Green Supermix (BIO-RAD) or THUNDERBIRD SYBR qPCR Mix (TOYOBO) diluted to 10 ⁇ M in advance is diluted with Milli-Q water to make 20 ⁇ L, and CFX96 Real-Time System (BIO- RAD) performed real-time PCR.
  • Example 7 Knockdown efficiency of Ct-SLCO1B3 siRNA in A549 cells
  • Method (1) Cell culture FCS (Life Technologies) deactivated by incubation for 30 minutes at 56 ° C in 500 mL of DMEM (Wako Pure Chemical Industries) and RPMI1640 (Wako Pure Chemical Industries) in 55 mL of PBS Diluted 100 mg / mL kanamycin sulfate was added after sterilization of 500 ⁇ L. Cell culture was performed at 37 ° C. and 5% CO 2 .
  • siRNA transfection The reverse transfection method was used.
  • a transfection complex of LipofectamineRNAiMAX (Life Technologies), Control siRNA (Cosmo Bio), Ct-SLCO1B3 siRNA (GeneDesign), and Opti-MEM (Life Technologies) was prepared, and A549 cell suspension was prepared so that the final siRNA concentration was 10 nM. Added to the suspension.
  • the sequence of the siRNA used is shown below.
  • Ct-SLCO1B3 siRNA # 1 ACCUGACAGUGGCAAUGUAtt (SEQ ID NO: 37) UACAUUGCCACUGUCAGGUtt (SEQ ID NO: 38)
  • Ct-SLCO1B3 siRNA # 2 CUGACAGUGGCAAUGUAUGtt (SEQ ID NO: 39)
  • the above siRNA was requested for synthesis from GeneDesign.
  • cDNA synthesis was performed using 500 ng of total RNA, and the obtained cDNA solution was diluted 10-fold with Milli-Q water and used for real-time PCR.
  • Example 8 Effect of Ct-SLCO1B3 siRNA on anchorage-dependent growth of A549 cells
  • Method Cells on the second day after siRNA transfection were collected and seeded on a 96 well plate at 1000 cells / well. After that, every 24 hours, WST-1 (DOJINDO) solution dissolved in 20 mM HEPES (pH 7.4) and 1-methoxy PMS (DOJINDO) solution mixed at a ratio of 9: 1 was added at 10 ⁇ L / well for 1 hour 30 Absorbance was measured after 450 minutes at 450 nm. A reference wavelength of 630 nm was used. result: The results are shown in FIG. Ct-SLCO1B3 siRNA (using # 4 in FIG. 8) had no effect on anchorage-dependent growth of A549 cells.
  • Example 9 Effect of Ct-SLCO1B3 siRNA on anchorage-independent growth of A549 cells
  • FCeM-D Non Chemical Co., Ltd.
  • FCS kanamycin
  • the cells on day 2 after siRNA transfection were replated on a 96-well plate at 500 cells / well, cultured for 48 hours, WST-1 reagent was added in the same manner as in Example 8, and the absorbance was measured.
  • FIG. Ct-SLCO1B3 siRNA (using # 4 in FIG. 9) suppressed anchorage-independent growth of A549 cells.
  • Example 10 Effect of Ct-SLCO1B3 siRNA on the migration ability of A549 cells
  • Method Cells on day 2 after siRNA transfection were collected and seeded so that the 24-well plate was confluent. After 24 hours, a portion of the proliferating cells was scraped with the tip of a pipette tip, and 0, 6, 12, 24, 36, and 48 hours later, the cell migration area was quantitatively evaluated using Image J. FSX100 (Olympus) was used for cell photography. result: The results are shown in FIG. Ct-SLCO1B3 siRNA (using # 4 in FIG. 10) suppressed the migration ability of A549 cells.
  • Example 11 Effect of Ct-SLCO1B3 siRNA on invasive ability of A549 cells
  • Method BD Matrigel Basement Membrane Matrix High Concentration (BD Biosciences) was diluted 40-fold with serum-free DMEM and coated onto CIM-Plate 16 (Roche). Collect cells on day 2 after siRNA transfection, dilute to 4 ⁇ 10 4 cells / well, seed on CIM-Plate, leave at room temperature for 1 hour, set to xCELLigence (Roche) and start measurement did. result: The results are shown in FIG. In control siRNA transfection, A549 was significantly infiltrated, whereas Ct-SLCO1B3 siRNA (# 4 was used in FIG. 11) remarkably suppressed its invasion effect.
  • Example 12 Immune cell staining of cell adhesion molecule expression in Ct-SLCO1B3 knockdown A549 cells
  • Method Cells 48 hours after transfection were replated at 5 ⁇ 10 4 cells / well in a 12-well plate with a cover glass, and incubated at 37 ° C. for a further 48 hours. Thereafter, ⁇ 20 ° C. methanol (Wako 137-01823) was added in an amount of 500 ⁇ L / well instead of the medium, and fixation was performed at ⁇ 20 ° C. for 15 minutes.
  • Example 13 Expression analysis of cell adhesion molecule and epithelial-mesenchymal transition regulatory molecule in Ct-SLCO1B3 knockdown A549 cells
  • E-cadherin, snail, slug hereinafter Cell Signaling Technology
  • occludin occludin
  • ⁇ -actin hereinafter SIGMA-ALDRICH
  • Immobilon-P Transfer Membrane (Millipore) was soaked in methanol, soaked in transfer buffer, and blended on a shaker for about 20 minutes. Thereafter, a transfer of 25 V and 60 minutes was performed by a submarine method. After completion of the transfer, the membrane was washed several times with TBS-T (1 M Tris-HCl (pH 7.4) 10 mL, 5 M NaCl 30 mL, Tween 20 1 mL dissolved in Milli-Q water to 1000 mL), Blocking was performed for about 20 minutes with skim milk (Morinaga) diluted to 3% with TBS-T. After washing several times with TBS-T, incubation was performed overnight at 4 ° C.
  • TBS-T Tris-HCl (pH 7.4) 10 mL, 5 M NaCl 30 mL, Tween 20 1 mL dissolved in Milli-Q water to 1000 mL
  • Blocking was performed for about 20 minutes with skim milk (Morinaga) diluted
  • Knockdown of Ct-SLCO1B3 showed a decrease in the expression of snail and slug, transcription factors involved in epithelial-mesenchymal transition, and an increase in the expression of adhesion molecules E-cadherin and occludin, which are presumed to be caused by them.
  • Example 14 Expression analysis of matrix metalloprotease 9 (MMP9) in Ct-SLCO1B3 knockdown A549 cells
  • Method (1) real-time PCR The method is the same as in Example 7.
  • Concentrated supernatant is added with equal volume of 6 ⁇ Sample Buffer (Milli-Q 1.6mL, 625mM Tris 2.4mL, Glycerol 2.4mL, 10% SDS, 4.8mL BPB (bromophenol blue)) and left at room temperature for several minutes did.
  • Sample Buffer Milli-Q 1.6mL, 625mM Tris 2.4mL, Glycerol 2.4mL, 10% SDS, 4.8mL BPB (bromophenol blue)
  • a 0.1% Gelatin (Sigma) / 10% acrylamide gel was prepared, and the sample was applied so that the number of cells was the same, and electrophoresed in the same manner as Western Blot. At this time, the applied amount of the supernatant sample was defined by the number of cells at the time of supernatant.
  • the gel was infiltrated with Zymogram Renaturating Buffer (life technology) for 30 minutes and then infiltrated with Zymogram Developing Buffer (life technology) for 30 minutes. Thereafter, the Zymogram Developing Buffer was further replaced, followed by overnight incubation at 37 ° C.
  • the gel was soaked in a CBB staining solution (composition methanol 250 mL / acetic acid 50 mL / MQ 200 mL / Coomassie Brilliant Blue (CBB)) and shaken for about 1 hour.
  • CBB staining solution composition methanol 250 mL / acetic acid 50 mL / MQ 200 mL / Coomassie Brilliant Blue (CBB)
  • Example 15 Establishment of Ct-SLCO1B3 and Lt-SLCO1B3 highly expressing cell lines using lung cancer cell line NCI-H23 in which Ct-SLCO1B3 is not expressed
  • a PCR reaction of full length Ct-SLCO1B3 and Lt-SLCO1B3 was performed using KOD-plus (TOYOBO). See Example 3 for KOD-plus-PCR reaction solution composition and Lt-SLCO1B3 primer.
  • PCR conditions and Ct-SLCO1B3 full length primer are as follows.
  • PCR product was subjected to agarose gel electrophoresis and then excised and purified by Wizard SV Gel and PCR Clean-up System (Promega). Using this product 90 ng and Perfectly Blunt Cloning Kit (Millipore), ligation was performed to pT7 Blue vector (Millipore). This was transformed into DH5 ⁇ (Life Technologies) and then inoculated on an ampicillin-containing LB agar medium and incubated overnight at 37 ° C.
  • the formed colonies were picked and cultured overnight in 3 mL of ampicillin-containing LB liquid medium, and the plasmid was purified from the resulting Escherichia coli solution by Wizard Plus SV Minipreps DNA Purification System (Promega) to confirm sequence matching.
  • the obtained plasmid and pcDNA 3.0 were made up to 44 ⁇ L with Milli-Q water, 5 ⁇ L of 10 ⁇ buffer K and 1 ⁇ L BamHI (TaKaRa) were added, and enzyme treatment was performed at 30 ° C. for 16 hours. Then, it refine
  • Example 16 Anchorage-independent growth of Ct-SLCO1B3, Lt-SLCO1B3 high expression NCI-H23 cell line
  • NCI-H23 cells with high expression of Ct-SLCO1B3 and Lt-SLCO1B3 are collected, diluted in FCeM-R medium (Nissan Chemical Industry Co., Ltd.) to 6000 cells / 90 ⁇ L, and seeded at 90 ⁇ L in a non coating 96 well plate (IWAKI) did.
  • FCeM-R medium Non Chemical Industry Co., Ltd.
  • WST-1 solution and 1-methoxy PMS solution both DOJINDO
  • absorb the absorbance at 450 nm It was measured.
  • a reference wavelength of 630 nm was used. result: The results are shown in FIG. In CCI-SLCO1B3 highly expressing NCI-H23 cells, an anchorage-independent growth promoting action was observed.
  • Example 17 Ct-SLCO1B3, Lt-SLCO1B3 high expression NCI-H23 cell line migration ability Method: Cells were seeded in a 48 well plate at 1 ⁇ 10 5 cells / well. After 24 hours, the tip of the chip was scratched, and after 0, 6, 12, 24, 36, and 48 hours, the migration area of the cells was measured using Image J software. Photographing was performed using FSX100 (Olympus). result: The results are shown in FIG. In Ct-SLCO1B3 highly expressing NCI-H23 cells, enhanced migration ability was observed.
  • Example 18 Generation of Ct-SLCO1B3 expression-suppressed A549 cells using CRISPR / Cas9 system Method: (1) Ct-SLCO1B3 exon1 * guide sequence introduced px330 production 5 ⁇ g of pX330-U6-Chimeric_BB-CBh-hSpCas9 (Addgene) was digested overnight at 37 ° C with BbsI (Thermo Scientific), then Wizard SV Gel and PCR Purified with Clean-up System (Promega). This was dephosphorylated overnight at 37 ° C. with Alkaline Phosphatase and Calf Intestinal (CIP) (NEB).
  • CIP Calf Intestinal
  • the resulting product was subjected to a ligation reaction using ligation high (TOYOBO).
  • the obtained plasmid was transformed into Competent Quick DH5 ⁇ (TOYOBO), and the plasmid was purified by Wizard Plus SV Minipreps DNA Purification System (Promega).
  • (3) Preparation of Ct-SLCO1B3 knockout A549 A549 cells were seeded on a 24-well plate at 2 ⁇ 10 4 cells / well. Next day Ct-SLCO1B3 exon1 * guide sequence introduced px330 vector 500ng, puromycin resistant vector 50ng, Lipofectamine 2000 (Life Technologies) 1 ⁇ L in Opti-MEM (Life Technologies), co-transfection, 6 hours later The medium was changed.
  • Example 19 Inhibition of Ct-SLCO1B3 expression Inhibition of anchorage-independent growth of A549 cells
  • Example 20 Ct-SLCO1B3 expression-suppressed A549 cells were subcutaneously transplanted to the back of the mouse to reduce tumor formation Method: 0.025% EDTA / PBS was added dropwise to Ct-SLCO1B3 knockout A549 clone No. 1-1 and control-1 cultured in a 15 cm dish, allowed to stand for 5 minutes, and then cells were collected using a scraper. Washing with PBS was performed twice, and the number of cells and viability of the cells were measured with a Countess TM Automated Cell Counter (Invitrogen).
  • Example 21 siRNA target candidate sequence within exon1 * of Ct-SLCO1B3
  • Method For the purpose of determining the optimal siRNA sequence for Ct-SLCO1B3, the siRNA sequences shown in Table 8 were set and synthesized.
  • Example 22 Knockdown efficiency of Ct-SLCO1B3 siRNA Method: Perform siRNA transfection in a 96-well plate so that A549 cells become 4000 cells / well (method is the same as in Example 7), medium change after 1 day, CellAmp Direct RNA Prep Kit for RT-PCR (Real Time) after 2 days RNA was recovered with (TaKaRa). Using 2 ⁇ L of this RNA, cDNA was synthesized with PrimeScript RT-PCR Kit (Perfect Real Time) (TaKaRa) and diluted 10-fold with Milli-Q water. Real-time PCR was performed in duplicate using Light Cycler 96 (Roche) (the method is the same as in Example 7). The results are shown in FIG.
  • Example 23 Evaluation of the effect of Ct-SLCO1B3 siRNA on A549 cell anchorage-dependent proliferation Method: The siRNA transfection was performed in a 96-well plate to give A549 cells of 500 cells / well (the method was the same as in Example 7), the medium was changed after 1 day, the WST-1 reagent was added after 4 days, and the absorbance was measured ( The method is the same as in Example 8.) result: The results are shown in FIG.
  • Example 25 Evaluation of the effect of Ct-SLCO1B3 siRNA on cell proliferation of non-Ct-SLCO1B3-expressing lung cancer cells NCI-H1975 Method: The siRNA transfection was performed in a 96-well plate so that A549 cells were 1500 cells / well (the method was the same as in Example 7). After 1 day, the medium was changed. The method is the same as in Example 8.)
  • Example 26 Secondary evaluation of knockdown efficiency of Ct-SLCO1B3 siRNA in A549 cells
  • Example 27 Secondary evaluation of the effect of Ct-SLCO1B3 siRNA on A549 cell anchorage-dependent proliferation Method: Perform siRNA transfection on 6-well plate so that A549 cells become 1 ⁇ 10 5 cells / well (method is the same as in Example 7), medium change after 1 day, and 500 cells / well to 96-well plate after 2 days Then, after culturing for 5 days under adhesion conditions, WST-1 reagent was added and the absorbance was measured (the method was the same as in Example 8). result: The results are shown in FIG.
  • Example 28 Secondary evaluation of the effect of Ct-SLCO1B3 siRNA on A549 cell anchorage-independent growth Method: Perform siRNA transfection in a 6-well plate to achieve A549 cells of 1 ⁇ 10 5 cells / well (the method is the same as in Example 7), medium change after 1 day, 10% FCS / FCeM-D (Nissan Chemical) after 2 days The cell suspension diluted to 500 cells / well in (Industry) was re-seeded on a Hydro Cell plate (Cell Seed Co., Ltd.), cultured for 9 days under floating conditions, added with WST-1 reagent, and the absorbance was measured. (The method is the same as in Example 9). result: The results are shown in FIG.
  • Example 29 Determination of nucleic acid drug target sequence for Ct-SLCO1B3 Based on the secondary evaluation results of Examples 26-28, an siRNA target sequence exhibiting a specific action on Ct-SLCO1B3 was determined. The sequence was found to be concentrated in the region containing ACGTTACTGAATCTACATGTTGCAAGAAGAAAAA (SEQ ID NO: 140) (FIG. 29). Results of the secondary evaluation # 26, 27, 29, 30, 32, 33 and # 4 showed a significant knockdown effect on A549 cells. For anchorage-independent growth, # 29 showed no inhibitory action and # 30 showed a weak inhibitory action, while # 26, 27, 32, 33 and # 4 showed a remarkable inhibitory action. On the other hand, # 27 had a weak anchorage-dependent growth inhibitory effect of A549 cells. Therefore, it was shown that # 4, 26, 32, and 33 are more specific and effective sequences for Ct-SLCO1B3.
  • Example 30 Quantitative analysis of Ct-SLCO1B3 expression in esophageal cancer, hepatocellular carcinoma or pancreatic cancer
  • cDNA synthesis was performed using 500 ng of total RNA extracted from clinical specimens of esophageal cancer, hepatocellular carcinoma or pancreatic cancer, and the expression level of Ct-SLCO1B3 was compared by real-time PCR. The results are shown in FIG. Although Ct-SLCO1B3 did not show significant differences in esophageal cancer, hepatocellular carcinoma, and pancreatic cancer, it was found that there were samples that were highly expressed in the cancer area compared to the non-cancer area.
  • Example 31 Anti-tumor effect by administration of Ct-SLCO1B3 siRNA Method: A549 cells were detached from the dish using 0.05% EDTA and washed twice with phosphate buffered saline (PBS). Cells were diluted 1: 1 with DMEM (Code No.041-29775, Wako Pure Chemical Industries, Ltd.) and BD Matrigel Basement Membrane Matrix High Concentration (Cat. No. 354248, BD Bioscience), 4 ⁇ 10 6 cells / Mouse / 100 ⁇ l was subcutaneously transplanted into 6 BALB / c Slc-nu mice in each group (5 weeks old, Japan SLC).
  • PBS phosphate buffered saline
  • Control siRNA and Ct-SLCO1B3 siRNA # 4 is adjusted to a final siRNA concentration of 2 nmol / 200 ⁇ l according to the AteloGene Local Use “Quick Gelation” (Product No. # 1490, Koken) protocol. Every 4 days, a total of 4 doses were administered around the tumor. A caliper was used to measure the tumor diameter. The formula for calculating the tumor volume was tumor major axis ⁇ minor axis ⁇ minor axis ⁇ 2.
  • Ct-SLCO1B3 siRNA # 4 5'-ACGUUACUGAAUCUACAUGtt (SEQ ID NO: 43), 5'-CAUGUAGAUUCAGUAACGUtt (SEQ ID NO: 44)
  • Control siRNA 5'-AUCCGCGCGAUAGUACGUAtt (SEQ ID NO: 141), 5'-UACGUACUAUCGCGCGGAUtt (SEQ ID NO: 142) result: The results are shown in FIG. Ct-SLCO1B3 siRNA administration showed an antitumor effect.
  • the present invention provides a nucleic acid having Ct-SLCO1B3 expression inhibitory activity, a pharmaceutical composition containing the nucleic acid as an active ingredient, and the like.
  • the nucleic acid and pharmaceutical composition of the present invention suppress Ct-SLCO1B3 expression and are useful for the treatment and prevention of diseases characterized by Ct-SLCO1B3 expression.
  • the nucleic acid drug for the cancer-specific exon region of Ct-SLCO1B3 can suppress the expression and function of Ct-SLCO1B3, it suppresses the growth of lung cancer and has an antitumor effect. Therefore, the nucleic acid drug according to the present invention is an innovative nucleic acid drug for carcinomas such as lung cancer that express Ct-SLCO1B3. Lung cancer is the disease with the highest cancer mortality. In addition to lung cancer, refractory cancer in which Ct-SLCO1B3 is expressed is also observed, so the effect is great.

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Abstract

The purpose of the present invention is to: identify a molecule that is expressed in cancer cells but not expressed or only expressed at a very low level in normal cells, and that contributes toward the onset and/or progress of cancer; and provide a substance or the like that inhibits the function or expression of the molecule. The present invention addresses the problem by providing a double-stranded nucleic acid or the like that reduces the expression of the cancer-type solute carrier organic anion transporter family member 1B3.

Description

癌型ＳＬＣＯ１Ｂ３を標的とする核酸医薬Nucleic acid medicine targeting cancer type SLCO1B3
　本発明は、癌型SLCO1B3の発現抑制に用いるための核酸又は該核酸を含む医薬組成物に関する。 The present invention relates to a nucleic acid for use in suppressing the expression of cancer-type SLCO1B3 or a pharmaceutical composition containing the nucleic acid.
　肺癌、膵癌等の難治性癌に対する有効な治療薬の創製は急務の課題である。そのためにはそれらの癌細胞に発現し、正常細胞に発現しない或いは発現が非常に低い分子であって、癌の発症又は進行に寄与する分子を同定し、その分子の機能や発現を阻害する物質の研究開発を行うことが必要となる。 The creation of effective therapeutic agents for refractory cancers such as lung cancer and pancreatic cancer is an urgent issue. To that end, molecules that are expressed in those cancer cells and not expressed in normal cells or very low in expression, identify molecules that contribute to the development or progression of cancer, and inhibit the function and expression of the molecules It is necessary to conduct research and development.
　肺癌では、上皮成長因子受容体(EGFR)の変異や、EML4(Echinoderm microtubule-associated protein-like 4)-ALK(anaplastic lymphoma kinase)などの融合タンパク質などがその発症に関わることが知られており、有望な分子標的治療薬が臨床使用されている。しかしながらそれらの発現がみられず、治療標的分子が確定できない肺癌も多く存在する。また標的分子が同定され治療薬が開発されているものでも、癌細胞のゲノム不安定性等の性質により耐性変異体が出現し、再発を見ることも問題となっている。そこで新たな癌の標的分子に対して特徴的な治療薬の創製が切望されている。 In lung cancer, epidermal growth factor receptor (EGFR) mutations and fusion proteins such as EML4 (Echinoderm microtubule-associated protein-like 4) -ALK (anaplastic lymphoma kinase) are known to be involved in the pathogenesis. Promising molecular targeted therapeutics are in clinical use. However, there are many lung cancers in which their expression is not observed and the therapeutic target molecule cannot be determined. In addition, even if a target molecule has been identified and a therapeutic drug has been developed, resistance mutants appear due to properties such as genomic instability of cancer cells, and recurrence is also a problem. Therefore, creation of a therapeutic agent characteristic for a new target molecule of cancer is eagerly desired.
　ところで、溶質キャリヤー有機アニオントランスポーターファミリーメンバー１Ｂ３（SLCO1B3;Solute carrier organic anion transporter family member 1B3）は、細胞膜上に発現する12回膜貫通型トランスポーターであり、幅広い基質認識性を示すことが知られている。SLCO1B3は通常肝臓に特異的に発現するが、癌化に伴い他の臓器でも発現が認められるようになることが報告されている。 By the way, the solute carrier organic anion transporter family member 1B3 (SLCO1B3; Solute carrier organic anion transporter family member 1B3) is a 12-transmembrane transporter expressed on the cell membrane and is known to show a wide range of substrate recognition properties. ing. SLCO1B3 is normally expressed specifically in the liver, but it has been reported that expression is also observed in other organs with canceration.
　特許文献１には、OATP1B3（SLCO1B3の別名）の選択的スプライシングバリアントが癌細胞に特異的に発現することが記載されている。該文献には、OATP1B3のexon2及びexon3の間にこれまで知られていなかった新たな転写開始点が存在し新たなexonが存在すること、このスプライシングバリアントは癌細胞に特異的に発現することが開示されている。
　また非特許文献１には、大腸癌及び膵臓癌細胞において、野生型SLCO1B3のexon1及びexon2の代わりに、exon様配列を有するSLCO1B3変異体が発現していることが記載されている。さらに、Thakkar Nらは、該変異体は、翻訳後修飾を経てプロテアソーム分解されること、及び該変異体は、SLCO1B3の基質の１つであるcholecystokin-8の取り込みが野生型と比較して少ないことを報告している。野生型と比較してこの変異体は細胞膜への局在が少なく、ほとんどが細胞内に局在している（非特許文献１）。 Patent Document 1 describes that an alternative splicing variant of OATP1B3 (also known as SLCO1B3) is specifically expressed in cancer cells. In this document, there is a new transcription initiation point between exon2 and exon3 of OATP1B3 and there is a new exon, and this splicing variant is expressed specifically in cancer cells. It is disclosed.
Non-Patent Document 1 describes that SLCO1B3 mutants having exon-like sequences are expressed in colon cancer and pancreatic cancer cells instead of exon1 and exon2 of wild-type SLCO1B3. Furthermore, Thakkar N et al. Show that the mutant is proteasomally degraded through post-translational modification, and that the mutant has less uptake of cholecystokin-8, one of the substrates of SLCO1B3, compared to the wild type. It is reported that. Compared to the wild type, this mutant has less localization to the cell membrane, and most of them are localized in the cell (Non-patent Document 1).
　このように、野生型SLCO1B3のexon2及びexon3の間に転写開始点を有するSLCO1B3バリアントは、癌細胞で特異的に発現することが報告され、癌のバイオマーカーとして機能し得ることが示された。しかしながら、これらのバリアントが、癌の発症及び／又は進行に寄与するのか否かについては全く明らかとなっていない。 Thus, the SLCO1B3 variant having a transcription initiation site between exon2 and exon3 of wild-type SLCO1B3 was reported to be specifically expressed in cancer cells, and was shown to be able to function as a cancer biomarker. However, it is not clear at all whether these variants contribute to the onset and / or progression of cancer.
特開2011-188853号公報JP 2011-188853
　本発明の目的は、癌細胞に発現し、正常細胞に発現しない或いは発現が非常に低い分子であって、癌の発症及び／又は進行に寄与する分子を同定し、その分子の機能や発現を阻害する物質を提供することであり、それを用いた癌の予防又は治療用医薬組成物を提供することである。さらに本発明は、癌の予防及び／又は治療作用を有する物質のスクリーニング方法、被検動物における癌の悪性度又は悪性化リスクを試験する方法、並びに癌の予防又は治療方法も提供する。 The object of the present invention is to identify a molecule that is expressed in cancer cells and not expressed in normal cells or very low in expression, and that contributes to the onset and / or progression of cancer. It is to provide a substance that inhibits, and to provide a pharmaceutical composition for preventing or treating cancer using the same. Furthermore, the present invention also provides a method for screening a substance having an effect of preventing and / or treating cancer, a method for testing the malignancy or risk of malignancy of cancer in a test animal, and a method for preventing or treating cancer.
　本発明者らは、上記目的を達成すべく鋭意研究を重ねた結果、肺癌術後検体を用いたexonの網羅的発現解析により、非癌部分とくらべ癌部分で特異的なexon（exon1*）を使用している遺伝子としてSLCO1B3を同定することに成功した。さらに本発明者らは、exon1*を使用するSLCO1B3である癌型SLCO1B3（Ct-SLCO1B3）が肺癌細胞において増殖や遊走、浸潤を促進する分子であることを見出した。また、本発明者らは、Ct-SLCO1B3は、snail、slugの発現を上昇させ、E-cadherinやoccludinの発現を抑制し、マトリックスメタロプロテアーゼ９（MMP9）の発現を誘導することを見出した。
　そこで、本発明者らは、Ct-SLCO1B3の発現と機能を効果的に抑制できる、標的となるCt-SLCO1B3の領域を探り、Ct-SLCO1B3の発現と機能を効果的に抑制できる核酸を見出した。
　これらの知見から、本発明者らは、Ct-SLCO1B3の発現抑制により癌細胞の増殖や遊走、浸潤を抑制し得ることを実証して、本発明を完成するに至った。 As a result of intensive studies to achieve the above object, the present inventors have conducted a comprehensive exon expression analysis using exon specimens after lung cancer, and exon (exon1 *) specific to the cancer part compared to the non-cancer part. We succeeded in identifying SLCO1B3 as a gene using. Furthermore, the present inventors have found that a cancer type SLCO1B3 (Ct-SLCO1B3), which is SLCO1B3 using exon1 *, is a molecule that promotes proliferation, migration, and invasion in lung cancer cells. The present inventors have also found that Ct-SLCO1B3 increases the expression of snail and slug, suppresses the expression of E-cadherin and occludin, and induces the expression of matrix metalloproteinase 9 (MMP9).
Therefore, the present inventors searched for a target Ct-SLCO1B3 region that can effectively suppress the expression and function of Ct-SLCO1B3, and found a nucleic acid that can effectively suppress the expression and function of Ct-SLCO1B3. .
Based on these findings, the present inventors have demonstrated that suppression of Ct-SLCO1B3 expression can suppress the proliferation, migration, and invasion of cancer cells, thereby completing the present invention.
　すなわち、本発明は以下の通りのものである。
1）センス鎖及びアンチセンス鎖から成り、少なくとも11個の塩基対の二重鎖領域を含む二本鎖核酸であって、前記アンチセンス鎖中の、少なくとも17個のヌクレオチドかつ多くとも30個のヌクレオチドの鎖長のオリゴヌクレオチド鎖において、下記（I）～（II）のいずれかに記載のDNAの塩基配列と完全に相補する標的Ct-SLCO1B3（癌型溶質キャリヤー有機アニオントランスポーターファミリーメンバー１Ｂ３）RNA配列と相補的である、Ct-SLCO1B3の発現を減少させる二本鎖核酸：
（I）配列番号1の配列からなるDNA、
（II）（I）記載のDNAと実質的に同一の配列を有する核酸。
2）標的Ct-SLCO1B3 RNA配列が、配列番号2の配列に含まれる、1）記載の二本鎖核酸。
3）標的Ct-SLCO1B3 RNA配列が、配列番号6～13からなる群より選択される、1）又は2）に記載の二本鎖核酸。
4）前記アンチセンス鎖は、配列番号14～21からなる群より選択される配列を含む、1）～3）のいずれかに記載の二本鎖核酸。
5）前記センス鎖は、配列番号22～29からなる群より選択される配列を含む、1）～4）のいずれかに記載の二本鎖核酸。
6）表１記載の配列番号14／配列番号22、配列番号15／配列番号23、配列番号16／配列番号24、配列番号17／配列番号25、配列番号18／配列番号26、配列番号19／配列番号27、配列番号20／配列番号28、及び配列番号21／配列番号29から成る群から選択される1対のアンチセンス鎖／センス鎖の配列を含む、1）～5）のいずれかに記載の二本鎖核酸。
7）1）～6）のいずれかに記載の二本鎖核酸の、アンチセンス鎖のみからなる一本鎖核酸。
8）Ct-SLCO1B3の発現を抑制する核酸を有効成分として含む医薬組成物。
9）1）～7）のいずれかに記載の核酸を有効成分として含む、医薬組成物。
10）癌の治療又は予防用である、8）又は9）記載の医薬組成物。
11）癌がCt-SLCO1B3を発現する非小細胞肺癌、肝癌、膵癌、又は食道癌である、10）記載の医薬組成物。
12）以下の（1）～（3）の工程を含む、癌の予防及び／又は治療作用を有する物質のスクリーニング方法：
（1）Ct-SLCO1B3を発現する細胞に、被検物質を接触させる工程、
（2）前記細胞におけるCt-SLCO1B3の発現量又は機能を測定する工程、及び
（3）被検物質の非存在下において測定した場合と比較して、Ct-SLCO1B3の発現量又は機能を低下させる化合物を、癌の予防及び／又は治療作用を有する物質の候補として選択する工程。
13）（1）及び（2）の工程を含む、被験動物より採取した試料中のCt-SLCO1B3の発現量を測定することを特徴とする癌の悪性度又は悪性化リスクを試験する方法：
（1）被験動物より採取した試料中の、Ct-SLCO1B3の発現量を測定する工程、
（2）正常動物由来の試料において測定した場合と比較して、前記発現量が上昇している被験動物を、癌の悪性度が高いか、又は将来悪性化するリスクが高いと判定する工程。
14）癌の予防又は治療用の医薬組成物を製造するための、1）～7）のいずれかに記載の核酸の使用。
15）癌が、Ct-SLCO1B3を発現する非小細胞肺癌、肝癌、膵癌、又は食道癌である、14）に記載の使用。
16）癌を予防又は治療する方法であって、予防上又は治療上有効量の1）～7）のいずれかに記載の核酸、又は8）～11）のいずれかに記載の医薬組成物を、該予防又は治療を必要とする動物に投与する工程を含む方法。
17）癌が、Ct-SLCO1B3を発現する非小細胞肺癌、肝癌、膵癌、又は食道癌である、16）に記載の方法。
18）癌の予防又は治療のための、1）～7）のいずれかに記載の核酸の使用。
19）癌が、Ct-SLCO1B3を発現する非小細胞肺癌、肝癌、膵癌、又は食道癌である、18）に記載の使用。 That is, the present invention is as follows.
1) a double-stranded nucleic acid consisting of a sense strand and an antisense strand, and comprising a double-stranded region of at least 11 base pairs, wherein the antisense strand has at least 17 nucleotides and at most 30 Target Ct-SLCO1B3 (cancer-type solute carrier organic anion transporter family member 1B3) that is completely complementary to the nucleotide sequence of the DNA described in any of (I) to (II) below in the oligonucleotide chain of nucleotide length Double-stranded nucleic acid that reduces the expression of Ct-SLCO1B3 that is complementary to the RNA sequence:
(I) DNA comprising the sequence of SEQ ID NO: 1,
(II) A nucleic acid having substantially the same sequence as the DNA described in (I).
2) The double-stranded nucleic acid according to 1), wherein the target Ct-SLCO1B3 RNA sequence is contained in the sequence of SEQ ID NO: 2.
3) The double-stranded nucleic acid according to 1) or 2), wherein the target Ct-SLCO1B3 RNA sequence is selected from the group consisting of SEQ ID NOs: 6 to 13.
4) The double-stranded nucleic acid according to any one of 1) to 3), wherein the antisense strand comprises a sequence selected from the group consisting of SEQ ID NOs: 14 to 21.
5) The double-stranded nucleic acid according to any one of 1) to 4), wherein the sense strand includes a sequence selected from the group consisting of SEQ ID NOs: 22 to 29.
6) SEQ ID NO: 14 / SEQ ID NO: 22, SEQ ID NO: 15 / SEQ ID NO: 23, SEQ ID NO: 16 / SEQ ID NO: 24, SEQ ID NO: 17 / SEQ ID NO: 25, SEQ ID NO: 18 / SEQ ID NO: 26, SEQ ID NO: 19 / 1) to 5), comprising a pair of antisense / sense strand sequences selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 20 / SEQ ID NO: 28, and SEQ ID NO: 21 / SEQ ID NO: 29 The double-stranded nucleic acid described.
7) A single-stranded nucleic acid comprising only the antisense strand of the double-stranded nucleic acid according to any one of 1) to 6).
8) A pharmaceutical composition comprising a nucleic acid that suppresses the expression of Ct-SLCO1B3 as an active ingredient.
9) A pharmaceutical composition comprising the nucleic acid according to any one of 1) to 7) as an active ingredient.
10) The pharmaceutical composition according to 8) or 9), which is used for treatment or prevention of cancer.
11) The pharmaceutical composition according to 10), wherein the cancer is non-small cell lung cancer, liver cancer, pancreatic cancer, or esophageal cancer that expresses Ct-SLCO1B3.
12) A screening method for a substance having a preventive and / or therapeutic action for cancer, comprising the following steps (1) to (3):
(1) a step of contacting a test substance with a cell expressing Ct-SLCO1B3,
(2) a step of measuring the expression level or function of Ct-SLCO1B3 in the cell, and (3) a decrease in the expression level or function of Ct-SLCO1B3 as compared to the case of measurement in the absence of the test substance. A step of selecting a compound as a candidate for a substance having a preventive and / or therapeutic action for cancer.
13) A method for testing malignancy or risk of malignant cancer characterized by measuring the expression level of Ct-SLCO1B3 in a sample collected from a test animal, including the steps (1) and (2):
(1) a step of measuring the expression level of Ct-SLCO1B3 in a sample collected from a test animal,
(2) A step of determining that a test animal having an increased expression level has a high cancer malignancy or a high risk of becoming malignant in the future, as compared with a case of measuring in a sample derived from a normal animal.
14) Use of the nucleic acid according to any one of 1) to 7) for producing a pharmaceutical composition for preventing or treating cancer.
15) The use according to 14), wherein the cancer is non-small cell lung cancer, liver cancer, pancreatic cancer, or esophageal cancer that expresses Ct-SLCO1B3.
16) A method for preventing or treating cancer, comprising a prophylactically or therapeutically effective amount of the nucleic acid according to any one of 1) to 7), or the pharmaceutical composition according to any one of 8) to 11). A method comprising administering to an animal in need of the prevention or treatment.
17) The method according to 16), wherein the cancer is non-small cell lung cancer, liver cancer, pancreatic cancer, or esophageal cancer that expresses Ct-SLCO1B3.
18) Use of the nucleic acid according to any one of 1) to 7) for prevention or treatment of cancer.
19) The use according to 18), wherein the cancer is non-small cell lung cancer, liver cancer, pancreatic cancer, or esophageal cancer that expresses Ct-SLCO1B3.
　本発明の核酸、又は該核酸を含む医薬組成物を投与することによって、Ct-SLCO1B3の発現を抑制することが可能となる。従って、Ct-SLCO1B3の発現に関連する疾患、特に癌の治療・予防が可能となる。 It is possible to suppress the expression of Ct-SLCO1B3 by administering the nucleic acid of the present invention or the pharmaceutical composition containing the nucleic acid. Accordingly, it is possible to treat or prevent diseases associated with the expression of Ct-SLCO1B3, particularly cancer.
肺癌臨床検体を用いたExon array解析による、SLCO1B3高発現肺癌症例の同定の結果を示す図である。It is a figure which shows the result of the identification of the SLCO1B3 high expression lung cancer case by Exon-array analysis using a lung cancer clinical specimen. 肺癌細胞株を用いたExon array解析によるSLCO1B3高発現肺癌症例の同定の結果を示す図である。It is a figure which shows the result of identification of the lung cancer case which highly expresses SLCO1B3 by Exon-array analysis using a lung cancer cell line. ヒト正常組織におけるSLCO1B3の発現を示す図である。上段：全長SLCO1B3、下段：GAPDH（コントロール）の発現。It is a figure which shows the expression of SLCO1B3 in a human normal tissue. Upper: full-length SLCO1B3, lower: GAPDH (control) expression. Ct-SLCO1B3、Lt-SLCO1B3のsplicing formを示す図である。It is a figure which shows splicing form of Ct-SLCO1B3 and Lt-SLCO1B3. 正常肝臓組織と非小細胞肺癌組織におけるSLCO1B3の免疫組織化学染色の結果を示す図である。It is a figure which shows the result of the immunohistochemical staining of SLCO1B3 in a normal liver tissue and a non-small cell lung cancer tissue. 非小細胞肺癌組織におけるCt-SLCO1B3発現の定量的解析の結果を示す図である。It is a figure which shows the result of the quantitative analysis of Ct-SLCO1B3 expression in a non-small cell lung cancer tissue. A549細胞におけるCt-SLCO1B3　siRNAのノックダウン効率を示す図である。It is a figure which shows the knockdown efficiency of Ct-SLCO1B3 siRNA in A549 cell. A549細胞の足場依存性増殖に対するCt-SLCO1B3　siRNAの効果を示す図である。X軸：トランスフェクション後の日数。Y軸：2日目の細胞数を1とした時の細胞増殖率。It is a figure which shows the effect of Ct-SLCO1B3 siRNA with respect to the anchorage-dependent proliferation of A549 cells. X axis: days after transfection. Y axis: Cell growth rate when the number of cells on the second day is 1. A549細胞の足場非依存性増殖に対するCt-SLCO1B3　siRNAの効果を示す図である。X軸：トランスフェクション後の日数。Y軸：2日目の細胞数を1とした時の細胞増殖率。It is a figure which shows the effect of Ct-SLCO1B3 siRNA with respect to the anchorage-independent proliferation of A549 cells. X axis: days after transfection. Y axis: Cell growth rate when the number of cells on the second day is 1. A549細胞の遊走能に対するCt-SLCO1B3 siRNAの効果を示す図である。It is a figure which shows the effect of Ct-SLCO1B3 * siRNA with respect to the migration ability of A549 cell. A549細胞の浸潤能に対するCt-SLCO1B3 siRNAの効果を示す図である。It is a figure which shows the effect of Ct-SLCO1B3 * siRNA with respect to the invasion ability of A549 cell. Ct-SLCO1B3ノックダウンA549細胞における細胞接着分子occludin（左）、E-cadherin（右）の免疫細胞染色の結果を示す図である。It is a figure which shows the result of the immune cell staining of the cell adhesion molecule occludin (left) and E-cadherin (right) in Ct-SLCO1B3 knockdown A549 cells. Ct-SLCO1B3ノックダウンA549細胞における細胞接着分子（occludin、E-cadherin）、上皮間葉転換制御分子（snail、slug）の発現解析の結果を示す図である。It is a figure which shows the result of the expression analysis of the cell adhesion molecule (occludin, E-cadherin) and epithelial-mesenchymal transition control molecule (snail, slug) in Ct-SLCO1B3 knockdown A549 cell. Ct-SLCO1B3ノックダウンA549細胞におけるmatrix metalloprotease 9（MMP9）の発現解析の結果を示す図である。It is a figure which shows the result of the expression analysis of matrix | metalloprotease-9 (MMP9) in Ct-SLCO1B3 knockdown A549 cell. Ct-SLCO1B3が発現していない肺癌細胞株NCI-H23を用いたCt-SLCO1B3、Lt-SLCO1B3高発現細胞株の樹立の結果を示す図である。樹立したCt-SLCO1B3、Lt-SLCO1B3高発現細胞株における、各SLCO1B3の発現。It is a figure which shows the result of the establishment of Ct-SLCO1B3 and Lt-SLCO1B3 high expression cell lines using lung cancer cell line NCI-H23 in which Ct-SLCO1B3 is not expressed. Expression of each SLCO1B3 in the established Ct-SLCO1B3 and Lt-SLCO1B3 high expression cell lines. Ct-SLCO1B3、Lt-SLCO1B3高発現NCI-H23細胞株の足場非依存性増殖を示す図である。It is a figure which shows the anchorage independent proliferation of Ct-SLCO1B3 and Lt-SLCO1B3 high expression NCI-H23 cell line. Ct-SLCO1B3、Lt-SLCO1B3高発現NCI-H23細胞株の遊走能を示す図である。It is a figure which shows the migration ability of CCI-SLCO1B3 and Lt-SLCO1B3 high expression NCI-H23 cell line. CRISPR/Cas9システムを利用したCt-SLCO1B3発現抑制A549細胞の作製を示す図である。It is a figure which shows preparation of the C549-SLCO1B3 expression suppression A549 cell using a CRISPR / Cas9 system. Ct-SLCO1B3発現抑制A549細胞の足場非依存性増殖抑制を示す図である。It is a figure which shows the anchorage independent proliferation suppression of Ct-SLCO1B3 expression suppression A549 cell. Ct-SLCO1B3発現抑制A549細胞のマウス背部皮下移植による腫瘍形成低下を示す図である。It is a figure which shows the tumor formation fall by the mouse | mouth back | dorsal transplantation of the mouse | mouth back of C549-SLCO1B3 expression suppression A549 cell. Ct-SLCO1B3作用機序のまとめを示す図である。It is a figure which shows the summary of Ct-SLCO1B3 action mechanism. Ct-SLCO1B3 siRNAのノックダウン効率を示す図である。It is a figure which shows the knockdown efficiency of Ct-SLCO1B3 siRNA. Ct-SLCO1B3 siRNAのA549細胞足場依存性増殖に対する作用評価の結果を示す図である。It is a figure which shows the result of the effect | action evaluation with respect to A549 cell anchorage-dependent proliferation of Ct-SLCO1B3 * siRNA. Ct-SLCO1B3 siRNAのA549細胞足場非依存性増殖に対する作用評価の結果を示す図である。It is a figure which shows the result of the effect | action evaluation with respect to A549 cell anchorage independent proliferation of Ct-SLCO1B3 (TM) siRNA. Ct-SLCO1B3 siRNAのCt-SLCO1B3非発現肺癌細胞NCI-H1975の細胞増殖に対する作用評価の結果を示す図である。It is a figure which shows the result of the effect | action with respect to the cell proliferation of the Ct-SLCO1B3 non-expression lung cancer cell NCI-H1975 of Ct-SLCO1B3 siRNA. Ct-SLCO1B3 siRNAのA549細胞におけるノックダウン効率の2次評価の結果を示す図である。It is a figure which shows the result of the secondary evaluation of the knockdown efficiency in A549 cell of Ct-SLCO1B3 siRNA. Ct-SLCO1B3 siRNAのA549細胞足場依存性増殖に対する作用2次評価の結果を示す図である。It is a figure which shows the result of the effect | action secondary evaluation with respect to A549 cell anchorage-dependent proliferation of Ct-SLCO1B3 (pi) siRNA. Ct-SLCO1B3 siRNAのA549細胞足場非依存性増殖に対する作用2次評価の結果を示す図である。It is a figure which shows the result of the secondary evaluation of the effect | action with respect to A549 cell anchorage independent proliferation of Ct-SLCO1B3 (TM) siRNA. Ct-SLCO1B3に対する核酸医薬標的配列の決定を示す図である。It is a figure which shows the determination of the nucleic acid pharmaceutical target sequence with respect to Ct-SLCO1B3. 食道癌、肝細胞癌又は膵癌におけるCt-SLCO1B3発現の定量的解析の結果を示す図である。It is a figure which shows the result of the quantitative analysis of Ct-SLCO1B3 expression in esophageal cancer, hepatocellular carcinoma, or pancreatic cancer. Ct-SLCO1B3 siRNAによる抗腫瘍作用を示す図である。X軸：A549細胞移植後の日数、Y軸：腫瘍体積。矢印はsiRNAの投与時を示す。It is a figure which shows the anti-tumor effect by Ct-SLCO1B3BsiRNA. X axis: days after A549 cell transplantation, Y axis: tumor volume. Arrows indicate when siRNA is administered.
１．Ct-SLCO1B3
　溶質キャリヤー有機アニオントランスポーターファミリーメンバー１Ｂ３（SLCO1B3；Solute carrier organic anion transporter family member 1B3）は、公知のタンパク質である。
　SLCO1B3は、有機アニオン輸送ポリペプチド１Ｂ３（OATP1B3; organic anion-transporting polypeptide 1B3）又はLST-2（Liver specific organic anion transporter-2）とも称される。
　SLCO1B3タンパク質は、SLCO1B3遺伝子によってコードされているタンパク質であり、そのアミノ酸配列及びヌクレオチド配列は公知である。例えば、ヒトSLCO1B3タンパク質のアミノ酸配列は、GenBank Accession No.NP_062818（配列番号3）として登録されている。ヒトSLCO1B3タンパク質をコードするヌクレオチド配列としては、GenBank Accession No.NM_019844（配列番号4）として登録されている。 1. Ct-SLCO1B3
Solute carrier organic anion transporter family member 1B3 (SLCO1B3; Solute carrier organic anion transporter family member 1B3) is a known protein.
SLCO1B3 is also referred to as organic anion-transporting polypeptide 1B3 (OATP1B3; organic anion-transporting polypeptide 1B3) or LST-2 (Liver specific organic anion transporter-2).
The SLCO1B3 protein is a protein encoded by the SLCO1B3 gene, and its amino acid sequence and nucleotide sequence are known. For example, the amino acid sequence of human SLCO1B3 protein is registered as GenBank Accession No. NP — 062818 (SEQ ID NO: 3). The nucleotide sequence encoding human SLCO1B3 protein is registered as GenBank Accession No. NM_019844 (SEQ ID NO: 4).
　本発明において、SLCO1B3は、哺乳動物の分子である。哺乳動物としては、ヒト及びヒトを除く哺乳動物が挙げられ、ヒトを除く哺乳動物としては、例えば、マウス、ラット、ハムスター、モルモット等のげっ歯類やウサギ等の実験動物、ブタ、ウシ、ヤギ、ウマ、ヒツジ等の家畜、イヌ、ネコ等のペット、サル、オランウータン、チンパンジー等の霊長類が挙げられる。配列番号1と配列類似性の高い配列を有するという観点から、好ましくは、SLCO1B3は、ヒト、チンパンジー及びオランウータンの分子である。また、ヒトの疾患の予防又は治療のためには、ヒト由来のSLCO1B3が好ましい。 In the present invention, SLCO1B3 is a mammalian molecule. Examples of mammals include humans and mammals other than humans. Examples of mammals other than humans include rodents such as mice, rats, hamsters, and guinea pigs, and laboratory animals such as rabbits, pigs, cows, and goats. , Primates such as domestic animals such as horses and sheep, pets such as dogs and cats, monkeys, orangutans and chimpanzees. From the viewpoint of having a sequence having high sequence similarity to SEQ ID NO: 1, SLCO1B3 is preferably a human, chimpanzee or orangutan molecule. In addition, human-derived SLCO1B3 is preferred for the prevention or treatment of human diseases.
　本明細書中、ヒト肝臓型SLCO1B3（Lt-SLCO1B3）タンパク質とは、ヒトSLCO1B3遺伝子のexon1、exon2、exon3、exon4、exon5、exon6、exon7、exon8、exon9、exon10、exon11、exon12、exon13、exon14及びexon15からなるDNAによりコードされるアミノ酸配列からなるタンパク質を指す。肝臓型Lt-SLCO1B3タンパク質のアミノ酸配列の例としては、配列番号3が挙げられる。
　本明細書中、肝臓型SLCO1B3タンパク質は、配列番号3のアミノ酸配列からなるタンパク質と同一もしくは実質的に同一のアミノ酸配列を含むタンパク質を指す。 In the present specification, human liver-type SLCO1B3 (Lt-SLCO1B3) protein means human SLCO1B3 gene exon1, exon2, exon3, exon4, exon5, exon6, exon7, exon8, exon9, exon10, exon11, exon12, exon13, exon14 and A protein consisting of an amino acid sequence encoded by DNA consisting of exon15. An example of the amino acid sequence of liver-type Lt-SLCO1B3 protein is SEQ ID NO: 3.
In the present specification, the liver-type SLCO1B3 protein refers to a protein comprising the same or substantially the same amino acid sequence as the protein consisting of the amino acid sequence of SEQ ID NO: 3.
　本明細書中、ヒト癌型SLCO1B3（Ct-SLCO1B3）タンパク質とは、ヒトLt-SLCO1B3遺伝子のexon2及びexon3の間の転写開始点から転写されるSLCO1B3のRNAによりコードされるタンパク質であって、配列番号1がコードするアミノ酸配列と同一又は実質的に同一のアミノ酸配列を含むタンパク質である。
　ヒトCt-SLCO1B3タンパク質の例としては、配列番号5のアミノ酸配列からなるタンパク質が挙げられる。
　本明細書中、Ct-SLCO1B3タンパク質は、配列番号5のアミノ酸配列からなるタンパク質と同一又は実質的に同一のアミノ酸配列を含むタンパク質を指す。 In this specification, human cancer-type SLCO1B3 (Ct-SLCO1B3) protein is a protein encoded by RNA of SLCO1B3 transcribed from the transcription start site between exon2 and exon3 of human Lt-SLCO1B3 gene, A protein comprising an amino acid sequence identical or substantially identical to the amino acid sequence encoded by No. 1.
Examples of human Ct-SLCO1B3 protein include a protein consisting of the amino acid sequence of SEQ ID NO: 5.
In the present specification, Ct-SLCO1B3 protein refers to a protein comprising the same or substantially the same amino acid sequence as the protein consisting of the amino acid sequence of SEQ ID NO: 5.
　本明細書において、アミノ酸配列が実質的に同一であるタンパク質としては、アミノ酸配列が約80％以上、好ましくは約90％以上、より好ましくは約95％以上、いっそう好ましくは約97％以上、特に好ましくは約98％以上、最も好ましくは約99％以上の同一性を有するタンパク質が挙げられる。
　あるいは、本明細書において、アミノ酸配列が実質的に同一であるタンパク質としては、アミノ酸配列において、1～10個、好ましくは1～8個、より好ましくは1～5個、いっそう好ましくは1～3個、特に好ましくは1又は2個、最も好ましくは1個のアミノ酸に、欠失、置換及び／又は付加の変異が生じたアミノ酸配列からなるタンパク質が挙げられる。 In the present specification, proteins having substantially the same amino acid sequence include amino acid sequences of about 80% or more, preferably about 90% or more, more preferably about 95% or more, more preferably about 97% or more, particularly A protein having an identity of preferably about 98% or more, most preferably about 99% or more is mentioned.
Alternatively, in the present specification, the protein having substantially the same amino acid sequence is preferably 1 to 10, preferably 1 to 8, more preferably 1 to 5, and more preferably 1 to 3 in the amino acid sequence. A protein having an amino acid sequence in which deletion, substitution, and / or addition mutation occurs in one, particularly preferably 1 or 2, and most preferably 1 amino acid.
　好ましい態様において、実質的に同一のアミノ酸配列を有するタンパク質同士は、共通する機能を1つ以上有する。
　例えば、Ct-SLCO1B3の機能としては、（1）細胞の足場非依存性増殖促進、（2）細胞の遊走能促進、（3）細胞の浸潤能促進、（4）snail発現量の上昇、（5）slug発現量の上昇、（6）E-cadherin発現量の低下、（7）occludin発現量の低下、（8）MMP9発現量の上昇が挙げられ、好ましい態様において、配列番号5のアミノ酸配列からなるヒトCt-SLCO1B3タンパク質と実質的に同一なアミノ酸配列を有するタンパク質は、上記（1）～（8）のいずれか1つ以上の機能を有する。タンパク質が上記（1）～（8）の機能を有するか否かは、実施例記載の方法又は本発明のスクリーニング法に記載の方法に準じて検証することができる。 In a preferred embodiment, proteins having substantially the same amino acid sequence have one or more common functions.
For example, the functions of Ct-SLCO1B3 include (1) promotion of cell anchorage-independent growth, (2) promotion of cell migration ability, (3) promotion of cell invasion ability, (4) increase in snail expression level, ( 5) Increase in slug expression level, (6) Decrease in E-cadherin expression level, (7) Decrease in occludin expression level, (8) Increase in MMP9 expression level. In a preferred embodiment, the amino acid sequence of SEQ ID NO: 5 A protein having substantially the same amino acid sequence as the human Ct-SLCO1B3 protein comprising any one of the above functions (1) to (8). Whether or not a protein has the functions (1) to (8) can be verified according to the method described in the examples or the method described in the screening method of the present invention.
　本明細書中、ヒトCt-SLCO1B3遺伝子のexon1*とは、Lt-SLCO1B3遺伝子のexon2及びexon3の間に存在する転写開始点から、Lt-SLCO1B3遺伝子のexon3領域の5’と転写開始点の間に位置するスプライシングドナーサイトまでの領域を指す。具体的には、ヒトCt-SLCO1B3遺伝子のexon1*の塩基配列としては、配列番号1が挙げられる。 In the present specification, exon1 * of the human Ct-SLCO1B3 gene refers to a region between the transcription start point existing between exon2 and exon3 of the Lt-SLCO1B3 gene and 5 ′ of the exon3 region of the Lt-SLCO1B3 gene and the transcription start point. Refers to the region up to the splicing donor site. Specifically, SEQ ID NO: 1 can be mentioned as the base sequence of exon1 * of the human Ct-SLCO1B3 gene.
２．Ct-SLCO1B3の発現を抑制する核酸
　本発明の核酸の標的となる、Ct-SLCO1B3遺伝子の塩基配列と完全に相補するRNA配列（本明細書中、単に「標的Ct-SLCO1B3 RNA配列」又は、「本発明の核酸の標的Ct-SLCO1B3 RNA配列」とも称する）について以下に説明する。 2. Nucleic acid that suppresses the expression of Ct-SLCO1B3 An RNA sequence that is completely complementary to the base sequence of the Ct-SLCO1B3 gene, which is the target of the nucleic acid of the present invention (herein, simply referred to as “target Ct-SLCO1B3 RNA sequence” or “ The “target Ct-SLCO1B3 RNA sequence of the nucleic acid of the present invention” is also described below.
　本発明の核酸は、以下の（I）～（II）のDNAの塩基配列と完全に相補する塩基配列を有するRNAを標的とする：
（I）配列番号1の配列からなるDNA、
（II）（I）記載のDNAと実質的に同一の配列を有する核酸。 The nucleic acid of the present invention targets RNA having a base sequence that is completely complementary to the base sequences of the following DNAs (I) to (II):
(I) DNA comprising the sequence of SEQ ID NO: 1,
(II) A nucleic acid having substantially the same sequence as the DNA described in (I).
　本明細書中、DNA配列は、DNAの塩基配列と同義に使用され、RNA配列は、RNAの塩基配列と同義に使用される。 In this specification, a DNA sequence is used synonymously with a DNA base sequence, and an RNA sequence is used synonymously with an RNA base sequence.
　本明細書において、塩基配列が実質的に同一である核酸としては、塩基配列が約80％以上、好ましくは約90％以上、より好ましくは約95％以上、いっそう好ましくは約97％以上、特に好ましくは約98％以上、最も好ましくは約99％以上の同一性を有する核酸が挙げられる。
　あるいは、塩基配列が実質的に同一である核酸としては、塩基配列において、1～10個、好ましくは1～8個、より好ましくは1～5個、いっそう好ましくは1～3個、特に好ましくは1又は2個、最も好ましくは1個のヌクレオチドに、欠失、置換及び／又は付加の変異が生じた塩基配列からなる核酸が挙げられる。 In the present specification, nucleic acids having substantially the same base sequence include a base sequence of about 80% or more, preferably about 90% or more, more preferably about 95% or more, more preferably about 97% or more, particularly Preferably, the nucleic acid has about 98% or more, most preferably about 99% or more identity.
Alternatively, the nucleic acids having substantially the same nucleotide sequence are preferably 1 to 10, preferably 1 to 8, more preferably 1 to 5, even more preferably 1 to 3, particularly preferably nucleotide sequences. Examples include a nucleic acid having a base sequence in which deletion, substitution and / or addition mutation has occurred in one or two nucleotides, most preferably one nucleotide.
　本発明の核酸の、標的Ct-SLCO1B3 RNA配列は、好ましくは配列番号2に含まれる配列であり、さらに好ましくは後述する配列番号6～13のいずれかであり、特に好ましくは配列番号6、7、8又は9である。 The target Ct-SLCO1B3 RNA sequence of the nucleic acid of the present invention is preferably a sequence included in SEQ ID NO: 2, more preferably any one of SEQ ID NOS: 6 to 13 described below, and particularly preferably SEQ ID NOS: 6, 7 , 8 or 9.
　本発明において、標的Ct-SLCO1B3 RNA配列に対して相補的な塩基配列を含む核酸をアンチセンス鎖核酸と称し、アンチセンス鎖核酸の塩基配列に対して相補的な塩基配列を含む核酸をセンス鎖核酸とも称する。本明細書において「本発明の核酸」という場合、特にことわらない限り、アンチセンス鎖核酸、センス鎖核酸、並びにセンス鎖及びアンチセンス鎖核酸が対形成した二本鎖核酸を包含する意味で用いられる。 In the present invention, a nucleic acid containing a base sequence complementary to the target Ct-SLCO1B3 RNA sequence is called an antisense strand nucleic acid, and a nucleic acid containing a base sequence complementary to the base sequence of the antisense strand nucleic acid is the sense strand. Also called nucleic acid. In the present specification, unless otherwise specified, the term “nucleic acid of the present invention” is used to include an antisense strand nucleic acid, a sense strand nucleic acid, and a double-stranded nucleic acid paired with a sense strand and an antisense strand nucleic acid. It is done.
　本発明の核酸としては、ヌクレオチド又は該ヌクレオチドと同等の機能を有する分子が重合した分子であればいかなる分子であってもよく、例えばリボヌクレオチドの重合体であるRNA、デオキシリボヌクレオチドの重合体であるDNA、RNAとDNAとからなるキメラ核酸、及びこれらの核酸の少なくとも一つのヌクレオチドが該ヌクレオチドと同等の機能を有する分子で置換されたヌクレオチド重合体があげられる。また、これらの核酸内にヌクレオチドと同等の機能を有する分子を少なくとも一つ含む誘導体も、本発明の核酸に含まれる。またウラシル（U）は、チミン（T）に一義的に読み替えることができる。 The nucleic acid of the present invention may be any molecule as long as it is a molecule obtained by polymerizing nucleotides or molecules having functions equivalent to the nucleotides, for example, RNA that is a polymer of ribonucleotides, or a polymer of deoxyribonucleotides. Examples thereof include DNA, chimeric nucleic acids composed of RNA and DNA, and nucleotide polymers in which at least one nucleotide of these nucleic acids is substituted with a molecule having a function equivalent to that of the nucleotide. In addition, derivatives containing at least one molecule having a function equivalent to nucleotide in these nucleic acids are also included in the nucleic acid of the present invention. Uracil (U) can be uniquely read as thymine (T).
　ヌクレオチドと同等の機能を有する分子としては、例えばヌクレオチド誘導体等があげられる。ヌクレオチド誘導体としては、ヌクレオチドに修飾を施した分子であればいかなる分子であってもよいが、例えばRNA又はDNAと比較して、ヌクレアーゼ耐性の向上もしくは安定化させるため、相補鎖核酸とのアフィニティーをあげるため、細胞透過性をあげるため、又は可視化させるために、リボヌクレオチド又はデオキシリボヌクレオチドに修飾を施した分子等が好適に用いられる。 Examples of molecules having a function equivalent to nucleotides include nucleotide derivatives. The nucleotide derivative may be any molecule as long as it is a nucleotide-modified molecule.For example, in order to improve or stabilize nuclease resistance compared to RNA or DNA, affinity with a complementary strand nucleic acid is increased. In order to increase the cell permeability or to make it visible, a molecule in which ribonucleotides or deoxyribonucleotides are modified is preferably used.
　ヌクレオチドに修飾を施した分子としては、例えば糖部修飾ヌクレオチド、リン酸ジエステル結合修飾ヌクレオチド、塩基修飾ヌクレオチド、ならびに糖部、リン酸ジエステル結合及び塩基の少なくとも一つが修飾されたヌクレオチド等があげられる。 Examples of the molecule in which the nucleotide is modified include a sugar moiety-modified nucleotide, a phosphodiester bond-modified nucleotide, a base-modified nucleotide, and a nucleotide in which at least one of the sugar moiety, phosphodiester bond and base is modified.
　糖部修飾ヌクレオチドとしては、ヌクレオチドの糖の化学構造の一部あるいは全てに対し、任意の置換基で修飾もしくは置換したもの、又は任意の原子で置換したものであればいかなるものでもよいが、2’－修飾ヌクレオチドが好ましく用いられる。 As the sugar-modified nucleotide, any or all of the chemical structure of the sugar of the nucleotide may be modified or substituted with any substituent, or may be substituted with any atom. '-Modified nucleotides are preferably used.
　2’－修飾ヌクレオチドとしては、例えばリボースの2’－OH基がH、OR、R、R’OR、SH、SR、NH2、NHR、NR2、N3、CN、F、Cl、Br及びＩからなる群（Rはアルキル又はアリール、好ましくは炭素数1～6のアルキルであり、R’はアルキレン、好ましくは炭素数1～6のアルキレンである）から選択される置換基で置換されたヌクレオチド、好ましくは2’－OH基がH、F又はメトキシ基で置換されたヌクレオチド、より好ましくは2’－OH基がF又はメトキシ基で置換されたヌクレオチドがあげられる。また、2’－OH基が2-(methoxy)ethoxy基、3-aminopropoxy基、2-[(N,N-dimethylamino)oxy]ethoxy基、3-(N,N-dimethylamino)propoxy基、2-[2-(N,N-dimethylamino)ethoxy]ethoxy基、2-(methylamino)-2-oxoethoxy基、2-(N-methylcarbamoyl)ethoxy基及び2-cyanoethoxy基からなる群から選択される置換基で置換されたヌクレオチド等もあげられる。 Examples of 2′-modified nucleotides include those in which the 2′-OH group of ribose is H, OR, R, R′OR, SH, SR, NH 2 , NHR, NR 2 , N 3 , CN, F, Cl, Br, and Substituted with a substituent selected from the group consisting of I (R is alkyl or aryl, preferably alkyl having 1 to 6 carbon atoms and R ′ is alkylene, preferably alkylene having 1 to 6 carbon atoms) A nucleotide, preferably a nucleotide in which the 2′-OH group is substituted with H, F or a methoxy group, more preferably a nucleotide in which the 2′-OH group is substituted with F or a methoxy group. In addition, 2'-OH group is 2- (methoxy) ethoxy group, 3-aminopropoxy group, 2-[(N, N-dimethylamino) oxy] ethoxy group, 3- (N, N-dimethylamino) propoxy group, 2- A substituent selected from the group consisting of [2- (N, N-dimethylamino) ethoxy] ethoxy group, 2- (methylamino) -2-oxoethoxy group, 2- (N-methylcarbamoyl) ethoxy group and 2-cyanoethoxy group Examples thereof include substituted nucleotides.
　糖部修飾ヌクレオチドとしては、糖部に架橋構造を導入することにより2つの環状構造を有する架橋構造型人工核酸（Bridged Nucleic Acid）（BNA）があげられ、具体的には、2’位の酸素原子と4’位の炭素原子がメチレンを介して架橋したロックト人工核酸（Locked Nucleic Acid）（LNA）、エチレン架橋構造型人工核酸（Ethylene bridged nucleic acid）（ENA）［Nucleic Acid Research, 32,e175(2004)］等があげられ、さらにペプチド核酸（PNA）［Acc.Chem.Res.,32,624(1999)］、オキシペプチド核酸（OPNA）［J.Am.Chem.Soc.,123,4653(2001)］、ペプチドリボ核酸（PRNA）［J.Am.Chem.Soc.,122,6900(2000)］等もあげられる。 Examples of the sugar-modified nucleotide include a bridged structure type artificial nucleic acid (BNA) having two cyclic structures by introducing a crosslinked structure into the sugar part, specifically, an oxygen at the 2 ′ position. Locked 核酸 Nucleic Acid (LNA), ethylene bridged nucleic acid (ENA) [Nucleic Acid Research, 32, e175 (2004)] and the like, and peptide nucleic acids (PNA) [Acc. Chem. Res., 32, 624 (1999)], oxypeptide nucleic acids (OPNA) [J. Am. Chem. Soc., 123,4653 (2001) )], Peptide ribonucleic acid (PRNA) [J. Am. Chem. Soc., 122, 6900 (2000)] and the like.
　リン酸ジエステル結合修飾ヌクレオチドとしては、ヌクレオチドのリン酸ジエステル結合の化学構造の一部あるいは全てに対し、任意の置換基で修飾もしくは置換したもの、又は任意の原子で置換したものであればいかなるものでもよく、例えば、リン酸ジエステル結合がホスホロチオエート結合に置換されたヌクレオチド、リン酸ジエステル結合がホスホロジチオエート結合に置換されたヌクレオチド、リン酸ジエステル結合がアルキルホスホネート結合に置換されたヌクレオチド、リン酸ジエステル結合がホスホロアミデート結合に置換されたヌクレオチド等があげられる。 As the phosphodiester bond-modified nucleotide, any or all of the chemical structure of the nucleotide phosphodiester bond modified or substituted with any substituent or any atom may be used. For example, a nucleotide in which a phosphodiester bond is replaced with a phosphorothioate bond, a nucleotide in which a phosphodiester bond is replaced with a phosphorodithioate bond, a nucleotide in which a phosphodiester bond is replaced with an alkylphosphonate bond, a phosphate Examples thereof include nucleotides in which a diester bond is substituted with a phosphoramidate bond.
　塩基修飾ヌクレオチドとしては、ヌクレオチドの塩基の化学構造の一部あるいは全てに対し、任意の置換基で修飾もしくは置換したもの、又は任意の原子で置換したものであればいかなるものでもよく、例えば、塩基内の酸素原子が硫黄原子で置換されたもの、水素原子が炭素数1～6のアルキル基、ハロゲン等で置換されたもの、メチル基が水素、ヒドロキシメチル、炭素数2～6のアルキル基等で置換されたもの、アミノ基が炭素数1～6のアルキル基、炭素数1～6のアルカノイル基、オキソ基、ヒドロキシ基等に置換されたものがあげられる。 The base-modified nucleotide may be any nucleotide as long as it is part or all of the nucleotide base chemical structure modified or substituted with an arbitrary substituent, or substituted with an arbitrary atom. In which oxygen atoms are substituted with sulfur atoms, hydrogen atoms are substituted with alkyl groups having 1 to 6 carbon atoms, halogens, etc., methyl groups are hydrogen, hydroxymethyl, alkyl groups with 2 to 6 carbon atoms, etc. And those in which the amino group is substituted with an alkyl group having 1 to 6 carbon atoms, an alkanoyl group having 1 to 6 carbon atoms, an oxo group, a hydroxy group, or the like.
　ヌクレオチド誘導体としては、ヌクレオチド又は糖部、リン酸ジエステル結合もしくは塩基の少なくとも一つが修飾されたヌクレオチド誘導体に、ペプチド、蛋白質、糖、脂質、リン脂質、フェナジン、フォレート、フェナントリジン、アントラキノン、アクリジン、フルオレセイン、ローダミン、クマリン、色素など、別の化学物質を、直接又はリンカーを介して付加したものもあげられ、具体的には、5’－ポリアミン付加ヌクレオチド誘導体、コレステロール付加ヌクレオチド誘導体、ステロイド付加ヌクレオチド誘導体、胆汁酸付加ヌクレオチド誘導体、ビタミン付加ヌクレオチド誘導体、Cy5付加ヌクレオチド誘導体、Cy3付加ヌクレオチド誘導体、6－FAM付加ヌクレオチド誘導体、及びビオチン付加ヌクレオチド誘導体等があげられる。
　ヌクレオチド誘導体は、核酸内の他のヌクレオチド又はヌクレオチド誘導体とアルキレン構造、ペプチド構造、ヌクレオチド構造、エーテル構造、エステル構造、及びこれらの少なくとも一つを組み合わせた構造等の架橋構造を形成してもよい。 Examples of the nucleotide derivative include a peptide, protein, sugar, lipid, phospholipid, phenazine, folate, phenanthridine, anthraquinone, acridine, a nucleotide derivative in which at least one of nucleotide or sugar moiety, phosphodiester bond or base is modified. Examples include those obtained by adding another chemical substance such as fluorescein, rhodamine, coumarin, and dye, directly or via a linker. Specific examples include 5'-polyamine-added nucleotide derivatives, cholesterol-added nucleotide derivatives, and steroid-added nucleotide derivatives. And bile acid addition nucleotide derivatives, vitamin addition nucleotide derivatives, Cy5 addition nucleotide derivatives, Cy3 addition nucleotide derivatives, 6-FAM addition nucleotide derivatives, and biotin addition nucleotide derivatives.
The nucleotide derivative may form a crosslinked structure such as an alkylene structure, a peptide structure, a nucleotide structure, an ether structure, an ester structure, and a structure obtained by combining at least one of these with other nucleotides or nucleotide derivatives in the nucleic acid.
　本発明の核酸は、核酸の分子中の一部あるいは全部の原子が質量数の異なる原子（同位体）で置換されたものも包含する。 The nucleic acid of the present invention includes those in which some or all atoms in the nucleic acid molecule are substituted with atoms (isotopes) having different mass numbers.
　本明細書において「相補」とは、2つの塩基間で塩基対合をし得る関係を意味し、例えば、アデニンとチミン又はウラシルとの関係、並びにグアニンとシトシンとの関係のように緩やかな水素結合を介して、二重鎖領域全体として2重螺旋構造をとるものをいう。 As used herein, “complementary” means a relationship that allows base pairing between two bases, for example, a moderate hydrogen such as a relationship between adenine and thymine or uracil, and a relationship between guanine and cytosine. It means a double-stranded structure as a whole double-stranded region through a bond.
　本明細書において「相補的」とは、2つのヌクレオチド配列が完全に相補する場合だけでなく、該ヌクレオチド配列間で0～30%、0～20%又は0～10%のミスマッチ塩基を有することができ、例えば、標的Ct-SLCO1B3 RNA配列に対して相補的なアンチセンス鎖は、該RNAの部分塩基配列と完全に相補する塩基配列において、1つ又は複数の塩基の置換を含んでよいことを意味する。具体的には、アンチセンス鎖は、標的遺伝子の標的配列に対して1～8個、好ましくは1～6個、1～4個、1～3個、特に2個又は1個のミスマッチ塩基を有していてもよい。例えば、アンチセンス鎖が21塩基長の場合には、標的遺伝子の標的配列に対して6個、5個、4個、3個、2個又は1個のミスマッチ塩基を有してもよく、そのミスマッチの位置は、それぞれの配列の5’末端又は3’末端であってもよい。
　また、「相補的」とは、一方のヌクレオチド配列が、他方のヌクレオチド配列と完全に相補する塩基配列において、1つ又は複数の塩基が付加及び／又は欠失した配列である場合を包含する。例えば、標的Ct-SLCO1B3 RNAと本発明のアンチセンス鎖核酸とは、アンチセンス鎖における塩基の付加及び／又は欠失により、アンチセンス鎖及び／又は標的Ct-SLCO1B3 RNAに1個又は2個のバルジ塩基を有してもよい。
　本明細書において、2つのヌクレオチド配列が「完全に相補する」とは、該ヌクレオチド配列間でミスマッチ塩基を0個有する、つまり全ての塩基において相補することを指す。 As used herein, “complementary” includes not only a case where two nucleotide sequences are completely complementary, but also 0-30%, 0-20%, or 0-10% mismatch bases between the nucleotide sequences. For example, the antisense strand complementary to the target Ct-SLCO1B3 RNA sequence may contain one or more base substitutions in the base sequence that is completely complementary to the partial base sequence of the RNA. Means. Specifically, the antisense strand contains 1 to 8, preferably 1 to 6, 1 to 4, 1 to 3, particularly 2 or 1 mismatch bases to the target sequence of the target gene. You may have. For example, when the antisense strand is 21 bases long, it may have 6, 5, 4, 3, 2 or 1 mismatch bases with respect to the target sequence of the target gene. The position of the mismatch may be the 5 ′ end or 3 ′ end of each sequence.
In addition, “complementary” includes a case where one nucleotide sequence is a sequence in which one or a plurality of bases are added and / or deleted in a nucleotide sequence that is completely complementary to the other nucleotide sequence. For example, the target Ct-SLCO1B3 RNA and the antisense strand nucleic acid of the present invention include one or two of the antisense strand and / or the target Ct-SLCO1B3 RNA due to the addition and / or deletion of a base in the antisense strand. You may have a bulge base.
In the present specification, “completely complementary” of two nucleotide sequences refers to having 0 mismatch bases between the nucleotide sequences, that is, complementary in all bases.
　本発明の核酸は、標的Ct-SLCO1B3 RNAの一部の塩基配列に対して相補的な塩基配列を含む核酸及び／又は該核酸の塩基配列に対して相補的な塩基配列を含む核酸であれば、いずれのヌクレオチド又はその誘導体から構成されていてもよい。本発明の二本鎖核酸は、標的Ct-SLCO1B3 RNA配列に対して相補的な塩基配列を含む核酸と、該核酸の塩基配列に対して相補的な塩基配列を含む核酸とが、二重鎖を形成することができればいずれの長さでもよいが、二重鎖を形成できる配列の長さは、通常11～35塩基であり、15～30塩基が好ましく、17～25塩基がより好ましく、17～23塩基がさらに好ましく、19～23塩基が特に好ましい。 The nucleic acid of the present invention is a nucleic acid containing a base sequence complementary to a part of the base sequence of the target Ct-SLCO1B3 RNA and / or a nucleic acid containing a base sequence complementary to the base sequence of the nucleic acid. , May be composed of any nucleotide or derivative thereof. In the double-stranded nucleic acid of the present invention, a nucleic acid containing a base sequence complementary to a target Ct-SLCO1B3 RNA sequence and a nucleic acid containing a base sequence complementary to the base sequence of the nucleic acid are double-stranded. However, the length of the sequence capable of forming a duplex is usually 11 to 35 bases, preferably 15 to 30 bases, more preferably 17 to 25 bases, -23 bases are more preferred, and 19-23 bases are particularly preferred.
　本発明のアンチセンス鎖核酸としては、標的Ct-SLCO1B3 RNA配列に対して相補的な塩基配列を含む核酸が用いられるが、該核酸のうち1～3塩基、好ましくは1～2塩基、より好ましくは1塩基が欠失、置換又は付加したものを用いてもよい。 As the antisense strand nucleic acid of the present invention, a nucleic acid containing a base sequence complementary to the target Ct-SLCO1B3 RNA sequence is used. Among these nucleic acids, 1 to 3 bases, preferably 1 to 2 bases, more preferably May be one in which one base is deleted, substituted or added.
　Ct-SLCO1B3の発現を抑制する核酸としては、標的Ct-SLCO1B3 RNA配列に対して相補的な塩基配列を含む核酸であって、かつCt-SLCO1B3の発現を抑制する一本鎖核酸、もしくは標的Ct-SLCO1B3 RNA配列に対して相補的な塩基配列を含む核酸と、該核酸の塩基配列に対して相補的な塩基配列を含む核酸とからなり、かつCt-SLCO1B3の発現を抑制する二本鎖核酸が好適に用いられる。 The nucleic acid that suppresses the expression of Ct-SLCO1B3 is a single-stranded nucleic acid that includes a base sequence complementary to the target Ct-SLCO1B3 RNA sequence and suppresses the expression of Ct-SLCO1B3, or the target Ct -Double-stranded nucleic acid consisting of a nucleic acid containing a base sequence complementary to the SLCO1B3 RNA sequence and a nucleic acid containing a base sequence complementary to the base sequence of the nucleic acid, and suppressing the expression of Ct-SLCO1B3 Are preferably used.
　本発明において二本鎖核酸とは、二本のヌクレオチド鎖が対合し二重鎖領域を有する核酸をいう。二重鎖領域とは、二本鎖核酸を構成するヌクレオチド又はその誘導体が塩基対を構成して二重鎖を形成している部分をいう。二重鎖領域は、通常11～27塩基対であり、15～25塩基対が好ましく、15～23塩基対がより好ましく、17～21塩基対がさらに好ましく、17～19塩基対が特に好ましい。 In the present invention, a double-stranded nucleic acid refers to a nucleic acid having a double-stranded region in which two nucleotide chains are paired. A double-stranded region refers to a portion where nucleotides constituting a double-stranded nucleic acid or a derivative thereof constitute a base pair to form a double strand. The double-stranded region is usually 11 to 27 base pairs, preferably 15 to 25 base pairs, more preferably 15 to 23 base pairs, still more preferably 17 to 21 base pairs, and particularly preferably 17 to 19 base pairs.
　二本鎖核酸を構成する一本鎖の核酸は、通常11～30塩基からなるが、15～29塩基からなることが好ましく、15～27塩基からなることがより好ましく、15～25塩基からなることがさらに好ましく、17～23塩基からなることが特に好ましく、19～21塩基からなることが最も好ましい。 The single-stranded nucleic acid constituting the double-stranded nucleic acid usually consists of 11 to 30 bases, preferably 15 to 29 bases, more preferably 15 to 27 bases, and 15 to 25 bases. More preferably, it consists of 17 to 23 bases, particularly preferably 19 to 21 bases.
　本発明の二本鎖核酸において、二重鎖領域に続く3’側又は5’側に二重鎖を形成しない追加のヌクレオチド又はヌクレオチド誘導体を有する場合には、これを突出部（オーバーハング）と呼ぶ。突出部を有する場合には、突出部を構成するヌクレオチドはリボヌクレオチド、デオキシリボヌクレオチド又はこれらの誘導体であってもよい。 When the double-stranded nucleic acid of the present invention has an additional nucleotide or nucleotide derivative that does not form a duplex on the 3 ′ side or 5 ′ side following the double-stranded region, this is referred to as an overhang. Call. In the case of having an overhang, the nucleotide constituting the overhang may be ribonucleotide, deoxyribonucleotide or a derivative thereof.
　突出部を有する二本鎖核酸としては、少なくとも一方の鎖の3’末端又は5’末端に1～6塩基、通常は1～3塩基からなる突出部を有するものが用いられるが、2塩基からなる突出部を有するものが好ましく用いられ、例えばdTdT又はUUからなる突出部を有するものがあげられる。突出部は、アンチセンス鎖のみ、センス鎖のみ、及びアンチセンス鎖とセンス鎖の両方に有することができるが、本発明において、アンチセンス鎖とセンス鎖の両方に突出部を有する二本鎖核酸が好ましく用いられる。なお、アンチセンス鎖は、二重鎖領域とそれに続く突出部とを含む、少なくとも17個のヌクレオチドかつ多くとも30個のヌクレオチドからなるオリゴヌクレオチド鎖において、標的Ct-SLCO1B3 RNAと十分に相補的である。さらに、本発明の二本鎖核酸としては、例えばDicer等のリボヌクレアーゼの作用により上記の二本鎖核酸を生成する核酸分子（WO2005/089287）や、3’末端や5’末端の突出部を有さず平滑末端を形成する二本鎖核酸、センス鎖のみが突出した二本鎖核酸（US2012/0040459）などを用いることもできる。 As the double-stranded nucleic acid having a protruding portion, one having a protruding portion consisting of 1 to 6 bases, usually 1 to 3 bases at the 3 ′ end or 5 ′ end of at least one strand is used. Those having protrusions are preferably used, and examples thereof include those having protrusions made of dTdT or UU. In the present invention, a double-stranded nucleic acid having a protruding portion on both the antisense strand and the sense strand can be provided on the antisense strand only, the sense strand only, and both the antisense strand and the sense strand. Is preferably used. It should be noted that the antisense strand is sufficiently complementary to the target Ct-SLCO1B3 RNA in an oligonucleotide strand consisting of at least 17 nucleotides and at most 30 nucleotides, including the double-stranded region and the subsequent overhang. is there. Furthermore, the double-stranded nucleic acid of the present invention has, for example, a nucleic acid molecule (WO2005 / 089287) that generates the above-mentioned double-stranded nucleic acid by the action of a ribonuclease such as Dicer, or a 3′-end or 5′-end overhang. Alternatively, a double-stranded nucleic acid that forms a blunt end, a double-stranded nucleic acid in which only the sense strand protrudes (US2012 / 0040459), and the like can also be used.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　好ましい態様において、本発明の二本鎖核酸のアンチセンス鎖は、配列番号14～21からなる群より選択される配列を含み、より好ましくは配列番号14～17からなる群より選択される配列を含む。別の好ましい態様として、本発明の二本鎖核酸のセンス鎖は、配列番号22～29からなる群より選択される配列を含み、より好ましくは配列番号22～25からなる群より選択される配列を含む。
　さらに、好ましい態様において、本発明の二本鎖核酸は、表１記載の配列番号14／配列番号22、配列番号15／配列番号23、配列番号16／配列番号24、配列番号17／配列番号25、配列番号18／配列番号26、配列番号19／配列番号27、配列番号20／配列番号28、及び配列番号21／配列番号29から成る群より選択される1対のアンチセンス鎖／センス鎖の配列を含む。
　最も好ましい態様において、本発明の二本鎖核酸は、表１記載の配列番号14／配列番号22、配列番号15／配列番号23、配列番号16／配列番号24、及び配列番号17／配列番号25から成る群より選択される1対のアンチセンス鎖／センス鎖の配列を含む。 In a preferred embodiment, the antisense strand of the double-stranded nucleic acid of the present invention comprises a sequence selected from the group consisting of SEQ ID NOs: 14-21, more preferably a sequence selected from the group consisting of SEQ ID NOs: 14-17. Including. In another preferred embodiment, the sense strand of the double-stranded nucleic acid of the present invention comprises a sequence selected from the group consisting of SEQ ID NOs: 22-29, more preferably a sequence selected from the group consisting of SEQ ID NOs: 22-25 including.
Further, in a preferred embodiment, the double-stranded nucleic acid of the present invention comprises SEQ ID NO: 14 / SEQ ID NO: 22, SEQ ID NO: 15 / SEQ ID NO: 23, SEQ ID NO: 16 / SEQ ID NO: 24, SEQ ID NO: 17 / SEQ ID NO: 25 described in Table 1. A pair of antisense strands / sense strands selected from the group consisting of: SEQ ID NO: 18 / SEQ ID NO: 26, SEQ ID NO: 19 / SEQ ID NO: 27, SEQ ID NO: 20 / SEQ ID NO: 28, and SEQ ID NO: 21 / SEQ ID NO: 29 Contains an array.
In a most preferred embodiment, the double-stranded nucleic acid of the present invention comprises SEQ ID NO: 14 / SEQ ID NO: 22, SEQ ID NO: 15 / SEQ ID NO: 23, SEQ ID NO: 16 / SEQ ID NO: 24, and SEQ ID NO: 17 / SEQ ID NO: 25 described in Table 1. A pair of antisense / sense strand sequences selected from the group consisting of:
　本発明の二本鎖核酸としては、標的遺伝子の塩基配列又はその相補鎖の塩基配列と同一の配列からなる核酸を用いてもよいが、該核酸の少なくとも一方の鎖の5’末端又は3’末端が1～4塩基削除された核酸と、該核酸の塩基配列に対して相補的な塩基配列を含む核酸とからなる二本鎖核酸を用いてもよい。 As the double-stranded nucleic acid of the present invention, a nucleic acid having the same sequence as the base sequence of the target gene or its complementary strand may be used, but the 5 ′ end or 3 ′ of at least one strand of the nucleic acid may be used. A double-stranded nucleic acid comprising a nucleic acid from which one to four bases have been deleted and a nucleic acid containing a base sequence complementary to the base sequence of the nucleic acid may be used.
　本発明の二本鎖核酸は、RNA同士が二重鎖を形成した二本鎖RNA（dsRNA）、DNA同士が二重鎖を形成した二本鎖DNA（dsDNA）、又はRNAとDNAが二重鎖を形成したハイブリッド核酸であってもよい。あるいは、二本鎖のうちの一方もしくは両方の鎖がDNAとRNAとのキメラ核酸であってもよい。好ましくは二本鎖RNA（dsRNA）である。 The double-stranded nucleic acid of the present invention is a double-stranded RNA (dsRNA) in which RNAs form a double strand, a double-stranded DNA (dsDNA) in which DNAs form a double strand, or a double-stranded RNA and DNA. It may be a hybrid nucleic acid that forms a strand. Alternatively, one or both of the double strands may be a chimeric nucleic acid of DNA and RNA. Double-stranded RNA (dsRNA) is preferred.
　本発明のアンチセンス鎖の5’末端から2番目のヌクレオチドは、標的Ct-SLCO1B3 RNA配列の3’末端から2番目のデオキシリボヌクレオチドと相補であることが好ましく、アンチセンス鎖の5’末端から2～7番目のヌクレオチドが、標的Ct-SLCO1B3 RNA配列の3’末端から2～7番目のデオキシリボヌクレオチドと完全に相補であることがより好ましく、アンチセンス鎖の5’末端から2～11番目のヌクレオチドが、標的Ct-SLCO1B3 RNA配列の3’末端から2～11番目のデオキシリボヌクレオチドと完全に相補であることがさらに好ましい。また、本発明の核酸におけるアンチセンス鎖の5’末端から11番目のヌクレオチドが、標的Ct-SLCO1B3 RNA配列の3’末端から11番目のデオキシリボヌクレオチドと相補であることが好ましく、アンチセンス鎖の5’末端から9～13番目のヌクレオチドが、標的Ct-SLCO1B3 RNA配列の3’末端から9～13番目のデオキシリボヌクレオチドと完全に相補であることがより好ましく、アンチセンス鎖の5’末端から7～15番目のヌクレオチドが、標的Ct-SLCO1B3 RNA配列の3’末端から7～15番目のデオキシリボヌクレオチドと完全に相補であることがさらに好ましい。 The second nucleotide from the 5 ′ end of the antisense strand of the present invention is preferably complementary to the second deoxyribonucleotide from the 3 ′ end of the target Ct-SLCO1B3 RNA sequence, and 2 nucleotides from the 5 ′ end of the antisense strand. More preferably, the seventh nucleotide is completely complementary to the second to seventh deoxyribonucleotides from the 3 ′ end of the target Ct-SLCO1B31RNA sequence, and the second to eleventh nucleotides from the 5 ′ end of the antisense strand Is more preferably completely complementary to deoxyribonucleotides 2 to 11 from the 3 ′ end of the target Ct-SLCO1B3 RNA sequence. Further, the 11th nucleotide from the 5 ′ end of the antisense strand in the nucleic acid of the present invention is preferably complementary to the 11th deoxyribonucleotide from the 3 ′ end of the target Ct-SLCO1B3 RNA sequence, More preferably, the 9th to 13th nucleotides from the end are completely complementary to the 9th to 13th deoxyribonucleotides from the 3 'end of the target Ct-SLCO1B3BRNA sequence, and 7 to 7 from the 5' end of the antisense strand. More preferably, the 15th nucleotide is completely complementary to the 7th to 15th deoxyribonucleotides from the 3 ′ end of the target Ct-SLCO1B3 RNA sequence.
　本発明の核酸を製造する方法としては、特に限定されず、公知の化学合成を用いる方法、あるいは、酵素的転写法等があげられる。公知の化学合成を用いる方法として、ホスホロアミダイト法、ホスホロチオエート法、ホスホトリエステル法、CEM法［Nucleic Acid Research,35,3287(2007)］等をあげることができ、例えば、ABI3900ハイスループット核酸合成機（アプライドバイオシステムズ社製）により合成することができる。合成が終了した後は、固相からの脱離、保護基の脱保護及び目的物の精製等を行う。精製により、純度90%以上、好ましくは95%以上の核酸を得るのが望ましい。二本鎖核酸の場合には、合成及び精製したセンス鎖、アンチセンス鎖を適当な比率、例えば、アンチセンス鎖１当量に対して、センス鎖0.1～10当量、好ましくは0.5～2当量、より好ましくは0.9～1.1当量、さらに好ましくは等モル量で混合した後、アニーリングを行って用いてもよいし、又は、混合したものをアニーリングする工程を省いて直接用いてもよい。アニーリングは、二本鎖核酸を形成できる条件であればいかなる条件で行ってもよいが、通常、センス鎖、アンチセンス鎖をほぼ等モル量で混合した後、94℃程度で５分程度加熱したのち、室温まで徐冷することにより行われる。本発明の核酸を製造する酵素的転写法としては、目的の塩基配列を有したプラスミド又はDNAを鋳型としてファージRNAポリメラーゼ、例えば、T7、T3、又はSP6 RNAポリメラーゼを用いた転写による方法があげられる。 The method for producing the nucleic acid of the present invention is not particularly limited, and examples thereof include a method using known chemical synthesis or an enzymatic transcription method. Examples of methods using known chemical synthesis include phosphoramidite method, phosphorothioate method, phosphotriester method, CEM method [Nucleic® Acid® Research, 35, 3287 (2007)]. For example, ABI3900 high-throughput nucleic acid synthesis Can be synthesized by a machine (Applied Biosystems). After the synthesis is completed, elimination from the solid phase, deprotection of the protecting group, purification of the target product, and the like are performed. It is desirable to obtain a nucleic acid having a purity of 90% or more, preferably 95% or more by purification. In the case of a double-stranded nucleic acid, the synthesized and purified sense strand and antisense strand are in an appropriate ratio, for example, 0.1 to 10 equivalents, preferably 0.5 to 2 equivalents of sense strand to 1 equivalent of antisense strand. Preferably, 0.9 to 1.1 equivalents, more preferably equimolar amounts are mixed and then annealed, or the mixed product may be used directly without the step of annealing. Annealing may be performed under any conditions that can form a double-stranded nucleic acid. Usually, the sense strand and the antisense strand are mixed in approximately equimolar amounts, and then heated at about 94 ° C. for about 5 minutes. Then, it is performed by slowly cooling to room temperature. Examples of the enzymatic transcription method for producing the nucleic acid of the present invention include a method by transcription using a phage RNA polymerase, for example, T7, T3, or SP6 RNA polymerase, using a plasmid or DNA having the target base sequence as a template. .
　本発明の核酸は、トランスフェクション用の担体、好ましくはカチオン性リポソーム等のカチオン性担体を用いて細胞内に導入することができる。また、リン酸カルシウム法、エレクトロポレーション法又はマイクロインジェクション法などにより、直接細胞内に導入することもできる。 The nucleic acid of the present invention can be introduced into cells using a transfection carrier, preferably a cationic carrier such as a cationic liposome. It can also be directly introduced into cells by the calcium phosphate method, electroporation method or microinjection method.
　本発明の核酸は、5’末端、3’末端及び／又は配列内部が1つ以上のリガンドや蛍光団により修飾されていてもよく、リガンドや蛍光団により修飾された核酸をコンジュゲート核酸とも呼ぶ。固相上での伸張反応時に、固相上で反応可能な修飾剤を反応させることで、5’末端、3’末端及び／又は配列内部に修飾を施すことができる。また、アミノ基、メルカプト基、アジド基又は3重結合などの官能基を導入した核酸をあらかじめ合成及び精製しておき、それらに修飾化剤を作用させることでコンジュゲート核酸を得ることもできる。リガンドとしては、生体分子と親和性のある分子であれば良いが、例えば、コレステロール、脂肪酸、トコフェロール、レチノイドなどの脂質類、N－アセチルガラクトサミン（GalNAc）、ガラクトース（Gal）、マンノース（Man）などの糖類、フル抗体、Fab、VHHなどの抗体、低密度リポタンパク質（LDL）、ヒト血清アルブミンなどのタンパク質、RGD、NGR、R9、CPPなどのペプチド類、葉酸などの低分子、合成ポリアミノ酸などの合成ポリマー、あるいは核酸アプタマーなどがあげられ、これらを組み合わせて用いることもできる。蛍光団としてはCy3シリーズ、Alexaシリーズ、ブラックホールクエンチャーなどがあげられる。 In the nucleic acid of the present invention, the 5 ′ end, 3 ′ end and / or the inside of the sequence may be modified with one or more ligands or fluorophores, and a nucleic acid modified with a ligand or fluorophore is also called a conjugate nucleic acid. . At the time of extension reaction on the solid phase, the 5 'end, 3' end and / or the inside of the sequence can be modified by reacting a modifying agent capable of reacting on the solid phase. Alternatively, a conjugated nucleic acid can be obtained by previously synthesizing and purifying a nucleic acid into which a functional group such as an amino group, a mercapto group, an azide group, or a triple bond has been introduced, and allowing a modifying agent to act on them. The ligand may be a molecule having affinity for a biomolecule. For example, lipids such as cholesterol, fatty acid, tocopherol, and retinoid, N-acetylgalactosamine (GalNAc), galactose (Gal), mannose (Man), etc. Saccharides, full antibodies, antibodies such as Fab and VHH, low density lipoprotein (LDL), proteins such as human serum albumin, peptides such as RGD, NGR, R9, and CPP, small molecules such as folic acid, synthetic polyamino acids, etc. These synthetic polymers or nucleic acid aptamers can be used, and these can be used in combination. Examples of fluorophores include the Cy3 series, Alexa series, and black hole quenchers.
　本発明の核酸の代わりに、細胞内に導入してこれらが発現されるようなベクターを用いてもよい。具体的には、本発明の核酸をコードする配列を発現ベクター内のプロモーター下流に挿入して発現ベクターを構築し、細胞に導入することにより該核酸等を発現させることができる。発現ベクターとしては、pCDNA6.2-GW/miR（Invitrogen社製）、pSilencer 4.1-CMV（Ambion社製）、pSINsi-hH1 DNA（タカラバイオ社製）、pSINsi-hU6 DNA（タカラバイオ社製）、pENTR/U6（Invitrogen社製）等をあげることができる。 In place of the nucleic acid of the present invention, a vector that can be introduced into cells and expressed can be used. Specifically, the nucleic acid or the like can be expressed by inserting the sequence encoding the nucleic acid of the present invention downstream of the promoter in the expression vector, constructing the expression vector, and introducing it into a cell. Expression vectors include pCDNA6.2-GW / miR (Invitrogen), pSilencer® 4.1-CMV (Ambion), pSINsi-hH1 DNA (Takara Bio), pSINsi-hU6 DNA (Takara Bio), pENTR / U6 (Invitrogen) etc. can be mentioned.
　また、本発明の核酸をコードする配列をウイルスベクター内のプロモーター下流に挿入し、該ベクターをパッケージング細胞に導入して生産した組換えウイルスベクターを用いることもできる。ウイルスベクターとしては、レトロウイルスベクター、レンチウイルスベクター、アデノウイルスベクター、アデノ随伴ウイルスベクターなどがあげられる。 It is also possible to use a recombinant viral vector produced by inserting a sequence encoding the nucleic acid of the present invention downstream of a promoter in a viral vector and introducing the vector into a packaging cell. Examples of virus vectors include retrovirus vectors, lentivirus vectors, adenovirus vectors, adeno-associated virus vectors, and the like.
　これらの二本鎖核酸を細胞に導入することにより、Ct-SLCO1B3の発現を抑制することができる。例えば本発明の二本鎖核酸は、数pM～数nMの濃度で、細胞に導入した後、24時間以上、例えば48時間培養した段階でCt-SLCO1B3のmRNAの発現を抑制することができる。 By introducing these double-stranded nucleic acids into cells, the expression of Ct-SLCO1B3 can be suppressed. For example, the double-stranded nucleic acid of the present invention can suppress the expression of Ct-SLCO1B3 mRNA after being introduced into cells at a concentration of several pM to several nM and then cultured for 24 hours or more, for example 48 hours.
　また、本発明の二本鎖核酸のCt-SLCO1B3 mRNAの発現抑制活性の評価は、該核酸等をヒト細胞株などにカチオン性リポソームなどを用いてトランスフェクションし、一定時間培養した後、当該ヒト細胞株におけるCt-SLCO1B3のmRNAの発現量を定量することにより行うことができる。 In addition, the evaluation of the Ct-SLCO1B3 mRNA expression inhibitory activity of the double-stranded nucleic acid of the present invention was carried out by transfecting the nucleic acid or the like into a human cell line using a cationic liposome, etc. This can be done by quantifying the expression level of Ct-SLCO1B3 mRNA in the cell line.
　Ct-SLCO1B3の発現抑制活性を有する核酸としては、上記二本鎖核酸以外にも、Ct-SLCO1B3 mRNAの一部の塩基配列に対して相補的な塩基配列を含む核酸であって、かつCt-SLCO1B3の発現を抑制する一本鎖核酸があげられる。該核酸を構成する一本鎖の核酸は、通常8～30塩基からなるが、12～30塩基からなることが好ましく、12～20塩基からなることがより好ましい。
　Ct-SLCO1B3 mRNAの一部の塩基配列は、好ましくは配列番号1と完全に相補するRNA配列に含まれる連続する塩基配列の一部であり、より好ましくは配列番号2に含まれる配列である。 In addition to the above double-stranded nucleic acid, the nucleic acid having a Ct-SLCO1B3 expression-suppressing activity is a nucleic acid containing a base sequence complementary to a part of the base sequence of Ct-SLCO1B3 mRNA, and Ct-SLCO1B3 A single-stranded nucleic acid that suppresses the expression of SLCO1B3. The single-stranded nucleic acid constituting the nucleic acid usually consists of 8 to 30 bases, preferably 12 to 30 bases, more preferably 12 to 20 bases.
The partial base sequence of Ct-SLCO1B3 mRNA is preferably a part of a continuous base sequence included in an RNA sequence completely complementary to SEQ ID NO: 1, and more preferably a sequence included in SEQ ID NO: 2.
　これらの一本鎖核酸も細胞に導入することにより、Ct-SLCO1B3の発現を抑制することができる。例えば本発明の一本鎖核酸は、数pM～数nMの濃度で、細胞に導入した後、24時間以上、例えば48時間培養した段階でCt-SLCO1B3のmRNAの発現を抑制することができる。 By introducing these single-stranded nucleic acids into cells, the expression of Ct-SLCO1B3 can be suppressed. For example, the single-stranded nucleic acid of the present invention can suppress the expression of Ct-SLCO1B3 mRNA after being introduced into cells at a concentration of several pM to several nM and then cultured for 24 hours or more, for example 48 hours.
　また、本発明の一本鎖核酸のCt-SLCO1B3のmRNAの発現抑制活性の評価は、該核酸等をヒト細胞株などにカチオン性リポソームなどを用いてトランスフェクションし、一定時間培養した後、当該ヒト細胞株におけるCt-SLCO1B3のmRNAの発現量を定量することにより行うことができる。 In addition, the evaluation of the Ct-SLCO1B3 mRNA expression inhibitory activity of the single-stranded nucleic acid of the present invention was carried out by transfecting the nucleic acid or the like into a human cell line using a cationic liposome or the like, culturing for a certain time, This can be done by quantifying the expression level of Ct-SLCO1B3 mRNA in a human cell line.
３．Ct-SLCO1B3の発現を抑制する核酸を含む医薬組成物
　本発明の医薬組成物は、Ct-SLCO1B3の発現を抑制する核酸を含有することを特徴とする。 3. Pharmaceutical Composition Containing Nucleic Acid that Suppresses Ct-SLCO1B3 Expression The pharmaceutical composition of the present invention comprises a nucleic acid that suppresses Ct-SLCO1B3 expression.
　本発明の医薬組成物に有効成分として含まれる、Ct-SLCO1B3の発現を抑制する核酸は、Ct-SLCO1B3の発現によって特徴づけられる種々の癌細胞に対して増殖抑制作用、浸潤転移抑制作用などを有し、癌の悪性化を抑制することが可能となる。従って、Ct-SLCO1B3の発現によって特徴づけられる癌の、治療、予防、進行防止、悪性化抑制に用いることができる。これらの作用から、本発明の医薬組成物は、哺乳動物（例、ヒト、チンパンジー、ゴリラ、サル、ネコ、ブタ、ウマ、ウシ、マウス、ラット、モルモット、イヌ、ウサギ等、好ましくはヒト、チンパンジー及びゴリラ、特に好ましくはヒト）に対し、Ct-SLCO1B3の発現によって特徴づけられる癌の、治療剤又は予防剤として有用である。 A nucleic acid that suppresses the expression of Ct-SLCO1B3, which is contained as an active ingredient in the pharmaceutical composition of the present invention, has a growth inhibitory action, an invasion metastasis inhibitory action, etc. on various cancer cells characterized by the expression of Ct-SLCO1B3. It becomes possible to suppress malignant transformation of cancer. Therefore, it can be used for treatment, prevention, progression prevention, and malignant inhibition of cancer characterized by the expression of Ct-SLCO1B3. From these actions, the pharmaceutical composition of the present invention can be used in mammals (eg, humans, chimpanzees, gorillas, monkeys, cats, pigs, horses, cows, mice, rats, guinea pigs, dogs, rabbits, etc., preferably humans, chimpanzees). And gorillas, particularly preferably humans, are useful as therapeutic or prophylactic agents for cancer characterized by the expression of Ct-SLCO1B3.
　本発明の医薬組成物に有効成分として含まれる、Ct-SLCO1B3の発現を抑制する核酸は、Ct-SLCO1B3の高発現に起因する、snail、slugの発現の上昇、E-cadherinやoccludinの発現抑制及びMMP9の発現誘導を阻害する効果を有する。従って、Ct-SLCO1B3の発現を阻害する物質は、Ct-SLCO1B3の発現に特徴づけられる癌の悪性化を抑制する作用を有する。snail及びslugの発現上昇は、上皮間葉転換を促進することが知られている。 The nucleic acid that suppresses the expression of Ct-SLCO1B3, which is contained in the pharmaceutical composition of the present invention, is an increase in the expression of snail and slug due to the high expression of Ct-SLCO1B3, and the expression suppression of E-cadherin and occludin And has the effect of inhibiting the induction of MMP9 expression. Therefore, a substance that inhibits the expression of Ct-SLCO1B3 has an action of suppressing malignant cancer characterized by the expression of Ct-SLCO1B3. It is known that increased expression of snail and slug promotes epithelial-mesenchymal transition.
　細胞がCt-SLCO1B3を発現しているか否かは、自体公知の方法によって検証することができる。例えば、Ct-SLCO1B3の発現量は、Ct-SLCO1B3をコードするDNAとストリンジェントな条件下でハイブリダイズし得る核酸又はそれと相補的な塩基配列とストリンジェントな条件下でハイブリダイズし得る核酸（DNA）を用いて、Ct-SLCO1B3遺伝子のmRNAを検出することにより、RNAレベルで測定することができる。あるいは、Ct-SLCO1B3の発現量は、Ct-SLCO1B3に対する抗体を用いて、Ct-SLCO1B3タンパク質を検出することにより、タンパク質レベルで測定することもできる。
　実施例に記載するとおり、Ct-SLCO1B3の発現が疑われる細胞からtotal RNAを抽出し、逆転写反応によりcDNAを合成して、配列番号30及び配列番号31のプライマーを用いてPCR反応を行ってもよい。 Whether or not a cell expresses Ct-SLCO1B3 can be verified by a method known per se. For example, the expression level of Ct-SLCO1B3 is determined based on the nucleic acid (DNA) that can hybridize under stringent conditions with a nucleic acid that can hybridize with DNA encoding Ct-SLCO1B3 under a stringent condition or with a complementary base sequence. ) To detect the mRNA of the Ct-SLCO1B3 gene, it can be measured at the RNA level. Alternatively, the expression level of Ct-SLCO1B3 can also be measured at the protein level by detecting Ct-SLCO1B3 protein using an antibody against Ct-SLCO1B3.
As described in the Examples, total RNA was extracted from cells suspected of expressing Ct-SLCO1B3, cDNA was synthesized by reverse transcription reaction, and PCR was performed using primers of SEQ ID NO: 30 and SEQ ID NO: 31. Also good.
　ストリンジェントな条件とは、例えば、Current Protocols in Molecular Biology,John Wiley&Sons,6.3.1-6.3.6,1999に記載される条件、例えば、6×SSC（sodium chloride/sodium citrate）／45℃でのハイブリダイゼーション、次いで0.2×SSC／0.1% SDS／50～65℃での一回以上の洗浄等が挙げられるが、当業者であれば、これと同等のストリンジェンシーを与えるハイブリダイゼーションの条件を適宜選択することができる。 The stringent conditions include, for example, the conditions described in Current Protocols in Molecular Biology, John Wiley & Sons, 6.3.1-6.3.6, 1999, for example, 6 × SSC (sodium chloride / sodium citrate) / 45 ° C. Hybridization, followed by one or more washings at 0.2 × SSC / 0.1% SDS / 50 to 65 ° C., etc., but those skilled in the art will appropriately select the hybridization conditions that give the same stringency. can do.
　本発明の医薬組成物の対象となる癌としては、癌細胞がCt-SLCO1B3を発現する限り特に制限されないが、例えば、固形癌、移行上皮癌、大腸癌、結腸直腸癌、結腸癌、肺癌（小細胞癌）、肺癌（非小細胞肺癌）、腎癌（腎細胞癌）、腎盂尿管癌、胆道癌、肝癌（肝細胞癌）、脳腫瘍、神経膠腫（グリオーマ）、膠芽腫、多型性神経膠芽腫、膵臓癌、頭頸部癌（扁平上皮癌）、多発性骨髄腫、骨軟部腫瘍、前立腺癌、陰茎癌、精巣癌、卵巣癌、消化管間質腫瘍（GIST）、胃癌、女性生殖器癌、子宮頸癌、乳癌、黒色腫（メラノーマ）、リンパ腫（非ホジキン）、リンパ腫（ホジキン）、リンパ腫（びまん性大細胞型）、白血病（急性骨髄性）、白血病（慢性リンパ性）、食道癌、口腔癌、舌癌、咽頭癌、喉頭癌、耳下腺癌、顎下腺癌、唾液腺癌、甲状腺癌、副腎皮質癌、線維性組織球腫、髄膜腫、膀胱癌、ユーイング肉腫、カポジ肉腫、中皮腫又は***性皮膚線維肉腫等が挙げられ、好ましい対象疾患は、肺癌、肝癌、食道癌、大腸癌又は膵癌であり、より好ましくは非小細胞肺癌、肝癌、膵癌又は食道癌であり、さらに好ましくは非小細胞肺癌である。 The target cancer of the pharmaceutical composition of the present invention is not particularly limited as long as cancer cells express Ct-SLCO1B3. For example, solid cancer, transitional cell cancer, colon cancer, colorectal cancer, colon cancer, lung cancer ( Small cell carcinoma), lung cancer (non-small cell lung cancer), renal cancer (renal cell carcinoma), renal pelvic and ureteral cancer, biliary tract cancer, liver cancer (hepatocellular carcinoma), brain tumor, glioma (glioma), glioblastoma, multi Type glioblastoma, pancreatic cancer, head and neck cancer (squamous cell carcinoma), multiple myeloma, bone soft tissue tumor, prostate cancer, penile cancer, testicular cancer, ovarian cancer, gastrointestinal stromal tumor (GIST), gastric cancer , Female genital cancer, cervical cancer, breast cancer, melanoma, lymphoma (non-Hodgkin), lymphoma (Hodgkin), lymphoma (diffuse large cell type), leukemia (acute myeloid), leukemia (chronic lymphoid) , Esophageal cancer, oral cancer, tongue cancer, laryngeal cancer, laryngeal cancer, parotid gland cancer, submandibular gland cancer, salivary gland cancer, Thyroid cancer, adrenocortical cancer, fibrous histiocytoma, meningioma, bladder cancer, Ewing sarcoma, Kaposi sarcoma, mesothelioma, or elevated dermatofibrosarcoma, etc., preferable target diseases are lung cancer, liver cancer, Esophageal cancer, colon cancer or pancreatic cancer, more preferably non-small cell lung cancer, liver cancer, pancreatic cancer or esophageal cancer, and further preferably non-small cell lung cancer.
　本発明の医薬組成物及び本発明の核酸は、上記に例示した癌の予防又は治療用に好適に用いることができる。本明細書中、「癌の予防」とは、癌に罹患するリスクを有する動物（患者）に投与することにより癌の発生を抑制する又は遅延させること、癌の治療後の動物（患者）に投与することにより癌の再発を防止することも包含する用語として使用される。本明細書中「癌の治療」とは、癌に罹患している動物（患者）に投与することにより、癌の進行を抑制する若しくは遅延させること、腫瘍の成長を抑制する若しくは遅延させる、腫瘍を縮小若しくは消失させること等を包含する用語として使用される。 The pharmaceutical composition of the present invention and the nucleic acid of the present invention can be suitably used for the prevention or treatment of cancer exemplified above. In the present specification, “prevention of cancer” refers to suppressing or delaying the occurrence of cancer by administering to an animal (patient) who is at risk of suffering from cancer, or to an animal (patient) after cancer treatment. It is also used as a term encompassing prevention of cancer recurrence by administration. In the present specification, "treatment of cancer" refers to a tumor that suppresses or delays the progression of cancer or suppresses or delays the growth of a tumor by administration to an animal (patient) suffering from cancer. Is used as a term encompassing reducing or eliminating the like.
　本発明の医薬組成物に有効成分として含まれるCt-SLCO1B3の発現を抑制する核酸は、転写されたCt-SLCO1B3のRNAに作用してその発現を特異的に阻害する物質であれば特に限定されるものではない。Ct-SLCO1B3は、二本鎖RNAであってもよく、一本鎖RNAであってもよい。また、本発明の医薬組成物に有効成分として含まれるCt-SLCO1B3の発現を抑制する核酸は、リボザイム核酸であってもよい。
　本発明の医薬組成物に有効成分として含まれる核酸は、所望の効果を有する限り、Ct-SLCO1B3のexon1*以外の領域を含む、Ct-SLCO1B3をコードするRNAのいずれの領域も標的とし得る。Ct-SLCO1B3のexon1*以外の領域を標的とする核酸を、本発明の医薬組成物に使用する場合、本発明の医薬組成物は、肝臓型SLCO1B3等の、Ct-SLCO1B3以外のSLCO1B3バリアントが発現していない組織に好適に投与される。
　好ましくは、本発明の医薬組成物は、本発明の核酸を有効成分として含む。 The nucleic acid that suppresses the expression of Ct-SLCO1B3 contained as an active ingredient in the pharmaceutical composition of the present invention is particularly limited as long as it acts on the transcribed Ct-SLCO1B3 RNA and specifically inhibits the expression. It is not something. Ct-SLCO1B3 may be a double-stranded RNA or a single-stranded RNA. The nucleic acid that suppresses the expression of Ct-SLCO1B3 contained as an active ingredient in the pharmaceutical composition of the present invention may be a ribozyme nucleic acid.
The nucleic acid contained as an active ingredient in the pharmaceutical composition of the present invention can target any region of RNA encoding Ct-SLCO1B3 including a region other than exon1 * of Ct-SLCO1B3 as long as it has a desired effect. When a nucleic acid that targets a region other than exon1 * of Ct-SLCO1B3 is used in the pharmaceutical composition of the present invention, the pharmaceutical composition of the present invention expresses an SLCO1B3 variant other than Ct-SLCO1B3, such as liver-type SLCO1B3. It is preferably administered to non-tissue.
Preferably, the pharmaceutical composition of the present invention contains the nucleic acid of the present invention as an active ingredient.
　本発明の医薬組成物に有効成分として含まれる核酸の製造方法は、前述の本発明の核酸の製造方法に準じて製造される。 The method for producing a nucleic acid contained as an active ingredient in the pharmaceutical composition of the present invention is produced according to the above-described method for producing a nucleic acid of the present invention.
　本発明の医薬組成物に有効成分として含有される、Ct-SLCO1B3の発現を抑制する核酸は、所望の効果を有する限り、複数種類のCt-SLCO1B3の発現を抑制する核酸を含んでもよい。 The nucleic acid that suppresses the expression of Ct-SLCO1B3 contained as an active ingredient in the pharmaceutical composition of the present invention may contain a nucleic acid that suppresses the expression of multiple types of Ct-SLCO1B3 as long as it has a desired effect.
　本発明の医薬組成物に有効成分として含有される、Ct-SLCO1B3の発現を抑制する核酸の含有量は、所望の効果を有する限り特に制限はされないが、製剤全体に対して通常、約0.01～約99.9重量%、好ましくは約0.1～約50重量%である。 The content of the nucleic acid that suppresses the expression of Ct-SLCO1B3, which is contained as an active ingredient in the pharmaceutical composition of the present invention, is not particularly limited as long as it has a desired effect. About 99.9% by weight, preferably about 0.1 to about 50% by weight.
　本発明の医薬組成物は、所望の効果を得るために、単独で投与することもでき、又他の抗癌剤及び／又は放射線療法と適宜組み合わせて用いることができる。
　他の抗癌剤としては、例えば、代謝拮抗剤（例、メソトレキセート、５－フルオロウラシル等）、アルキル化剤（例、サイクロフォスファミド、イフォスファミド等）、白金系抗癌剤（例、シスプラチン、カルボプラチン等）、トポイソメラーゼ阻害剤（例、エトポシド等）、抗癌性抗生物質（例、マイトマイシン、アドリアマイシン等）、植物由来抗癌剤（例、ビンクリスチン、ビンデシン、タキソール等）、チロシンキナーゼ阻害剤（例、ゲフィニチブ、イマニチブ等）、ヒト化抗体（例、ハーセプチン等）等が挙げられるが、これらに限定されない。 In order to obtain a desired effect, the pharmaceutical composition of the present invention can be administered alone, or can be used in appropriate combination with other anticancer agents and / or radiation therapy.
Examples of other anticancer agents include antimetabolites (eg, methotrexate, 5-fluorouracil, etc.), alkylating agents (eg, cyclophosphamide, ifosfamide, etc.), platinum anticancer agents (eg, cisplatin, carboplatin, etc.), topoisomerase, etc. Inhibitors (eg, etoposide, etc.), anticancer antibiotics (eg, mitomycin, adriamycin, etc.), plant-derived anticancer agents (eg, vincristine, vindesine, taxol, etc.), tyrosine kinase inhibitors (eg, gefitinib, imatinib, etc.), Examples include, but are not limited to, humanized antibodies (eg, Herceptin).
　本発明の核酸は、それ自体を投与してもよいし、又は適当な医薬組成物として投与してもよい。投与に用いられる医薬組成物としては、本発明の核酸と薬理学的に許容され得る担体、希釈剤もしくは賦形剤とを含むものであってよい。このような医薬組成物は、経口又は非経口投与に適する剤形として提供される。
　本発明の核酸を上記の医薬組成物として使用する場合、自体公知の方法に従って製剤化し、投与することができる。即ち、所望の効果を有する限り、本発明の核酸を、単独あるいはレトロウイルスベクター、アデノウイルスベクター、アデノウイルスアソシエーテッドウイルスベクターなどの適当な哺乳動物細胞用の発現ベクターに機能可能な態様で挿入した後、常套手段に従って製剤化することができる。該核酸は、そのままで、あるいは摂取促進のための補助剤とともに、遺伝子銃やハイドロゲルカテーテルのようなカテーテルによって投与することができる。あるいは、エアロゾル化して吸入剤として気管内に局所投与することもできるが、これらに限定されない。一実施形態において、本発明の核酸は、被験動物の有する腫瘍（及び／又は腫瘍周囲）への局所投与に適切な形態として製剤化される。例えば、本発明の核酸は、アテロコラーゲンゲル等のゲル、クリーム、リポソーム、エクソソーム等の形態として製剤化してもよい。 The nucleic acids of the invention may be administered per se or as a suitable pharmaceutical composition. The pharmaceutical composition used for administration may contain the nucleic acid of the present invention and a pharmacologically acceptable carrier, diluent or excipient. Such a pharmaceutical composition is provided as a dosage form suitable for oral or parenteral administration.
When the nucleic acid of the present invention is used as the above pharmaceutical composition, it can be formulated and administered according to a method known per se. That is, as long as it has a desired effect, the nucleic acid of the present invention is inserted alone or in a functional manner into an appropriate expression vector for mammalian cells such as a retrovirus vector, adenovirus vector, adenovirus associated virus vector, etc. Thereafter, it can be formulated according to conventional means. The nucleic acid can be administered as it is or together with an auxiliary agent for promoting intake by a catheter such as a gene gun or a hydrogel catheter. Alternatively, it can be aerosolized and locally administered into the trachea as an inhalant, but is not limited thereto. In one embodiment, the nucleic acid of the invention is formulated in a form suitable for local administration to a tumor (and / or surrounding tumor) of a subject animal. For example, the nucleic acid of the present invention may be formulated as a gel such as atelocollagen gel, cream, liposome, exosome or the like.
　非経口投与のための組成物としては、例えば、注射剤、坐剤等が用いられ、注射剤は静脈注射剤、皮下注射剤、皮内注射剤、筋肉注射剤、点滴注射剤等の剤形を包含しても良い。このような注射剤は、公知の方法に従って調製できる。注射剤の調製方法としては、例えば、上記本発明の核酸を通常注射剤に用いられる無菌の水性液、又は油性液に溶解、懸濁又は乳化することによって調製できる。注射用の水性液としては、例えば、生理食塩水、ブドウ糖やその他の補助薬を含む等張液等が用いられ、適当な溶解補助剤、例えば、アルコール（例、エタノール）、ポリアルコール（例、プロピレングリコール、ポリエチレングリコール）、非イオン界面活性剤〔例、ポリソルベート80、HCO-50（polyoxyethylene（50mol）adduct of hydrogenated castor oil）〕等と併用してもよい。油性液としては、例えば、ゴマ油、大豆油等が用いられ、溶解補助剤として安息香酸ベンジル、ベンジルアルコール等を併用してもよい。調製された注射液は、適当なアンプルに充填されることが好ましい。直腸投与に用いられる坐剤は、上記核酸を通常の坐薬用基剤に混合することによって調製されてもよい。非経口投与のための組成物としては、上記の組成物の他にゲル剤、エアロゾル剤等の吸入剤、クリーム剤、軟膏剤、ローション剤等が挙げられるが、これらに限定されない。 As a composition for parenteral administration, for example, injections, suppositories and the like are used. Injections are dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, infusions, and the like. May be included. Such an injection can be prepared according to a known method. As a method for preparing an injection, it can be prepared, for example, by dissolving, suspending or emulsifying the nucleic acid of the present invention in a sterile aqueous liquid or oily liquid usually used for injection. As an aqueous solution for injection, for example, an isotonic solution containing physiological saline, glucose and other adjuvants, and the like are used, and suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct-of-hydrogenated-castor-oil)) and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent. The prepared injection solution is preferably filled in a suitable ampoule. Suppositories used for rectal administration may be prepared by mixing the nucleic acid with a normal suppository base. Examples of compositions for parenteral administration include, but are not limited to, inhalants such as gels and aerosols, creams, ointments, lotions and the like in addition to the above compositions.
　経口投与のための組成物としては、固体又は液体の剤形、具体的には錠剤（糖衣錠、フィルムコーティング錠を含む）、丸剤、顆粒剤、散剤、カプセル剤（ソフトカプセル剤を含む）、シロップ剤、乳剤、懸濁剤等が挙げられる。このような組成物は公知の方法によって製造され、製剤分野において通常用いられる担体、希釈剤もしくは賦形剤を含有していても良い。錠剤用の担体、賦形剤としては、例えば、乳糖、でんぷん、蔗糖、ステアリン酸マグネシウムが用いられるが、これらに限定されない。 Compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), and syrups. Agents, emulsions, suspensions and the like. Such a composition is produced by a known method and may contain a carrier, a diluent or an excipient usually used in the pharmaceutical field. Examples of carriers and excipients for tablets include lactose, starch, sucrose, and magnesium stearate, but are not limited thereto.
　上記の非経口用又は経口用医薬組成物は、活性成分の投与量に適合するような投薬単位の剤形に調製されることが好都合である。このような投薬単位の剤形としては、例えば、錠剤、丸剤、カプセル剤、注射剤（アンプル）、坐剤が挙げられるが、これらに限定されない。 The above parenteral or oral pharmaceutical composition is conveniently prepared in a dosage unit form suitable for the dosage of the active ingredient. Examples of the dosage form of such a dosage unit include, but are not limited to, tablets, pills, capsules, injections (ampoules), and suppositories.
　本発明の医薬組成物は、さらに、体内動態の改良、半減期の長期化、細胞内取り込み効率の改善を目的に、前記核酸を単独又はリポソームなどの担体とともに製剤（注射剤）化し、静脈、皮下等に投与してもよい。 The pharmaceutical composition of the present invention is further formulated into a formulation (injection) with the above-mentioned nucleic acid alone or with a carrier such as a liposome for the purpose of improving pharmacokinetics, prolonging the half-life, and improving cellular uptake efficiency, It may be administered subcutaneously or the like.
　本発明の医薬組成物は、核酸を細胞内に移行させるのに有効な担体をさらに含むことができる。核酸を細胞内に移行させるのに有効な担体としては、例えばカチオン性担体があげられる。カチオン性担体としては、カチオン性リポソーム及びカチオン性ポリマーなどがあげられる。また、核酸を細胞内に移行させるのに有効な担体として、ウイルスエンベロープを利用した担体を用いてもよい。カチオン性ポリマーとしては、JetSI（Qbiogene社）、Jet-PEI（ポリエチレンイミン；Qbiogene社）などが好ましく用いられる。ウイルスエンベロープを利用した担体としては、GenomeOne（HVJ-Eリポソーム；石原産業社）などが好ましく用いられる。 The pharmaceutical composition of the present invention can further contain a carrier effective for transferring nucleic acid into cells. Examples of the carrier effective for transferring the nucleic acid into the cell include a cationic carrier. Examples of the cationic carrier include cationic liposomes and cationic polymers. Further, a carrier utilizing a viral envelope may be used as an effective carrier for transferring nucleic acids into cells. As the cationic polymer, JetSI (Qbiogene), Jet-PEI (polyethyleneimine; Qbiogene) and the like are preferably used. As a carrier using a viral envelope, GenomeOne (HVJ-E liposome; Ishihara Sangyo Co., Ltd.) and the like are preferably used.
　本発明の核酸と上記担体を含む組成物は、当業者に既知の方法により調製することができる。例えば、適当な濃度の担体分散液と核酸溶液とを混合して調製することができる。カチオン性担体を用いる場合、核酸は通常、水溶液中で負電荷を帯びているため、常法により水溶液中で混合することによって容易に調製することができる。該組成物を調製するために用いる水性溶媒としては、注射用水、注射用蒸留水、生理食塩水などの電解質液、ブドウ糖液、マルトース液などの糖液などがあげられる。また、該組成物を調製する際のpH及び温度などの条件は当業者が適宜選択できる。該組成物は、必要ならば超音波分散装置や高圧乳化装置などを用いて分散処理を行うことにより、均一な組成物とすることもできる。担体と核酸とを含む組成物の調製に最適な方法及び条件は、用いる担体に依存するので、上記の方法にとらわれることなく、当業者であれば用いる担体に最適な方法を選択できる。 The composition containing the nucleic acid of the present invention and the above carrier can be prepared by methods known to those skilled in the art. For example, it can be prepared by mixing a carrier dispersion having an appropriate concentration and a nucleic acid solution. When a cationic carrier is used, since the nucleic acid is usually negatively charged in an aqueous solution, it can be easily prepared by mixing in an aqueous solution by a conventional method. Examples of the aqueous solvent used for preparing the composition include electrolyte solutions such as water for injection, distilled water for injection, and physiological saline, and sugar solutions such as glucose solution and maltose solution. Moreover, those skilled in the art can appropriately select conditions such as pH and temperature when preparing the composition. If necessary, the composition can be made into a uniform composition by carrying out a dispersion treatment using an ultrasonic dispersing device or a high-pressure emulsifying device. The optimum method and conditions for the preparation of the composition containing the carrier and the nucleic acid depend on the carrier to be used, so that those skilled in the art can select the optimum method for the carrier to be used without being bound by the above method.
　また、本発明の医薬組成物としては、例えば核酸とリード粒子とを構成成分とする複合粒子及び該複合粒子を被覆する脂質膜から構成される組成物も、好適に用いられる。リード粒子としては、例えば、脂質集合体、リポソーム、エマルジョン粒子、高分子、金属コロイド、微粒子製剤等があげられ、好ましくはカチオン性リポソームが用いられる。本発明におけるリード粒子は、脂質集合体、リポソーム、エマルジョン粒子、高分子、金属コロイド、微粒子製剤等を２つ以上組み合わせた複合体を構成成分としていてもよく、脂質集合体、リポソーム、エマルジョン粒子、高分子、金属コロイド、微粒子製剤等と他の化合物（例えば糖、脂質、無機化合物等）とを組み合わせた複合体を構成成分としていてもよい。
　該複合粒子を被覆する脂質膜としては、例えば非カチオン性脂質、粒子の凝集を阻止する脂質及びカチオン性脂質等を構成成分とするものがあげられる。 In addition, as the pharmaceutical composition of the present invention, for example, a composition composed of composite particles containing nucleic acids and lead particles and a lipid membrane covering the composite particles is also preferably used. Examples of the lead particles include lipid aggregates, liposomes, emulsion particles, polymers, metal colloids, fine particle preparations, and the like, and cationic liposomes are preferably used. The lead particles in the present invention may be composed of a complex obtained by combining two or more lipid aggregates, liposomes, emulsion particles, polymers, metal colloids, fine particle formulations, etc., and lipid aggregates, liposomes, emulsion particles, A complex formed by combining a polymer, a metal colloid, a fine particle preparation, and the like with another compound (eg, sugar, lipid, inorganic compound, etc.) may be used as a constituent component.
Examples of the lipid membrane that coats the composite particles include non-cationic lipids, lipids that prevent aggregation of particles, and cationic lipids.
　該組成物は、例えば国際公開公報2006/080118号パンフレット等に記載の方法に従って調製することができる。 The composition can be prepared according to the method described in, for example, International Publication No. 2006/080118 Pamphlet.
　本発明の核酸は、例えば、投薬単位剤形当たり通常5～500mg、とりわけ注射剤では5～100mg、その他の剤形では10～250mg含有されていることが好ましい。 The nucleic acid of the present invention is preferably contained, for example, usually 5 to 500 mg per dosage unit dosage form, particularly 5 to 100 mg for injections and 10 to 250 mg for other dosage forms.
　本発明の核酸又は本発明の医薬組成物の投与方法は、所望の効果が得られる限り特に制限されないが、好ましい投与方法の一つとして、所望の部位（例えば、腫瘍部位及び／又は腫瘍周囲）への局所投与が挙げられる。局所投与の方法の例としては、注射等の自体公知の方法を適宜用いることができる。本発明の核酸は、癌組織特異的に発現するCt-SLCO1B3を標的としており、全身投与を行った場合においても癌組織でのみ作用することが予想されるため、全身投与を行ってもよい。また、本発明の核酸又は本発明の医薬組成物を全身投与する場合には、所望の部位（例えば、腫瘍部位及び／又は腫瘍周囲）への安定かつ高効率な送達を達成するために、自体公知の薬剤送達技術を適宜用いることもできる。 The administration method of the nucleic acid of the present invention or the pharmaceutical composition of the present invention is not particularly limited as long as the desired effect is obtained. As one of preferable administration methods, a desired site (for example, a tumor site and / or a tumor periphery) Topical administration. As an example of the local administration method, a method known per se such as injection can be appropriately used. Since the nucleic acid of the present invention targets Ct-SLCO1B3 expressed specifically in cancer tissues and is expected to act only in cancer tissues even when administered systemically, it may be administered systemically. In addition, when the nucleic acid of the present invention or the pharmaceutical composition of the present invention is administered systemically, in order to achieve stable and highly efficient delivery to a desired site (for example, tumor site and / or surrounding tumor) Known drug delivery techniques can also be used as appropriate.
　本発明の核酸を含有する上記医薬の投与量は、投与対象、対象疾患、症状、投与ルートなどによっても異なるが、例えば、癌の治療・予防のために使用する場合には、本発明の核酸を1回量として、通常0.01～20mg/kg体重程度、好ましくは0.1～10mg/kg体重程度、さらに好ましくは0.1～5mg/kg体重程度を、1日1～5回程度、好ましくは1日1～3回程度、静脈注射により投与するのが好都合である。他の非経口投与及び経口投与の場合もこれに準ずる量を投与することができる。症状が特に重い場合には、その症状に応じて増量してもよい。 The dosage of the above-mentioned pharmaceutical containing the nucleic acid of the present invention varies depending on the administration subject, target disease, symptom, administration route, etc., but for example, when used for the treatment / prevention of cancer, the nucleic acid of the present invention. Is usually about 0.01 to 20 mg / kg body weight, preferably about 0.1 to 10 mg / kg body weight, more preferably about 0.1 to 5 mg / kg body weight, about 1 to 5 times a day, preferably 1 day a day. It is convenient to administer about 3 times by intravenous injection. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If symptoms are particularly severe, the dose may be increased according to the symptoms.
　なお前記した各組成物は、本発明の核酸との配合により好ましくない相互作用を生じない限り適宜他の活性成分を含有してもよい。 Note that each of the above-described compositions may appropriately contain other active ingredients as long as an undesirable interaction is not caused by blending with the nucleic acid of the present invention.
４．疾病に対する医薬候補化合物のスクリーニング
　Ct-SLCO1B3の発現は、細胞の足場非依存性増殖を促進し、細胞の遊走能及び浸潤能を亢進させる。
　従って、Ct-SLCO1B3の発現及び／又は機能を抑制する化合物は、細胞の足場非依存性増殖、浸潤能亢進及び／又は遊走能亢進を抑制させる、癌の予防剤及び／又は治療剤として使用することができる。つまり、Ct-SLCO1B3を産生する細胞は、Ct-SLCO1B3の発現量及び／又は機能を指標とすることにより、癌の予防及び／治療作用を有する物質のスクリーニングのためのツールとして用いることができる。
　また、上述の通り、Ct-SLCO1B3の発現は上皮間葉転換に関わるsnail、slugの発現を上昇させる。Ct-SLCO1B3は、癌では発現が低下していることが知られているE-cadherin、occludinの発現を抑制し、浸潤に関わるMMP9の発現を誘導する。
　従って、Ct-SLCO1B3を産生する細胞は、Ct-SLCO1B3の発現に起因する、snail、slug、E-cadherin、occludin及びMMP9からなる群より選ばれる分子の発現量を変化させる活性を有する物質のスクリーニングのためのツールとして用いることができる。
　つまり、本発明は、以下の（1）～（3）の工程を含む、癌の予防及び／又は治療作用を有する物質のスクリーニング方法（本発明のスクリーニング方法とも称する）を提供する：
（1）Ct-SLCO1B3を発現する細胞に、被検物質を接触させる工程、
（2）前記細胞におけるCt-SLCO1B3の発現量又は機能を測定する工程、及び
（3）被検物質の非存在下において測定した場合と比較して、発現量又は機能を低下させる化合物を癌の予防及び／又は治療作用を有する物質の候補として選択する工程。 4). Screening of drug candidate compounds for disease Expression of Ct-SLCO1B3 promotes anchorage-independent growth of cells and enhances cell migration ability and invasion ability.
Therefore, a compound that suppresses the expression and / or function of Ct-SLCO1B3 is used as a prophylactic and / or therapeutic agent for cancer that suppresses anchorage-independent growth, increased invasive ability and / or enhanced migration ability of cells. be able to. That is, a cell producing Ct-SLCO1B3 can be used as a tool for screening a substance having a preventive and / or therapeutic action for cancer by using the expression level and / or function of Ct-SLCO1B3 as an index.
Moreover, as described above, the expression of Ct-SLCO1B3 increases the expression of snail and slug involved in epithelial-mesenchymal transition. Ct-SLCO1B3 suppresses the expression of E-cadherin and occludin, which are known to be down-regulated in cancer, and induces the expression of MMP9 related to invasion.
Therefore, cells that produce Ct-SLCO1B3 are screened for substances that have an activity to change the expression level of a molecule selected from the group consisting of snail, slug, E-cadherin, occludin and MMP9 due to the expression of Ct-SLCO1B3. Can be used as a tool for.
That is, the present invention provides a method for screening a substance having a preventive and / or therapeutic action for cancer (also referred to as the screening method of the present invention), comprising the following steps (1) to (3):
(1) a step of contacting a test substance with a cell expressing Ct-SLCO1B3,
(2) a step of measuring the expression level or function of Ct-SLCO1B3 in the cell, and (3) a compound that decreases the expression level or function compared to the case of measurement in the absence of the test substance. Selecting as a candidate for a substance having a preventive and / or therapeutic action.
　Ct-SLCO1B3の発現及び／又は機能を抑制する化合物又はその塩をスクリーニングする場合、該スクリーニング方法は、Ct-SLCO1B3を産生する能力を有する細胞を、被検物質の存在下又は非存在下で培養し、両条件下におけるCt-SLCO1B3の発現量及び／又は機能を比較することを含む。 When screening for a compound or a salt thereof that suppresses the expression and / or function of Ct-SLCO1B3, the screening method involves culturing cells capable of producing Ct-SLCO1B3 in the presence or absence of a test substance. And comparing the expression level and / or function of Ct-SLCO1B3 under both conditions.
　上記のスクリーニング方法において用いられるCt-SLCO1B3を産生する能力を有する細胞としては、Ct-SLCO1B3を生来発現しているヒト若しくは他の哺乳動物由来の細胞又はそれを含む生体試料（例えば、摘出した癌部、血液、組織、臓器等）であれば特に制限はない。非ヒト動物由来の血液、組織、臓器等の場合は、それらを生体から単離して培養してもよいし、あるいは生体自体に被検物質を投与し、一定時間経過後にそれら生体試料を単離してもよい。
　Ct-SLCO1B3を発現しているヒト由来の培養細胞株としては、ヒト肺胞基底上皮腺癌細胞であるA549細胞などが好ましく用いられる。
　また、Ct-SLCO1B3を産生する能力を有する細胞としては、公知慣用の遺伝子工学的手法により作製された各種の形質転換体が例示される。宿主としては、例えば、NCI-H2細胞、HEK293細胞、COS7細胞、CHO細胞などの動物細胞が好ましく用いられる。
　具体的には、Ct-SLCO1B3をコードするDNA（配列番号32）で表される塩基配列又は該塩基配列に対し相補性を有する塩基配列とストリンジェントな条件下でハイブリダイズし、且つ配列番号5で表されるアミノ酸配列からなるタンパク質と同質の機能を有するポリペプチドをコードする塩基配列を含むDNAを、適当な発現ベクター中のプロモーターの下流に連結して宿主動物細胞に導入することにより調製することができる。 Cells having the ability to produce Ct-SLCO1B3 used in the above screening method include cells derived from humans or other mammals that naturally express Ct-SLCO1B3 or biological samples containing the same (for example, excised cancer Part, blood, tissue, organ, etc.). In the case of blood, tissues, organs, etc. derived from non-human animals, they may be isolated from the living body and cultured, or the test substance is administered to the living body itself, and these biological samples are isolated after a certain period of time. May be.
As a human-derived cultured cell line expressing Ct-SLCO1B3, A549 cells which are human alveolar basal epithelial adenocarcinoma cells are preferably used.
Examples of cells having the ability to produce Ct-SLCO1B3 include various transformants prepared by known and commonly used genetic engineering techniques. As the host, animal cells such as NCI-H2 cells, HEK293 cells, COS7 cells, and CHO cells are preferably used.
Specifically, it hybridizes under stringent conditions with a base sequence represented by DNA encoding Ct-SLCO1B3 (SEQ ID NO: 32) or a base sequence complementary to the base sequence, and SEQ ID NO: 5 A DNA comprising a nucleotide sequence that encodes a polypeptide having the same function as the protein consisting of the amino acid sequence represented by is ligated downstream of a promoter in an appropriate expression vector and introduced into a host animal cell. be able to.
　Ct-SLCO1B3をコードする遺伝子の調製方法について、以下に説明する。
　Ct-SLCO1B3をコードする遺伝子は、通常の遺伝子工学的方法（例えば、Sambrook J.,FrischE.F.,Maniatis T.著、モレキュラークローニング第2版（Molecular Cloning 2nd edition）、コールドスプリングハーバーラボラトリー発行（Cold Spring Harbor Laboratory press）等に記載されている方法）に準じて取得することができる。すなわち、Ct-SLCO1B3をコードするDNAは、例えば、配列番号32で表される塩基配列に基づいて、適当なオリゴヌクレオチドをプローブもしくはプライマーとして合成し、前記したCt-SLCO1B3を産生する細胞・組織由来のcDNAもしくはcDNAライブラリーから、ハイブリダイゼーション法やPCR法を用いてクローニングすることができる。ハイブリダイゼーションは、例えば、モレキュラー・クローニング（Molecular Cloning）第2版（上記）に記載の方法などに従って行なうことができる。また、市販のライブラリーを使用する場合、ハイブリダイゼーションは、該ライブラリーに添付された使用説明書に記載の方法に従って行なうことができる。 A method for preparing a gene encoding Ct-SLCO1B3 is described below.
The gene encoding Ct-SLCO1B3 can be obtained by conventional genetic engineering methods (eg, Sambrook J., Frisch E.F., Maniatis T., Molecular Cloning 2nd edition), Cold Spring Harbor Laboratory ( The method can be obtained according to the method described in Cold Spring Harbor Laboratory press). That is, DNA encoding Ct-SLCO1B3 is derived from cells / tissues that produce Ct-SLCO1B3 as described above by synthesizing an appropriate oligonucleotide as a probe or primer based on the base sequence represented by SEQ ID NO: 32, for example. The cDNA or cDNA library can be cloned using a hybridization method or a PCR method. Hybridization can be performed, for example, according to the method described in Molecular Cloning 2nd edition (above). When a commercially available library is used, hybridization can be performed according to the method described in the instruction manual attached to the library.
　クローン化されたDNAは、目的によりそのまま、又は所望により制限酵素で消化するか、リンカーを付加した後に、使用することができる。該DNAはその5’末端側に翻訳開始コドンとしてのATGを有し、また3’末端側には翻訳終止コドンとしてのTAA、TGA又はTAGを有していてもよい。これらの翻訳開始コドンや翻訳終止コドンは、適当な合成DNAアダプターを用いて付加することができる。
　次いで、得られたCt-SLCO1B3遺伝子を用いて、通常の遺伝子工学的方法に準じてCt-SLCO1B3（タンパク質）を製造・取得することができる。
　例えば、Ct-SLCO1B3遺伝子が宿主細胞中で発現できるようなプラスミドを作製し、これを宿主細胞に導入して形質転換し、さらに形質転換された宿主細胞（形質転換体）を培養することで得られる培養物からCt-SLCO1B3を取得すればよい。上記プラスミドとしては、例えば、宿主細胞中で複製可能な遺伝情報を含み、自律的に複製できるものであって、宿主細胞からの単離・精製が容易であり、宿主細胞中で機能可能なプロモーターを有し、検出可能なマーカーをもつ発現ベクターに、Ct-SLCO1B3をコードする遺伝子が導入されたものを好ましく挙げることができる。 The cloned DNA can be used as it is or after digestion with a restriction enzyme or addition of a linker, if desired. The DNA may have ATG as a translation initiation codon on the 5 ′ end side and TAA, TGA or TAG as a translation termination codon on the 3 ′ end side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.
Next, using the obtained Ct-SLCO1B3 gene, Ct-SLCO1B3 (protein) can be produced and obtained according to an ordinary genetic engineering method.
For example, it can be obtained by preparing a plasmid that can express the Ct-SLCO1B3 gene in a host cell, introducing it into the host cell, transforming it, and then culturing the transformed host cell (transformant). Ct-SLCO1B3 may be obtained from the obtained culture. Examples of the plasmid include a promoter that can replicate autonomously in a host cell, can replicate autonomously, can be easily isolated and purified from the host cell, and can function in the host cell. Preferred examples include those in which a gene encoding Ct-SLCO1B3 is introduced into an expression vector having a detectable marker.
　また、動物細胞発現プラスミド（例：pA1-11、pXT1、pRc/CMV、pRc/RSV、pcDNAI/Neo）；λファージなどのバクテリオファージ；レトロウイルス、ワクシニアウイルス、アデノウイルスなどの動物ウイルスベクターなどを用いることもできる。プロモーターとしては、遺伝子の発現に用いる宿主に対応して適切なプロモーターであればいかなるものでもよい。例えば、SRαプロモーター、SV40プロモーター、LTRプロモーター、CMV（サイトメガロウイルス）プロモーター、RSV（ラウス肉腫ウイルス）プロモーター、MoMuLV（モロニーマウス白血病ウイルス）LTR、HSV-TK（単純ヘルペスウイルスチミジンキナーゼ）プロモーター、βアクチン遺伝子プロモーター、aP2遺伝子プロモーターなどが用いられる。なかでも、CMVプロモーター、SRαプロモーターなどが好ましい。
　発現ベクターとしては、上記の他に、所望によりエンハンサー、スプライシングシグナル、ポリＡ付加シグナル、選択マーカー、SV40複製起点（以下、SV40 oriと略称する場合がある）などを含有しているものを用いることができる。 In addition, animal cell expression plasmids (eg, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo); bacteriophages such as λ phage; animal virus vectors such as retrovirus, vaccinia virus, adenovirus, etc. It can also be used. The promoter may be any promoter as long as it is appropriate for the host used for gene expression. For example, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter, β-actin A gene promoter, aP2 gene promoter, etc. are used. Of these, CMV promoter, SRα promoter and the like are preferable.
In addition to the above, an expression vector containing an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), etc. is used as desired. Can do.
　選択マーカーとしては、例えば、ジヒドロ葉酸還元酵素遺伝子（以下、dhfrと略称する場合がある、メソトレキセート（MTX）耐性）、アンピシリン耐性遺伝子（以下、amprと略称する場合がある）、ネオマイシン耐性遺伝子（以下、neorと略称する場合がある、G418耐性）等が挙げられる。特に、dhfr遺伝子欠損チャイニーズハムスター細胞を用い、dhfr遺伝子を選択マーカーとして使用する場合、チミジンを含まない培地によって目的遺伝子を選択することもできる。 Examples of selectable markers include a dihydrofolate reductase gene (hereinafter abbreviated as dhfr, methotrexate (MTX) resistance), an ampicillin resistance gene (hereinafter abbreviated as ampr), a neomycin resistance gene (hereinafter abbreviated as amplicon). , G418 resistance, which may be abbreviated as neor). In particular, when dhfr gene-deficient Chinese hamster cells are used and the dhfr gene is used as a selection marker, the target gene can also be selected using a medium that does not contain thymidine.
　前記のようにして得られたプラスミドは、通常の遺伝子工学的方法により前記宿主細胞に導入することができる。形質転換体の培養は、自体公知の方法によって行うことができる。形質転換は、リン酸カルシウム共沈殿法、PEG法、エレクトロポレーション法、マイクロインジェクション法、リポフェクション法などにより行うことができる。 The plasmid obtained as described above can be introduced into the host cell by an ordinary genetic engineering method. The transformant can be cultured by a method known per se. Transformation can be performed by calcium phosphate coprecipitation method, PEG method, electroporation method, microinjection method, lipofection method and the like.
　上記のようにして得られる形質転換細胞や生来Ct-SLCO1B3を産生する能力を有する哺乳動物細胞又は該細胞を含む組織・臓器は、例えば、約5～20%の胎仔牛血清を含む最小必須培地（MEM）、ダルベッコ改変イーグル培地（DMEM）、RPMI 1640培地、199培地などの培地中で培養することができる。培地のpHは約6～8であるのが好ましい。培養は通常約30～40℃で行ない、必要に応じて通気や撹拌を加える。 The transformed cells obtained as described above, the mammalian cells having the ability to produce native Ct-SLCO1B3, or the tissues / organs containing the cells are, for example, the minimum essential medium containing about 5 to 20% fetal calf serum. (MEM), Dulbecco's modified Eagle's medium (DMEM), RPMI 培 地 1640 medium, 199 medium, and the like. The pH of the medium is preferably about 6-8. Cultivation is usually carried out at about 30-40 ° C, with aeration and agitation as necessary.
　本発明のスクリーニングを実施するに当たり、被検物質としては、例えばタンパク質、ペプチド、非ペプチド性化合物、合成化合物、発酵生産物、細胞抽出液、植物抽出液、動物組織抽出液などが挙げられ、これらの物質は新規なものであってもよいし、公知のものであってもよい。
　また、Ct-SLCO1B3もしくはCt-SLCO1B3遺伝子の発現量を低下させる物質、又はCt-SLCO1B3の機能を低下させる物質を選択する際に、被検物質を接触させない対照細胞を比較対照として用いることもできる。ここで「被検物質を接触させない」とは、被検物質の代わりに被検物質と同量の溶媒（ブランク）を添加する場合や、Ct-SLCO1B3もしくはCt-SLCO1B3遺伝子の発現量又はCt-SLCO1B3の機能に影響を与えないネガティブコントロール物質を添加する場合も含まれる。 In conducting the screening of the present invention, examples of the test substance include proteins, peptides, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. These substances may be novel or may be known ones.
In addition, when selecting a substance that decreases the expression level of Ct-SLCO1B3 or Ct-SLCO1B3 gene, or a substance that decreases the function of Ct-SLCO1B3, a control cell that does not contact the test substance can also be used as a comparative control. . Here, “do not contact the test substance” means that the same amount of solvent (blank) as the test substance is added instead of the test substance, or the expression level of Ct-SLCO1B3 or Ct-SLCO1B3 gene or Ct- This includes cases where a negative control substance that does not affect the function of SLCO1B3 is added.
　被検物質の上記細胞との接触は、例えば、上記の培地や各種緩衝液（例えば、HEPES緩衝液、リン酸緩衝液、リン酸緩衝生理食塩水、トリス塩酸緩衝液、ホウ酸緩衝液、酢酸緩衝液など）の中に被検物質を添加して、細胞を一定時間インキュベートすることにより実施することができる。添加される被検物質の濃度は化合物の種類（溶解度、毒性等）により異なるが、例えば、約0.1nM～約100μMの範囲で適宜選択される。インキュベート時間としては、例えば、約10分～約24時間が挙げられる。 The contact of the test substance with the cells may be performed by, for example, the above-mentioned medium or various buffers (for example, HEPES buffer, phosphate buffer, phosphate buffered saline, Tris-HCl buffer, borate buffer, acetic acid). The test substance can be added to a buffer solution or the like, and the cells can be incubated for a certain time. The concentration of the test substance to be added varies depending on the type of compound (solubility, toxicity, etc.), but is appropriately selected within the range of about 0.1 nM to about 100 μM, for example. Examples of the incubation time include about 10 minutes to about 24 hours.
　Ct-SLCO1B3を産生する細胞が、非ヒト哺乳動物個体の形態で提供される場合、該動物個体の状態は特に制限されないが、例えば、癌細胞を移植したモデルマウス（例えば、A549細胞をBalb/c slc-nu/nuマウスに背部皮下移植して作製したマウス）等のモデル動物であってもよい。使用される動物の飼育条件に特に制限はないが、SPFグレード以上の環境下で飼育されたものであることが好ましい。被検物質の該細胞との接触が該動物個体への被検物質の投与によって行われる場合、投与経路は特に制限されないが、例えば、静脈内投与、動脈内投与、皮下投与、皮内投与、腹腔内投与、経口投与、気道内投与、直腸投与等が挙げられる。投与量も特に制限はないが、例えば、１回量として約0.5～20mg/kgを、1日1～5回、好ましくは1日1～3回、1～14日間投与することができる。 When the cell producing Ct-SLCO1B3 is provided in the form of a non-human mammal individual, the state of the animal individual is not particularly limited. For example, a model mouse transplanted with cancer cells (for example, A549 cells in Balb / It may be a model animal such as a mouse produced by subcutaneous implantation in the back of a c slc-nu / nu mouse. There are no particular restrictions on the breeding conditions of the animals to be used, but it is preferable that the animals are raised in an environment of SPF grade or higher. When contact of the test substance with the cells is performed by administration of the test substance to the animal individual, the administration route is not particularly limited, for example, intravenous administration, intraarterial administration, subcutaneous administration, intradermal administration, Examples include intraperitoneal administration, oral administration, intratracheal administration, and rectal administration. The dose is not particularly limited. For example, a dose of about 0.5 to 20 mg / kg can be administered 1 to 5 times a day, preferably 1 to 3 times a day for 1 to 14 days.
（Ct-SLCO1B3の発現量の測定）
　本発明のスクリーニング方法は、Ct-SLCO1B3を産生する能力を有する細胞における該タンパク質（遺伝子）の発現を、被検物質の存在下と非存在下で比較することを特徴とする、癌の治療又は予防活性を有する物質のスクリーニング方法を提供する。本方法において用いられる細胞、被検物質の種類、被検物質と細胞との接触の態様などは、上記と同様である。 (Measurement of Ct-SLCO1B3 expression level)
The screening method of the present invention is characterized in that the expression of the protein (gene) in a cell capable of producing Ct-SLCO1B3 is compared in the presence and absence of a test substance, or a cancer treatment or A method for screening a substance having prophylactic activity is provided. The cells used in this method, the type of test substance, the mode of contact between the test substance and cells, etc. are the same as described above.
　遺伝子の発現量の比較は、自体公知の方法を用いて行うことができる。 The comparison of gene expression levels can be performed using a method known per se.
　より具体的には、本発明は、
（a）Ct-SLCO1B3を産生する能力を有する細胞を被検物質の存在下又は非存在下に培養し、両条件下における該タンパク質をコードするmRNAの量を、本発明の検出用核酸を用いて測定、比較することを特徴とする、癌の治療又は予防活性を有する物質のスクリーニング方法、及び
（b）Ct-SLCO1B3を産生する能力を有する細胞を被検物質の存在下又は非存在下に培養し、両条件下における該タンパク質の量を検出用抗体を用いて測定、比較することを特徴とする、癌の治療又は予防活性を有する物質のスクリーニング方法を提供する。 More specifically, the present invention provides:
(A) Cells having the ability to produce Ct-SLCO1B3 are cultured in the presence or absence of a test substance, and the amount of mRNA encoding the protein under both conditions is determined using the nucleic acid for detection of the present invention. A method for screening a substance having a therapeutic or prophylactic activity for cancer, and (b) a cell having the ability to produce Ct-SLCO1B3 in the presence or absence of the test substance. A method for screening a substance having a therapeutic or prophylactic activity for cancer, comprising culturing and measuring and comparing the amount of the protein under both conditions using a detection antibody.
　すなわち、Ct-SLCO1B3の発現量を変化させる物質のスクリーニングは、以下のようにして行うことができる。
（i）正常あるいは疾患（例えば、A549細胞をBalb/c slc-nu/nuマウスに背部皮下移植して作製したマウスなど）モデル非ヒト哺乳動物（例えば、マウス、ラット、ウサギ、ヒツジ、ブタ、ウシ、ネコ、イヌ、サルなど）に対して被検物質を投与し、一定時間経過した後（30分後～3日後、好ましくは1時間後～2日後、より好ましくは1時間後～24時間後）に、特定の臓器（例えば、肺等）、あるいは臓器から単離した組織又は細胞を得る。
　Ct-SLCO1B3のmRNAは、通常の方法により細胞等からmRNAを抽出して定量することができ、あるいは自体公知のノーザンブロット解析により定量することもできる。一方、Ct-SLCO1B3のタンパク質量は、自体公知の方法により定量することができる。例えば、Ct-SLCO1B3のタンパク質量は、ウェスタンブロット解析や以下に詳述する各種イムノアッセイ法を用いて定量することができるがこれらに限定されない。
（ii）Ct-SLCO1B3遺伝子を発現する細胞（例えば、Ct-SLCO1B3を導入した形質転換体）を上記の方法に従って作製し、常法に従って培養する際に被検物質を培地もしくは緩衝液中に添加し、一定時間インキュベート後（1日後～7日後、好ましくは1日後～3日後、より好ましくは2日後～3日後）、該細胞に含まれるCt-SLCO1B3あるいはそれをコードするmRNAを、上記（i）と同様にして定量、解析することができる。 That is, screening for a substance that changes the expression level of Ct-SLCO1B3 can be performed as follows.
(I) Normal or disease (for example, a mouse produced by subcutaneous implantation of A549 cells into Balb / c slc-nu / nu mice) Model non-human mammals (for example, mice, rats, rabbits, sheep, pigs, After the test substance is administered to cows, cats, dogs, monkeys, etc., after a certain period of time (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours) Later, a specific organ (for example, lung, etc.) or a tissue or cell isolated from the organ is obtained.
The mRNA of Ct-SLCO1B3 can be quantified by extracting mRNA from cells or the like by a usual method, or can be quantified by Northern blot analysis known per se. On the other hand, the protein amount of Ct-SLCO1B3 can be quantified by a method known per se. For example, the amount of Ct-SLCO1B3 protein can be quantified using Western blot analysis or various immunoassay methods described in detail below, but is not limited thereto.
(Ii) Ct-SLCO1B3 gene-expressing cells (for example, transformants into which Ct-SLCO1B3 has been introduced) are prepared according to the above method, and the test substance is added to the medium or buffer when culturing according to conventional methods After incubation for a certain period of time (1 day to 7 days, preferably 1 day to 3 days, more preferably 2 days to 3 days), Ct-SLCO1B3 contained in the cell or the mRNA encoding the same is added to the above (i ) Can be quantified and analyzed in the same manner.
　Ct-SLCO1B3遺伝子（mRNA）の発現レベルの検出及び定量は、前記細胞から調製したRNA又はそれから転写された相補的なポリヌクレオチドを用いて、ノーザンブロット法、RT-PCR法など公知の方法で実施できる。具体的には、Ct-SLCO1B3遺伝子の塩基配列において連続する少なくとも15塩基を有するポリヌクレオチド及び／又はその相補的なポリヌクレオチドをプライマー又はプローブとして用いることによって、RNA中のCt-SLCO1B3遺伝子の発現の有無やその発現レベルを検出、測定することができる。そのようなプローブもしくはプライマーは、Ct-SLCO1B3遺伝子の塩基配列をもとに、例えばprimer 3（http://www.genome.wi.mit.edu/cgi-bin/primer/primer3.cgi）あるいはベクターNTI（Infomax社製）を利用して設計することができる。 Detection and quantification of the expression level of Ct-SLCO1B3 gene (mRNA) is carried out by a known method such as Northern blotting or RT-PCR using RNA prepared from the cells or a complementary polynucleotide transcribed therefrom. it can. Specifically, by using a polynucleotide having at least 15 bases continuous in the base sequence of the Ct-SLCO1B3 gene and / or a complementary polynucleotide thereof as a primer or probe, the expression of the Ct-SLCO1B3 gene in RNA can be detected. Presence / absence and its expression level can be detected and measured. Such a probe or primer is based on the base sequence of the Ct-SLCO1B3 gene, for example, primer 3 (http://www.genome.wi.mit.edu/cgi-bin/primer/primer3.cgi) or a vector It can be designed using NTI (Infomax).
　ノーザンブロット法を利用する場合、前記プライマーもしくはプローブを放射性同位元素（32P、33Pなど：RI）や蛍光物質などで標識し、それを、常法に従ってナイロンメンブレン等にトランスファーした細胞由来のRNAとハイブリダイズさせた後、形成された前記プライマーもしくはプローブ（DNA又はRNA）とRNAとの二重鎖を、前記プライマーもしくはプローブの標識物（RI若しくは蛍光物質）に由来するシグナルとして放射線検出器（BAS-1800II、富士フィルム社製）又は蛍光検出器で検出、測定する方法を例示することができる。また、AlkPhos Direct Labelling and Detection System （Amersham PharamciaBiotech社製）を用いて、該プロトコールに従って前記プローブを標識し、細胞由来のRNAとハイブリダイズさせた後、前記プローブの標識物に由来するシグナルをマルチバイオイメージャーSTORM860（Amersham Pharmacia Biotech社製）で検出、測定する方法を使用することもできる。 When using Northern blotting, the primer or probe is labeled with a radioisotope (32P, 33P, etc .: RI) or a fluorescent substance and hybridized with cell-derived RNA transferred to a nylon membrane or the like according to a conventional method. After soy, the formed duplex of the primer or probe (DNA or RNA) and RNA is used as a signal from the primer or probe label (RI or fluorescent material) as a radiation detector (BAS- 1800II (manufactured by Fuji Film) or a method of detecting and measuring with a fluorescence detector can be exemplified. In addition, using AlkPhos Direct Labelling and Detection System (Amersham Pharamcia Biotech), the probe is labeled according to the protocol, hybridized with cell-derived RNA, and then the signal derived from the probe label is expressed in multibioin. A method of detecting and measuring with a major STORM860 (manufactured by Amersham Pharmacia Biotech) can also be used.
　RT-PCR法を利用する場合は、細胞由来のRNAから常法に従ってcDNAを調製して、これを鋳型として標的のCt-SLCO1B3遺伝子の領域が増幅できるように、Ct-SLCO1B3遺伝子の配列に基づき調製した一対のプライマーをこれとハイブリダイズさせて、常法に従ってPCR法を行い、得られた増幅二本鎖DNAを検出する方法を例示することができる。なお、増幅された二本鎖DNAの検出は、上記PCRを予めRIや蛍光物質で標識しておいたプライマーを用いて行うことによって産生される標識二本鎖DNAを検出する方法、産生された二本鎖DNAを常法に従ってナイロンメンブレン等にトランスファーさせて、標識した前記プライマーをプローブとして使用してこれとハイブリダイズさせて検出する方法などを用いることができる。なお、生成された標識二本鎖DNA産物はアジレント2100バイオアナライザ（横河アナリティカルシステムズ社製）などで測定することができる。また、SYBR Green RT-PCR Reagents （Applied Biosystems社製）で該プロトコールに従ってRT-PCR反応液を調製し、ABI PRIME 7900 Sequence Detection System （Applied Biosystems社製）で反応させて、該反応物を検出することもできる。 When using the RT-PCR method, cDNA is prepared from cell-derived RNA according to a conventional method, and based on this, the target Ct-SLCO1B3 gene region can be amplified using this as a template, based on the sequence of the Ct-SLCO1B3 gene. An example is a method in which a pair of prepared primers are hybridized with this, followed by PCR according to a conventional method, and detection of the resulting amplified double-stranded DNA. The detection of the amplified double-stranded DNA was performed by a method for detecting the labeled double-stranded DNA produced by performing the PCR using a primer previously labeled with RI or a fluorescent substance. For example, a method can be used in which double-stranded DNA is transferred to a nylon membrane or the like according to a conventional method, and the labeled primer is used as a probe to hybridize with this to detect it. The produced labeled double-stranded DNA product can be measured with an Agilent 2100 Bioanalyzer (manufactured by Yokogawa Analytical Systems). Also, prepare an RT-PCR reaction solution according to the protocol using SYBR Green RT-PCR Reagents (Applied Biosystems) and react with ABI PRIME 7900 Sequence Detection System (Applied Biosystems) to detect the reaction product. You can also.
　被検物質を添加した細胞におけるCt-SLCO1B3遺伝子の発現が被検物質を添加しない対照細胞での発現量と比較して約70％以下、好ましくは約50%以下、より好ましくは約30%以下、更に好ましくは約20%以下、特に好ましくは約10%以下であれば、該被検物質はCt-SLCO1B3遺伝子の発現抑制物質として選択することができる。 Expression of Ct-SLCO1B3 gene in cells added with test substance is about 70% or less, preferably about 50% or less, more preferably about 30% or less compared to the expression level in control cells not containing test substance Furthermore, if it is more preferably about 20% or less, particularly preferably about 10% or less, the test substance can be selected as a Ct-SLCO1B3 gene expression inhibitor.
　Ct-SLCO1B3のタンパク質量の測定方法としては、具体的には、例えば、
（i）検出用抗体と、試料液及び標識化されたCt-SLCO1B3とを競合的に反応させ、該抗体に結合した標識化されたタンパク質を検出することにより試料液中のCt-SLCO1B3を定量する方法や、
（ii）試料液と、担体上に不溶化した検出用抗体及び標識化された別の検出用抗体とを、同時あるいは連続的に反応させた後、不溶化担体上の標識剤の量（活性）を測定することにより、試料液中のCt-SLCO1B3を定量する方法等が挙げられる。 As a method for measuring the protein amount of Ct-SLCO1B3, specifically, for example,
(I) Ct-SLCO1B3 in the sample solution is quantified by competitively reacting the detection antibody with the sample solution and labeled Ct-SLCO1B3 and detecting the labeled protein bound to the antibody. How to do,
(Ii) After reacting the sample solution with the detection antibody insolubilized on the carrier and another labeled detection antibody simultaneously or successively, the amount (activity) of the labeling agent on the insolubilized carrier is determined. A method of quantifying Ct-SLCO1B3 in the sample solution by measuring it may be mentioned.
　Ct-SLCO1B3のタンパク質発現レベルの検出及び定量は、Ct-SLCO1B3を認識する抗体を用いたウェスタンブロット法等の公知方法に従って定量できる。ウェスタンブロット法は、一次抗体としてCt-SLCO1B3を認識する抗体を用いた後、二次抗体として125Iなどの放射性同位元素、蛍光物質、ホースラディッシュペルオキシダーゼ（HRP）等の酵素等で標識した一次抗体に結合する抗体を用いて標識し、これら標識物質由来のシグナルを放射線測定器（BAI-1800II：富士フィルム社製など）、蛍光検出器などで測定することによって実施できる。また、一次抗体としてCt-SLCO1B3を認識する抗体を用いた後、ECL Plus Western Blotting Detection System（アマシャム　ファルマシアバイオテク社製）を利用して該プロトコールに従って検出し、マルチバイオメージャーSTORM860（アマシャム　ファルマシアバイオテク社製）で測定することもできる。 Detection and quantification of the protein expression level of Ct-SLCO1B3 can be quantified according to a known method such as Western blotting using an antibody recognizing Ct-SLCO1B3. Western blotting uses an antibody that recognizes Ct-SLCO1B3 as the primary antibody, followed by a primary antibody labeled with a radioisotope such as 125 I, a fluorescent substance, or an enzyme such as horseradish peroxidase (HRP) as the secondary antibody. It can be carried out by labeling with an antibody that binds to, and measuring the signal derived from these labeling substances with a radiation measuring instrument (BAI-1800II: manufactured by Fuji Film Co., Ltd.), a fluorescence detector, or the like. In addition, after using an antibody that recognizes Ct-SLCO1B3 as a primary antibody, detection is performed according to the protocol using ECL Plus Western Blotting Detection System (Amersham Pharmacia Biotech), and multi-biomeasuring STORM860 (Amersham Pharmacia Biotech) ).
　上記の検出用抗体は、その形態に特に制限はなく、Ct-SLCO1B3を免疫抗原とするポリクローナル抗体であっても、またそのモノクローナル抗体であってもよく、さらにはCt-SLCO1B3を構成するアミノ酸配列のうち少なくとも連続する、通常8アミノ酸、好ましくは15アミノ酸、より好ましくは20アミノ酸からなるポリペプチドに対して抗原結合性を有する抗体を用いることもできる。
　これらの抗体の製造方法は、すでに周知であり、これらの常法に従って抗体を製造することができる（Current protocols in Molecular Biology edit. Ausubel et al.(1987)Publish. John Wiley and Sons. Section 11.12~11.13）。
　また、検出用抗体は、Ct-SLCO1B3及びLt-SLCO1B3のいずれをも認識する抗体であってもよい。Ct-SLCO1B3及びLt-SLCO1B3のいずれをも認識する抗体を用いる場合、Ct-SLCO1B3及びLt-SLCO1B3を区別するため、さらに生化学的性質、物理学的性質などにより、両者を区別する工程を含めても良い。例えば、試料液をSDS-PAGE電気泳動又はクロマトグラフィーなどを組み合わせることにより、タンパク質のサイズの違いからCt-SLCO1B3及びLt-SLCO1B3を区別することができる。 The form of the above-mentioned detection antibody is not particularly limited, and may be a polyclonal antibody having Ct-SLCO1B3 as an immunizing antigen or a monoclonal antibody thereof, and further an amino acid sequence constituting Ct-SLCO1B3 Among them, an antibody having an antigen binding property to a polypeptide consisting of at least continuous, usually 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids can also be used.
Methods for producing these antibodies are already well known, and antibodies can be produced according to these conventional methods (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.12- 11.13).
Further, the detection antibody may be an antibody that recognizes both Ct-SLCO1B3 and Lt-SLCO1B3. When using an antibody that recognizes both Ct-SLCO1B3 and Lt-SLCO1B3, in order to distinguish between Ct-SLCO1B3 and Lt-SLCO1B3, a process for distinguishing both of them according to biochemical and physical properties is included. May be. For example, Ct-SLCO1B3 and Lt-SLCO1B3 can be distinguished from the difference in protein size by combining the sample solution with SDS-PAGE electrophoresis or chromatography.
　上記（ii）の定量法においては、2種の抗体はCt-SLCO1B3の異なる部分を認識するものであることが望ましい。例えば、一方の抗体がCt-SLCO1B3のN端部を認識する抗体であれば、他方の抗体として該タンパク質のC端部と反応するものを用いることができる。
　標識物質を用いる測定法に用いられる標識剤としては、例えば、放射性同位元素、酵素、蛍光物質、発光物質などが用いられる。放射性同位元素としては、例えば、〔125I〕、〔131I〕、〔3H〕、〔14C〕などが用いられる。上記酵素としては、安定で比活性の大きなものが好ましく、例えば、β－ガラクトシダーゼ、β－グルコシダーゼ、アルカリフォスファターゼ、パーオキシダーゼ、リンゴ酸脱水素酵素などが用いられる。蛍光物質としては、例えば、フルオレスカミン、フルオレッセンイソチオシアネートなどが用いられる。発光物質としては、例えば、ルミノール、ルミノール誘導体、ルシフェリン、ルシゲニンなどが用いられる。さらに、抗体あるいは抗原と標識剤との結合にビオチン－（ストレプト）アビジン系を用いることもできる。 In the quantification method (ii) above, it is desirable that the two antibodies recognize different portions of Ct-SLCO1B3. For example, if one antibody recognizes the N-terminal part of Ct-SLCO1B3, one that reacts with the C-terminal part of the protein can be used as the other antibody.
As a labeling agent used in a measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or the like is used. As the radioisotope, for example, [ 125 I], [ 131 I], [ 3 H], [ 14 C] and the like are used. The enzyme is preferably stable and has a large specific activity. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used. Furthermore, a biotin- (strept) avidin system can be used for binding of an antibody or antigen to a labeling agent.
　検出用抗体を用いるCt-SLCO1B3の定量法は、特に制限されるべきものではなく、試料液中の抗原量に対応した、抗体、抗原もしくは抗体－抗原複合体の量を化学的又は物理的手段により検出し、これを既知量の抗原を含む標準液を用いて作製した標準曲線より算出する測定法であれば、いずれの測定法を用いてもよい。例えば、ネフロメトリー、競合法、イムノメトリック法及びサンドイッチ法が好適に用いられる。感度、特異性の点で、例えば、後述するサンドイッチ法を用いるのが好ましい。 The Ct-SLCO1B3 quantification method using the detection antibody is not particularly limited, and the amount of the antibody, antigen or antibody-antigen complex corresponding to the amount of antigen in the sample solution is chemically or physically measured. Any measurement method may be used as long as it is a measurement method that is detected from the above and calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, competition method, immunometric method and sandwich method are preferably used. In view of sensitivity and specificity, for example, the sandwich method described later is preferably used.
　抗原あるいは抗体の不溶化にあたっては、物理吸着を用いてもよく、また通常タンパク質あるいは酵素等を不溶化・固定化するのに用いられる化学結合を用いてもよい。担体としては、アガロース、デキストラン、セルロースなどの不溶性多糖類、ポリスチレン、ポリアクリルアミド、シリコン等の合成樹脂、あるいはガラス等があげられる。 In the insolubilization of the antigen or antibody, physical adsorption may be used, or a chemical bond usually used for insolubilizing / immobilizing proteins or enzymes may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or glass.
　サンドイッチ法においては不溶化した検出用抗体に試料液を反応させ（1次反応）、さらに標識化した別の検出用抗体を反応させた（2次反応）後、不溶化担体上の標識剤の量もしくは活性を測定することにより、試料液中のCt-SLCO1B3を定量することができる。1次反応と2次反応は逆の順序で行っても、また、同時に行ってもよいし、時間をずらして行ってもよい。標識化剤及び不溶化の方法は前記のそれらに準じることができる。また、サンドイッチ法による免疫測定法において、固相化抗体あるいは標識化抗体に用いられる抗体は必ずしも1種類である必要はなく、測定感度を向上させる等の目的で2種類以上の抗体の混合物を用いてもよい。 In the sandwich method, the sample solution is reacted with the insolubilized detection antibody (primary reaction), and further reacted with another labeled detection antibody (secondary reaction), and then the amount of labeling agent on the insolubilized carrier or By measuring the activity, Ct-SLCO1B3 in the sample solution can be quantified. The primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at different times. The labeling agent and the insolubilizing method can be the same as those described above. Also, in the immunoassay method by the sandwich method, the antibody used for the immobilized antibody or labeled antibody is not necessarily one type, and a mixture of two or more types of antibodies is used for the purpose of improving measurement sensitivity. May be.
　検出用抗体は、サンドイッチ法以外の測定システム、例えば、競合法、イムノメトリック法あるいはネフロメトリーなどにも用いることができる。
　競合法では、試料液中のCt-SLCO1B3と標識したCt-SLCO1B3とを抗体に対して競合的に反応させた後、未反応の標識抗原（F）と、抗体と結合した標識抗原（B）とを分離し（B/F分離）、B、Fいずれかの標識量を測定することにより、試料液中のCt-SLCO1B3を定量する。本反応法には、抗体として可溶性抗体を用い、ポリエチレングリコールや前記抗体（1次抗体）に対する2次抗体などを用いてＢ／Ｆ分離を行う液相法、及び、1次抗体として固相化抗体を用いるか（直接法）、あるいは１次抗体は可溶性のものを用い、2次抗体として固相化抗体を用いる（間接法）固相化法とが用いられる。
　イムノメトリック法では、試料液中のCt-SLCO1B3と固相化したCt-SLCO1B3とを一定量の標識化抗体に対して競合反応させた後、固相と液相を分離するか、あるいは試料液中のCt-SLCO1B3と過剰量の標識化抗体とを反応させ、次に固相化したCt-SLCO1B3を加えて未反応の標識化抗体を固相に結合させた後、固相と液相を分離する。次に、いずれかの相の標識量を測定し試料液中の抗原量を定量する。
　また、ネフロメトリーでは、ゲル内あるいは溶液中で抗原抗体反応の結果生じた不溶性の沈降物の量を測定する。試料液中のCt-SLCO1B3の量がわずかであり、少量の沈降物しか得られない場合にもレーザーの散乱を利用するレーザーネフロメトリーなどが好適に用いられる。 The detection antibody can also be used in measurement systems other than the sandwich method, such as a competitive method, an immunometric method, or nephrometry.
In the competitive method, Ct-SLCO1B3 in the sample solution and labeled Ct-SLCO1B3 are reacted with the antibody competitively, then the unreacted labeled antigen (F) and the labeled antigen bound to the antibody (B) Is separated (B / F separation), and the amount of labeling of either B or F is measured to quantify Ct-SLCO1B3 in the sample solution. In this reaction method, a soluble antibody is used as an antibody, B / F separation is performed using polyethylene glycol or a secondary antibody against the antibody (primary antibody), and a solid phase is used as the primary antibody. Either an antibody is used (direct method), or a primary antibody is soluble, and a solid phase antibody is used as a secondary antibody (indirect method).
In the immunometric method, Ct-SLCO1B3 in the sample solution and Ct-SLCO1B3 immobilized on the solid phase are allowed to compete with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated or the sample solution is separated. Ct-SLCO1B3 and an excess amount of labeled antibody are reacted, and then solid-phased Ct-SLCO1B3 is added to bind the unreacted labeled antibody to the solid phase. To separate. Next, the amount of label in any phase is measured to quantify the amount of antigen in the sample solution.
In nephrometry, the amount of insoluble precipitate produced as a result of the antigen-antibody reaction in a gel or solution is measured. Laser nephrometry using laser scattering is preferably used even when the amount of Ct-SLCO1B3 in the sample solution is small and only a small amount of precipitate is obtained.
　これら個々の免疫学的測定法を本発明の定量方法に適用するにあたっては、特別の条件、操作等の設定は必要とされない。それぞれの方法における通常の条件、操作法に当業者の通常の技術的配慮を加えてCt-SLCO1B3の測定系を構築すればよい。これらの一般的な技術手段の詳細については、総説、成書などを参照することができる。
　例えば、入江　寛編「ラジオイムノアッセイ」（講談社、昭和49年発行）、入江　寛編「続ラジオイムノアッセイ」（講談社、昭和54年発行）、石川栄治ら編「酵素免疫測定法」（医学書院、昭和53年発行）、石川栄治ら編「酵素免疫測定法」（第2版）（医学書院、昭和57年発行）、石川栄治ら編「酵素免疫測定法」（第3版）（医学書院、昭和62年発行）、「Methods in ENZYMOLOGY」Vol.70(Immunochemical Techniques(Part A))、同書Vol.73(Immunochemical Techniques(Part B))、同書Vol.74(Immunochemical Techniques (Part C))、同書Vol.84(Immunochemical Techniques(Part D:Selected Immunoassays))、同書Vol.92(Immunochemical Techniques(Part E:Monoclonal Antibodies and General Immunoassay Methods))、同書Vol.121(Immunochemical Techniques(Part I:Hybridoma Technology and Monoclonal Antibodies))（以上、アカデミックプレス社発行）などを参照することができる。
　以上のようにして、検出用抗体を用いることによって、細胞におけるCt-SLCO1B3の量を感度よく定量することができる。 In applying these individual immunological measurement methods to the quantification method of the present invention, special conditions, operations and the like are not required to be set. A measurement system for Ct-SLCO1B3 may be constructed by adding ordinary technical considerations to those skilled in the art to the usual conditions and operation methods in each method. For details of these general technical means, it is possible to refer to reviews, books and the like.
For example, Hiroshi Irie “Radioimmunoassay” (Kodansha, published in 1974), Hiroshi Irie “Continue Radioimmunoassay” (published in Kodansha, 1974), “Enzyme Immunoassay” edited by Eiji Ishikawa et al. 53), "Enzyme Immunoassay" edited by Eiji Ishikawa et al. (2nd edition) (Medical School, published in 1982), "Enzyme Immunoassay" edited by Eiji Ishikawa et al. (3rd edition) (Medical School, Showa) 62)), `` Methods in ENZYMOLOGY '' Vol. 70 (Immunochemical Techniques (Part A)), Id.Vol. 73 (Immunochemical Techniques (Part B)), Id. Vol. 74 (Immunochemical Techniques (Part C)) .84 (Immunochemical Techniques (Part D: Selected Immunoassays)), ibid.Vol.92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), ibid. )) (Above, published by Academic Press) wear.
As described above, the amount of Ct-SLCO1B3 in the cell can be quantified with high sensitivity by using the detection antibody.
　例えば、上記スクリーニング法において、被検物質の存在下におけるCt-SLCO1B3の発現量（mRNA量又はタンパク質量）が、被検物質の非存在下における場合に比べて、約20%以上、好ましくは約30%以上、より好ましくは約50%以上阻害された場合、該被検物質を、Ct-SLCO1B3の発現抑制物質、従って、癌の治療又は予防活性を有する物質の候補として選択することができる。 For example, in the above screening method, the expression level (mRNA level or protein level) of Ct-SLCO1B3 in the presence of the test substance is about 20% or more, preferably about 20% or more compared to that in the absence of the test substance. When inhibition is 30% or more, more preferably about 50% or more, the test substance can be selected as a Ct-SLCO1B3 expression-suppressing substance, and thus a candidate for a substance having cancer therapeutic or prophylactic activity.
（Ct-SLCO1B3の機能の測定）
　本発明のスクリーニング方法は、被検物質がCt-SLCO1B3の機能を抑制するか否かを指標として行うこともできる。
　例えば、Ct-SLCO1B3の有する下記（1）～（8）の機能を指標に、癌の悪性化の抑制、浸潤抑制など、Ct-SLCO1B3の発現に特徴づけられる癌の治療及び／又は予防活性を有する活性を有する物質のスクリーニングのためのツールとして用いることができる。
（1） 細胞の足場非依存性増殖促進
（2）細胞の遊走能促進
（3）細胞の浸潤能促進
（4）snail発現量の上昇
（5）slug発現量の上昇
（6）E-cadherin発現量の低下
（7）occludin発現量の低下
（8）MMP9発現量の上昇
　Ct-SLCO1B3の機能を阻害するとは、上述の（1）～（8）からなる群より選択されるCt-SLCO1B3の機能のうち、１つ以上の機能を阻害すればよく、好ましくは2つ以上、3つ以上、4つ以上、5つ以上、6つ以上、7つ以上、又は8つ全ての機能を阻害する。 (Measurement of Ct-SLCO1B3 function)
The screening method of the present invention can also be performed using as an index whether or not a test substance suppresses the function of Ct-SLCO1B3.
For example, using the following functions (1) to (8) of Ct-SLCO1B3 as indicators, the therapeutic and / or prophylactic activity of cancers characterized by the expression of Ct-SLCO1B3, such as suppression of cancer malignancy and suppression of invasion It can be used as a tool for screening a substance having an activity.
(1) Cell anchorage-independent growth promotion (2) Cell migration ability promotion (3) Cell invasion ability promotion (4) Snail expression increase (5) Slug expression increase (6) E-cadherin expression Reduced amount (7) Decreased occludin expression level (8) Increased MMP9 expression level To inhibit Ct-SLCO1B3 function, Ct-SLCO1B3 function selected from the group consisting of (1) to (8) above Of these, one or more functions may be inhibited, and preferably two or more, three or more, four or more, five or more, six or more, seven or more, or all eight functions are inhibited.
　（1）細胞の足場非依存性増殖促進
　具体的には、Ct-SLCO1B3を発現する細胞において、被検物質を添加することにより、該細胞の足場非依存性増殖が抑制されるか否かを測定することにより実施することができる。例えば、被検物質存在下におけるCt-SLCO1B3を発現する細胞の足場非依存性増殖が、被検物質の非存在下における細胞の足場非依存性増殖に比べて、約10%以上、好ましくは約20%以上、より好ましくは約30%以上、更に好ましくは約50%以上阻害された場合に、該被検物質を、Ct-SLCO1B3の機能抑制物質、従って、癌の治療及び／又は予防活性を有する物質の候補として選択することができる。
　Ct-SLCO1B3を発現する細胞としては、下記1）又は2）が好適に用いられる。
1）Ct-SLCO1B3非発現細胞にCt-SLCO1B3を発現させた細胞であって、親株であるCt-SLCO1B3非発現細胞と比較して、細胞の足場非依存性増殖が亢進した細胞
2）Ct-SLCO1B3発現細胞であって、Ct-SLCO1B3の発現を抑制させた場合に親株であるCt-SLCO1B3発現細胞と比較して、細胞の足場非依存性増殖が抑制される細胞
　細胞の足場非依存性増殖とは、コラーゲンなどの細胞外マトリックス非存在下における増殖能を意味する。細胞の足場非依存性増殖は、例えば、実施例９に記載の方法に準じて評価することができる。
　インビボにおいては細胞の足場非依存性増殖は、例えば、A549細胞をBalb/c slc-nu/nuマウスに背部皮下移植したマウスにおいて、A549細胞の形成する腫瘍の大きさを計測することによって測定することもできる。 (1) Promoting anchorage-independent growth of cells Specifically, in cells expressing Ct-SLCO1B3, whether or not anchorage-independent growth of the cells is suppressed by adding a test substance. It can be implemented by measuring. For example, the anchorage-independent growth of cells expressing Ct-SLCO1B3 in the presence of the test substance is about 10% or more, preferably about 10% or more compared to the anchorage-independent growth of cells in the absence of the test substance. When the test substance is inhibited by 20% or more, more preferably about 30% or more, and still more preferably about 50% or more, the test substance is treated with a Ct-SLCO1B3 function inhibitory substance, and thus has a therapeutic and / or prophylactic activity for cancer. It can be selected as a candidate for the substance to have.
As the cells expressing Ct-SLCO1B3, the following 1) or 2) is preferably used.
1) Cells in which Ct-SLCO1B3 is expressed in cells that do not express Ct-SLCO1B3, and cells whose anchorage-independent growth of cells is enhanced compared to cells that do not express Ct-SLCO1B3
2) Cells that are Ct-SLCO1B3-expressing cells, and when Ct-SLCO1B3 expression is suppressed, cells that inhibit the anchorage-independent growth of cells compared to the parent Ct-SLCO1B3-expressing cells Independent growth means the ability to grow in the absence of an extracellular matrix such as collagen. The anchorage-independent growth of cells can be evaluated according to the method described in Example 9, for example.
In vivo cell-independent growth of cells is measured, for example, by measuring the size of tumors formed by A549 cells in mice transplanted subcutaneously in the back to Balb / c slc-nu / nu mice. You can also.
　（2）遊走能促進
　具体的には、Ct-SLCO1B3を発現する細胞において、被検物質を添加することにより、該細胞の遊走能が抑制されるか否かを測定することにより実施することができる。例えば、被検物質存在下におけるCt-SLCO1B3を発現する細胞の遊走能が、被検物質の非存在下における細胞の遊走能に比べて、約10%以上、好ましくは約20%以上、より好ましくは約30%以上、更に好ましくは約50%以上阻害された場合に、該被検物質を、Ct-SLCO1B3の機能抑制物質、従って、癌の治療及び／又は予防活性を有する物質の候補として選択することができる。
　Ct-SLCO1B3を発現する細胞としては、下記1）又は2）が好適に用いられる。
1）Ct-SLCO1B3非発現細胞にCt-SLCO1B3を発現させた細胞であって、親株であるCt-SLCO1B3非発現細胞と比較して、細胞の遊走能が亢進した細胞
2）Ct-SLCO1B3発現細胞であって、Ct-SLCO1B3の発現を抑制させた場合に親株であるCt-SLCO1B3発現細胞と比較して、細胞の遊走能が抑制される細胞
　細胞の遊走能は、自体公知の方法によってアッセイすることができる。例えば、実施例１０に記載の方法に準じて評価することができる。細胞の遊走能は、例えば細胞を播種しコンフルエントになるまで培養した後に、ピペットチップの先で細胞の一部を剥ぎ取り、一定時間後（例えば、6時間後、12時間後、24時間後など）の細胞遊走面積を計測することで測定することができる。 (2) Promotion of migration ability Specifically, in cells expressing Ct-SLCO1B3, it can be carried out by measuring whether the migration ability of the cells is suppressed by adding a test substance. it can. For example, the migration ability of a cell expressing Ct-SLCO1B3 in the presence of a test substance is about 10% or more, preferably about 20% or more, more preferably compared to the migration ability of a cell in the absence of the test substance. Is selected as a candidate for a Ct-SLCO1B3 function inhibitor, and thus a substance having a therapeutic and / or prophylactic activity for cancer, when it is inhibited by about 30% or more, more preferably about 50% or more can do.
As the cells expressing Ct-SLCO1B3, the following 1) or 2) is preferably used.
1) Cells in which Ct-SLCO1B3 is expressed in Ct-SLCO1B3 non-expressing cells, which have enhanced cell migration ability compared to the parent strain Ct-SLCO1B3 non-expressing cells
2) Ct-SLCO1B3-expressing cells, and when Ct-SLCO1B3 expression is suppressed, compared to the parent cell line Ct-SLCO1B3-expressing cells It can be assayed by a method known per se. For example, evaluation can be performed according to the method described in Example 10. For example, after cell seeding and culturing until confluent, a part of the cells is peeled off at the tip of the pipette tip, and after a certain time (for example, 6 hours, 12 hours, 24 hours, etc.) ) To measure the cell migration area.
　（3）浸潤能促進
　具体的には、Ct-SLCO1B3を発現する細胞において、被検物質を添加することにより、該細胞の浸潤能が抑制されるか否かを測定することにより実施することができる。例えば、被検物質存在下におけるCt-SLCO1B3を発現する細胞の浸潤能が、被検物質の非存在下における細胞の浸潤能に比べて、約10%以上、好ましくは約20%以上、より好ましくは約30%以上、更に好ましくは約50%以上阻害された場合に、該被検物質を、Ct-SLCO1B3の機能抑制物質、従って、癌の治療及び／又は予防活性を有する物質の候補として選択することができる。
　Ct-SLCO1B3を発現する細胞としては、下記1）又は2）が好適に用いられる。
1）Ct-SLCO1B3非発現細胞にCt-SLCO1B3を発現させた細胞であって、親株であるCt-SLCO1B3非発現細胞と比較して、細胞の浸潤能が亢進した細胞
2）Ct-SLCO1B3発現細胞であって、Ct-SLCO1B3の発現を抑制させた場合に親株であるCt-SLCO1B3発現細胞と比較して、細胞の浸潤能が抑制される細胞 (3) Promotion of invasive ability Specifically, in cells expressing Ct-SLCO1B3, it can be carried out by measuring whether or not the invasive ability of the cells is suppressed by adding a test substance. it can. For example, the invasion ability of cells expressing Ct-SLCO1B3 in the presence of the test substance is about 10% or more, preferably about 20% or more, more preferably compared to the invasion ability of the cells in the absence of the test substance. Is selected as a candidate for a Ct-SLCO1B3 function inhibitor, and thus a substance having a therapeutic and / or prophylactic activity for cancer, when it is inhibited by about 30% or more, more preferably about 50% or more can do.
As the cells expressing Ct-SLCO1B3, the following 1) or 2) is preferably used.
1) Cells in which Ct-SLCO1B3 is expressed in cells that do not express Ct-SLCO1B3, and in which the invasion capacity of cells is enhanced compared to cells that do not express Ct-SLCO1B3
2) Ct-SLCO1B3-expressing cells that suppress the invasive ability of the cells when Ct-SLCO1B3 expression is suppressed compared to the parent Ct-SLCO1B3-expressing cells
　細胞の浸潤能とは、組織外へ移動する能力を意味する。細胞の浸潤能は、自体公知の方法によってアッセイすることができる。例えば、癌細胞のインビトロにおける浸潤能アッセイは、例えば、CIM-Plate16などのトランスウェルチャンバーを用いて行うことができる。細胞の播種前にあらかじめ上部チャンバーの膜上部をマトリゲルによって覆っておき、細胞を播種し培養後、下部チャンバーに浸潤した細胞数を計測すること等により測定ができる。
　細胞の浸潤能は、例えば、実施例１１に記載の方法に準じて評価することができる。 The invasive ability of cells means the ability to move out of the tissue. The invasive ability of cells can be assayed by a method known per se. For example, an in vitro invasive ability assay of cancer cells can be performed using a transwell chamber such as CIM-Plate16. Before the seeding of cells, the upper part of the membrane of the upper chamber is covered with Matrigel in advance, and after seeding and culturing the cells, the number of cells infiltrating the lower chamber can be measured.
The invasive ability of cells can be evaluated according to the method described in Example 11, for example.
　（4）snail発現量の上昇
　具体的には、Ct-SLCO1B3を発現する細胞において、被検物質を添加することにより、該細胞のsnail発現量が低下するか否かを測定することにより実施することができる。例えば、被検物質存在下におけるCt-SLCO1B3を発現する細胞のsnail発現量が、被検物質の非存在下における細胞の浸潤能に比べて、約70%以下、好ましくは約50%以下、より好ましくは約30%以下、更に好ましくは約20%以下、特に好ましくは約10%以下に低下した場合に、該被検物質を、Ct-SLCO1B3の機能抑制物質、従って、癌の治療及び／又は予防活性を有する物質の候補として選択することができる。
　Ct-SLCO1B3を発現する細胞としては、下記1) 又は2) が好適に用いられる。
1）Ct-SLCO1B3非発現細胞にCt-SLCO1B3を発現させた細胞であって、親株であるCt-SLCO1B3非発現細胞と比較して、snail発現量が約10%以上、好ましくは約20%以上、より好ましくは約30%以上、更に好ましくは約50%以上上昇した細胞
2）Ct-SLCO1B3発現細胞であって、Ct-SLCO1B3の発現を抑制させた場合に親株であるCt-SLCO1B3発現細胞と比較して、snail発現量が約70%以下、好ましくは約50%以下、より好ましくは約30%以下、更に好ましくは約20%以下、特に好ましくは約10%以下に低下した細胞
　遺伝子の発現量を比較する方法は上述のとおりである。例えば、snail発現量は実施例１３に記載の方法に準じて比較することができる。
　従って、より具体的には、本発明は、
（a）Ct-SLCO1B3を産生する能力を有する細胞を被検物質の存在下又は非存在下に培養し、両条件下におけるsnailタンパク質をコードするmRNAの量を、該mRNA検出用核酸を用いて測定、比較することを特徴とする、癌の治療及び／又は予防活性を有する物質のスクリーニング方法、及び
（b）Ct-SLCO1B3を産生する能力を有する細胞を被検物質の存在下又は非存在下に培養し、両条件下におけるsnailタンパク質の量を、snailタンパク質に対する抗体を用いて測定、比較することを特徴とする、癌の治療及び／又は予防活性を有する物質のスクリーニング方法を提供する。 (4) Increase in snail expression level Specifically, in cells expressing Ct-SLCO1B3, it is carried out by measuring whether or not the snail expression level in the cells decreases by adding a test substance be able to. For example, the snail expression level of cells expressing Ct-SLCO1B3 in the presence of the test substance is about 70% or less, preferably about 50% or less, compared to the invasion capacity of the cells in the absence of the test substance. Preferably, when the test substance is reduced to about 30% or less, more preferably about 20% or less, and particularly preferably about 10% or less, the test substance is treated as a Ct-SLCO1B3 function inhibitor, and thus cancer treatment and / or It can be selected as a candidate for a substance having prophylactic activity.
As the cell expressing Ct-SLCO1B3, the following 1) or 2) is preferably used.
1) A cell in which Ct-SLCO1B3 is expressed in a Ct-SLCO1B3 non-expressing cell, and the snail expression level is about 10% or more, preferably about 20% or more compared to the parent strain Ct-SLCO1B3 non-expressing cell More preferably about 30% or more, more preferably about 50% or more
2) Ct-SLCO1B3-expressing cells, when the expression of Ct-SLCO1B3 is suppressed, the snail expression level is about 70% or less, preferably about 50% or less compared to the parent Ct-SLCO1B3-expressing cell More preferably, the method for comparing the expression levels of cellular genes reduced to about 30% or less, more preferably about 20% or less, and particularly preferably about 10% or less is as described above. For example, the expression level of snail can be compared according to the method described in Example 13.
Therefore, more specifically, the present invention
(A) Cells having the ability to produce Ct-SLCO1B3 are cultured in the presence or absence of a test substance, and the amount of mRNA encoding the snail protein under both conditions is determined using the nucleic acid for mRNA detection. A method for screening a substance having a therapeutic and / or prophylactic activity for cancer, characterized by measuring and comparing, and (b) a cell capable of producing Ct-SLCO1B3 in the presence or absence of a test substance And a method for screening a substance having cancer therapeutic and / or prophylactic activity, comprising measuring and comparing the amount of snail protein under both conditions using an antibody against the snail protein.
　（5）slug発現量の上昇
　具体的には、Ct-SLCO1B3を発現する細胞において、被検物質を添加することにより、該細胞のslug発現量が低下するか否かを測定することにより実施することができる。例えば、被検物質存在下におけるCt-SLCO1B3を発現する細胞のslug発現量が、被検物質の非存在下における細胞の浸潤能に比べて、約70%以下、好ましくは約50%以下、より好ましくは約30%以下、更に好ましくは約20%以下、特に好ましくは約10%以下に低下した場合に、該被検物質を、Ct-SLCO1B3の機能抑制物質、従って、癌の治療及び／又は予防活性を有する物質の候補として選択することができる。
　Ct-SLCO1B3を発現する細胞としては、下記1）又は2）が好適に用いられる。
1）Ct-SLCO1B3非発現細胞にCt-SLCO1B3を発現させた細胞であって、親株であるCt-SLCO1B3非発現細胞と比較して、slug発現量が約10%以上、好ましくは約20%以上、より好ましくは約30%以上、更に好ましくは約50%以上上昇した細胞
2）Ct-SLCO1B3発現細胞であって、Ct-SLCO1B3の発現を抑制させた場合に親株であるCt-SLCO1B3発現細胞と比較して、slug発現量が約70%以下、好ましくは約50%以下、より好ましくは約30%以下、更に好ましくは約20%以下、特に好ましくは約10%以下に低下した細胞
　遺伝子の発現量を比較する方法は上述のとおりである。例えば、slug発現量は実施例１３に記載の方法に準じて比較することができる。
　従って、より具体的には、本発明は、
（a）Ct-SLCO1B3を産生する能力を有する細胞を被検物質の存在下又は非存在下に培養し、両条件下におけるslugタンパク質をコードするmRNAの量を、該mRNA検出用核酸を用いて測定、比較することを特徴とする、癌の治療及び／又は予防活性を有する物質のスクリーニング方法、及び
（b）Ct-SLCO1B3を産生する能力を有する細胞を被検物質の存在下又は非存在下に培養し、両条件下におけるslugタンパク質の量を、slugタンパク質に対する抗体を用いて測定、比較することを特徴とする、癌の治療及び／又は予防活性を有する物質のスクリーニング方法を提供する。 (5) Increase in slug expression level Specifically, in cells expressing Ct-SLCO1B3, it is carried out by measuring whether the slug expression level of the cells decreases by adding a test substance be able to. For example, the amount of slug expression of cells expressing Ct-SLCO1B3 in the presence of the test substance is about 70% or less, preferably about 50% or less, compared to the invasive ability of the cells in the absence of the test substance. Preferably, when the test substance is reduced to about 30% or less, more preferably about 20% or less, and particularly preferably about 10% or less, the test substance is treated as a Ct-SLCO1B3 function inhibitor, and thus cancer treatment and / or It can be selected as a candidate for a substance having prophylactic activity.
As the cells expressing Ct-SLCO1B3, the following 1) or 2) is preferably used.
1) A cell in which Ct-SLCO1B3 is expressed in a Ct-SLCO1B3 non-expressing cell, and the slug expression level is about 10% or more, preferably about 20% or more compared to the parent strain Ct-SLCO1B3 non-expressing cell More preferably about 30% or more, more preferably about 50% or more
2) Ct-SLCO1B3-expressing cells, and when Ct-SLCO1B3 expression is suppressed, the slug expression level is about 70% or less, preferably about 50% or less compared to the parent strain Ct-SLCO1B3-expressing cells More preferably, the method for comparing the expression levels of cellular genes reduced to about 30% or less, more preferably about 20% or less, and particularly preferably about 10% or less is as described above. For example, the expression level of slug can be compared according to the method described in Example 13.
Therefore, more specifically, the present invention
(A) Cells capable of producing Ct-SLCO1B3 are cultured in the presence or absence of a test substance, and the amount of mRNA encoding slug protein under both conditions is determined using the nucleic acid for mRNA detection. A method for screening a substance having a therapeutic and / or prophylactic activity for cancer, characterized by measuring and comparing, and (b) a cell capable of producing Ct-SLCO1B3 in the presence or absence of a test substance And a method for screening a substance having a therapeutic and / or prophylactic activity for cancer, characterized by measuring and comparing the amount of slug protein under both conditions using an antibody against the slug protein.
　（6）E-cadherin発現量の低下
　具体的には、Ct-SLCO1B3を発現する細胞において、被検物質を添加することにより、該細胞のE-cadherin発現量が上昇するか否かを測定することにより実施することができる。例えば、被検物質存在下におけるCt-SLCO1B3を発現する細胞のE-cadherin発現量が、被検物質の非存在下における細胞の浸潤能に比べて、約10%以上、好ましくは約20%以上、より好ましくは約30%以上、更に好ましくは約50%以上に上昇した場合に、該被検物質を、Ct-SLCO1B3の機能抑制物質、従って、癌の治療及び／又は予防活性を有する物質の候補として選択することができる。
　Ct-SLCO1B3を発現する細胞としては、下記1）又は2）が好適に用いられる。
1）Ct-SLCO1B3非発現細胞にCt-SLCO1B3を発現させた細胞であって、親株であるCt-SLCO1B3非発現細胞と比較して、E-cadherin発現量が約70%以下、好ましくは約50%以下、より好ましくは約30%以下、更に好ましくは約20%以下、特に好ましくは約10%以下に低下した細胞
2）Ct-SLCO1B3発現細胞であって、Ct-SLCO1B3の発現を抑制させた場合に親株であるCt-SLCO1B3発現細胞と比較して、E-cadherin発現量が、約10%以上、好ましくは約20%以上、より好ましくは約30%以上、更に好ましくは約50%以上に上昇する細胞
　遺伝子の発現量を比較する方法は上述のとおりである。例えば、E-cadherin発現量は実施例１２に記載の方法に準じて比較することができる。
　従って、より具体的には、本発明は、
（a）Ct-SLCO1B3を産生する能力を有する細胞を被検物質の存在下又は非存在下に培養し、両条件下におけるE-cadherinタンパク質をコードするmRNAの量を、該mRNA検出用核酸を用いて測定、比較することを特徴とする、癌の治療及び／又は予防活性を有する物質のスクリーニング方法、及び
（b）Ct-SLCO1B3を産生する能力を有する細胞を被検物質の存在下又は非存在下に培養し、両条件下におけるE-cadherinタンパク質の量を、E-cadherinタンパク質に対する抗体を用いて測定、比較することを特徴とする、癌の治療及び／又は予防活性を有する物質のスクリーニング方法を提供する。 (6) Decrease in E-cadherin expression level Specifically, in cells expressing Ct-SLCO1B3, it is measured whether the E-cadherin expression level in the cells increases by adding a test substance. Can be implemented. For example, the E-cadherin expression level of cells expressing Ct-SLCO1B3 in the presence of the test substance is about 10% or more, preferably about 20% or more, compared to the invasive ability of the cells in the absence of the test substance More preferably about 30% or more, and even more preferably about 50% or more, the test substance is a Ct-SLCO1B3 function inhibitory substance, and therefore a substance having a therapeutic and / or prophylactic activity for cancer. Can be selected as a candidate.
As the cells expressing Ct-SLCO1B3, the following 1) or 2) is preferably used.
1) A cell in which Ct-SLCO1B3 is expressed in a Ct-SLCO1B3 non-expressing cell, and the E-cadherin expression level is about 70% or less, preferably about 50, compared to the parent strain Ct-SLCO1B3 non-expressing cell. % Or less, more preferably about 30% or less, more preferably about 20% or less, particularly preferably about 10% or less
2) Ct-SLCO1B3-expressing cells, and when Ct-SLCO1B3 expression is suppressed, the amount of E-cadherin expressed is about 10% or more, preferably about The method for comparing the expression levels of cellular genes that rise to 20% or more, more preferably about 30% or more, and even more preferably about 50% or more is as described above. For example, the expression level of E-cadherin can be compared according to the method described in Example 12.
Therefore, more specifically, the present invention
(A) Cells having the ability to produce Ct-SLCO1B3 are cultured in the presence or absence of a test substance, and the amount of mRNA encoding E-cadherin protein under both conditions is determined using the nucleic acid for mRNA detection. A method for screening a substance having a therapeutic and / or preventive activity for cancer, characterized by using and measuring, and (b) a cell having the ability to produce Ct-SLCO1B3 in the presence or absence of the test substance Screening for a substance having therapeutic and / or prophylactic activity for cancer, characterized by measuring and comparing the amount of E-cadherin protein in both conditions using an antibody against E-cadherin protein Provide a method.
　（7）occludin発現量の低下
　具体的には、Ct-SLCO1B3を発現する細胞において、被検物質を添加することにより、該細胞のoccludin発現量が上昇するか否かを測定することにより実施することができる。例えば、被検物質存在下におけるCt-SLCO1B3を発現する細胞のoccludin発現量が、被検物質の非存在下における細胞の浸潤能に比べて、約10%以上、好ましくは約20%以上、より好ましくは約30%以上、更に好ましくは約50%以上に上昇した場合に、該被検物質を、Ct-SLCO1B3の機能抑制物質、従って、癌の治療及び／又は予防活性を有する物質の候補として選択することができる。
　Ct-SLCO1B3を発現する細胞としては、下記1）又は2）が好適に用いられる。
1）Ct-SLCO1B3非発現細胞にCt-SLCO1B3を発現させた細胞であって、親株であるCt-SLCO1B3非発現細胞と比較して、occludin発現量が約70%以下、好ましくは約50%以下、より好ましくは約30%以下、更に好ましくは約20%以下、特に好ましくは約10%以下に低下した細胞
2）Ct-SLCO1B3発現細胞であって、Ct-SLCO1B3の発現を抑制させた場合に親株であるCt-SLCO1B3発現細胞と比較して、occludin発現量が、約10%以上、好ましくは約20%以上、より好ましくは約30%以上、更に好ましくは約50%以上に上昇する細胞
　遺伝子の発現量を比較する方法は上述のとおりである。例えば、occludin発現量は実施例１２に記載の方法に準じて比較することができる。
　従って、より具体的には、本発明は、
（a）Ct-SLCO1B3を産生する能力を有する細胞を被検物質の存在下又は非存在下に培養し、両条件下におけるoccludinタンパク質をコードするmRNAの量を、該mRNA検出用核酸を用いて測定、比較することを特徴とする、癌の治療及び／又は予防活性を有する物質のスクリーニング方法、及び
（b）Ct-SLCO1B3を産生する能力を有する細胞を被検物質の存在下又は非存在下に培養し、両条件下におけるoccludinタンパク質の量を、occludinタンパク質に対する抗体を用いて測定、比較することを特徴とする、癌の治療及び／又は予防活性を有する物質のスクリーニング方法を提供する。 (7) Decrease in the expression level of occludin Specifically, in a cell expressing Ct-SLCO1B3, it is carried out by measuring whether or not the occludin expression level of the cell increases by adding a test substance be able to. For example, the expression level of occludin in cells expressing Ct-SLCO1B3 in the presence of the test substance is about 10% or more, preferably about 20% or more compared to the invasion ability of the cells in the absence of the test substance. Preferably, when it is increased to about 30% or more, more preferably about 50% or more, the test substance is regarded as a Ct-SLCO1B3 function inhibitory substance, and therefore a candidate for a substance having cancer therapeutic and / or prophylactic activity. You can choose.
As the cells expressing Ct-SLCO1B3, the following 1) or 2) is preferably used.
1) A cell in which Ct-SLCO1B3 is expressed in a Ct-SLCO1B3 non-expressing cell, and the occludin expression level is about 70% or less, preferably about 50% or less compared to the parent strain Ct-SLCO1B3 non-expressing cell More preferably about 30% or less, still more preferably about 20% or less, particularly preferably about 10% or less.
2) Ct-SLCO1B3-expressing cells, and when the expression of Ct-SLCO1B3 is suppressed, the expression level of occludin is about 10% or more, preferably about 20%, compared to the parent Ct-SLCO1B3-expressing cells The method for comparing the expression levels of cellular genes that rise to about 30% or more, more preferably about 50% or more is more as described above. For example, the expression level of occludin can be compared according to the method described in Example 12.
Therefore, more specifically, the present invention
(A) Cells having the ability to produce Ct-SLCO1B3 are cultured in the presence or absence of a test substance, and the amount of mRNA encoding the occludin protein under both conditions is determined using the nucleic acid for mRNA detection. A method for screening a substance having a therapeutic and / or prophylactic activity for cancer, characterized by measuring and comparing, and (b) a cell capable of producing Ct-SLCO1B3 in the presence or absence of a test substance And a method for screening a substance having a therapeutic and / or prophylactic activity for cancer, characterized by measuring and comparing the amount of occludin protein under both conditions using an antibody against occludin protein.
　（8）MMP9発現量の上昇
　具体的には、Ct-SLCO1B3を発現する細胞において、被検物質を添加することにより、該細胞のMMP9発現量が低下するか否かを測定することにより実施することができる。例えば、被検物質存在下におけるCt-SLCO1B3を発現する細胞のMMP9発現量が、被検物質の非存在下における細胞の浸潤能に比べて、約70%以下、好ましくは約50%以下、より好ましくは約30%以下、更に好ましくは約20%以下、特に好ましくは約10%以下に低下した場合に、該被検物質を、Ct-SLCO1B3の機能抑制物質、従って、癌の治療及び／又は予防活性を有する物質の候補として選択することができる。
　Ct-SLCO1B3を発現する細胞としては、下記1）又は2）が好適に用いられる。
1）Ct-SLCO1B3非発現細胞にCt-SLCO1B3を発現させた細胞であって、親株であるCt-SLCO1B3非発現細胞と比較して、MMP9発現量が約10%以上、好ましくは約20%以上、より好ましくは約30%以上、更に好ましくは約50%以上上昇した細胞
2）Ct-SLCO1B3発現細胞であって、Ct-SLCO1B3の発現を抑制させた場合に親株であるCt-SLCO1B3発現細胞と比較して、MMP9発現量が約70%以下、好ましくは約50%以下、より好ましくは約30%以下、更に好ましくは約20%以下、特に好ましくは約10%以下に低下した細胞
　遺伝子の発現量を比較する方法は上述のとおりである。例えば、MMP9発現量は実施例１３に記載の方法に準じて比較することができる。
　従って、より具体的には、本発明は、
（a）Ct-SLCO1B3を産生する能力を有する細胞を被検物質の存在下又は非存在下に培養し、両条件下におけるMMP9タンパク質をコードするmRNAの量を、該mRNA検出用核酸を用いて測定、比較することを特徴とする、癌の治療及び／又は予防活性を有する物質のスクリーニング方法、及び
（b）Ct-SLCO1B3を産生する能力を有する細胞を被検物質の存在下又は非存在下に培養し、両条件下におけるMMP9タンパク質の量を、MMP9タンパク質に対する抗体を用いて測定、比較することを特徴とする、癌の治療及び／又は予防活性を有する物質のスクリーニング方法を提供する。 (8) Increase in MMP9 expression level Specifically, in cells expressing Ct-SLCO1B3, it is carried out by measuring whether the MMP9 expression level in the cells decreases by adding a test substance be able to. For example, the expression level of MMP9 in cells expressing Ct-SLCO1B3 in the presence of the test substance is about 70% or less, preferably about 50% or less, compared to the invasion ability of the cells in the absence of the test substance. Preferably, when the test substance is reduced to about 30% or less, more preferably about 20% or less, and particularly preferably about 10% or less, the test substance is treated as a Ct-SLCO1B3 function inhibitor, and thus cancer treatment and / or It can be selected as a candidate for a substance having prophylactic activity.
As the cells expressing Ct-SLCO1B3, the following 1) or 2) is preferably used.
1) A cell in which Ct-SLCO1B3 is expressed in a Ct-SLCO1B3 non-expressing cell, and the expression level of MMP9 is about 10% or more, preferably about 20% or more compared to the parent strain Ct-SLCO1B3 non-expressing cell More preferably about 30% or more, more preferably about 50% or more
2) Ct-SLCO1B3-expressing cells, and when the expression of Ct-SLCO1B3 is suppressed, the MMP9 expression level is about 70% or less, preferably about 50% or less compared to the parent Ct-SLCO1B3-expressing cell More preferably, the method for comparing the expression levels of cellular genes reduced to about 30% or less, more preferably about 20% or less, and particularly preferably about 10% or less is as described above. For example, the expression level of MMP9 can be compared according to the method described in Example 13.
Therefore, more specifically, the present invention
(A) Cells having the ability to produce Ct-SLCO1B3 are cultured in the presence or absence of a test substance, and the amount of mRNA encoding MMP9 protein under both conditions is determined using the nucleic acid for mRNA detection. A method for screening a substance having a therapeutic and / or prophylactic activity for cancer, characterized by measuring and comparing, and (b) a cell capable of producing Ct-SLCO1B3 in the presence or absence of a test substance And a method for screening a substance having a therapeutic and / or prophylactic activity for cancer, which comprises measuring and comparing the amount of MMP9 protein under both conditions using an antibody against MMP9 protein.
　上記のスクリーニング方法において、コントロールとして、常法を用いて作製される、Ct-SLCO1B3遺伝子がノックアウトされた細胞を用いることにより、被検物質がCt-SLCO1B3遺伝子を発現していないコントロール細胞において上記機能を示さないことを確認できる。すなわち、上記のスクリーニング方法において得られる癌の治療又は予防活性を有する候補物質の作用機序が、Ct-SLCO1B3もしくはCt-SLCO1B3遺伝子の発現抑制又はCt-SLCO1B3の機能抑制に基づくものであることが確認できる。 In the above screening method, as a control, by using a cell in which the Ct-SLCO1B3 gene is knocked out, which is prepared using a conventional method, the test substance has the above function in a control cell not expressing the Ct-SLCO1B3 gene. Can be confirmed. That is, the mechanism of action of the candidate substance having cancer therapeutic or prophylactic activity obtained in the above screening method is based on suppression of Ct-SLCO1B3 or Ct-SLCO1B3 gene expression or Ct-SLCO1B3 function I can confirm.
　本発明の上記いずれかのスクリーニング方法を用いて得られる、Ct-SLCO1B3の発現又は機能を抑制する物質は、癌の予防及び／又は治療用に、医薬として有用である。
　本発明のスクリーニング方法を用いて得られる化合物を上述の予防・治療剤として使用する場合、上記Ct-SLCO1B3の発現又は機能を抑制する低分子化合物と同様に製剤化することができ、同様の投与経路及び投与量で、ヒト又は哺乳動物（例えば、マウス、ラット、ウサギ、ヒツジ、ブタ、ウシ、ウマ、ネコ、イヌ、サル、チンパンジーなど）に対して、経口的に又は非経口的に投与することができる。 A substance that suppresses the expression or function of Ct-SLCO1B3 obtained by using any one of the screening methods of the present invention is useful as a medicament for the prevention and / or treatment of cancer.
When the compound obtained using the screening method of the present invention is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated in the same manner as the low molecular weight compound that suppresses the expression or function of Ct-SLCO1B3, and the same administration Administered orally or parenterally to humans or mammals (eg, mice, rats, rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, chimpanzees, etc.) by route and dose be able to.
５．癌の悪性度又は悪性化リスクを試験する方法
　また、本発明は、被験動物より採取した試料中のCt-SLCO1B3の発現量を測定することを特徴とする癌の悪性度又は悪性化リスクを試験する方法（以下、本発明の試験方法とも称する）を提供する。当該方法は、以下の工程：
（a）被験動物より採取した試料中の、Ct-SLCO1B3の発現量を測定する工程、及び
（b）正常動物由来の試料において測定した場合と比較して、前記発現量が高い被験動物を、癌の悪性度が高いか、将来悪性化するリスクが高いと判定する工程、
を含む被験動物より採取した試料中のCt-SLCO1B3の発現量を測定することを特徴とする癌の悪性度又は悪性化リスクを試験する方法を提供する。 5). Method for testing malignancy or risk of malignancy of cancer Further , the present invention tests the malignancy or risk of malignancy of cancer characterized by measuring the expression level of Ct-SLCO1B3 in a sample collected from a test animal (Hereinafter also referred to as the test method of the present invention). The method comprises the following steps:
(A) a step of measuring the expression level of Ct-SLCO1B3 in a sample collected from the test animal, and (b) a test animal having a high expression level compared to the case of measuring in a sample derived from a normal animal, The process of determining that the malignancy of the cancer is high or that the risk of malignancy in the future is high,
A method for testing the malignancy or risk of malignancy of cancer, characterized by measuring the expression level of Ct-SLCO1B3 in a sample collected from a test animal containing.
　被験動物としては、ヒト又は他の哺乳動物が挙げられるが、好ましくはヒト、あるいは実験動物として汎用されるマウス、ラット、ウサギ、イヌ、サル等である。測定対象試料としては、生検サンプル（例、癌の可能性が疑われる動物の組織（肺、肝臓、膵臓、食道等）から摘出された細胞又は該組織の一部分）、血液、血漿、血清、脳脊髄液、リンパ液、唾液、粘膜、尿、涙、***、関節液等が挙げられる。測定対象試料としては、癌の可能性が疑われる組織から採取された試料が好ましい。 Examples of test animals include humans and other mammals, and preferred examples include humans, mice, rats, rabbits, dogs, monkeys, and the like that are widely used as experimental animals. Samples to be measured include biopsy samples (eg, cells or a part of the tissue extracted from animal tissues suspected of having cancer (lung, liver, pancreas, esophagus, etc.), blood, plasma, serum, Examples include cerebrospinal fluid, lymph fluid, saliva, mucous membrane, urine, tears, semen, and joint fluid. The sample to be measured is preferably a sample collected from a tissue suspected of having cancer.
　癌の悪性度は、当業者により、Ct-SLCO1B3と関連付けられ得る任意の癌悪性度を意味し、例えば、転移、増殖、浸潤の可能性及び／又は早さと関連するものであり得る。 Cancer malignancy refers to any cancer malignancy that can be associated with Ct-SLCO1B3 by those skilled in the art, and may be associated with, for example, the likelihood and / or the speed of metastasis, proliferation, invasion.
　試料中のCt-SLCO1B3の発現量は、Ct-SLCO1B3のmRNA量又はCt-SLCO1B3タンパク質の量により測定することができ、これらは、該遺伝子もしくは該タンパク質の発現量を指標とする上記スクリーニング法に記載されたのと同様の方法により測定することができる。
　上記測定の結果、被験動物より採取した試料中のCt-SLCO1B3遺伝子の発現量又はCt-SLCO1B3タンパク質の量が、正常動物より採取した試料中のCt-SLCO1B3遺伝子の発現量又はCt-SLCO1B3タンパク質の量と比較して有意に高かった場合、該被験動物は、癌を発症しているか、将来発症するリスクが高いと判定することができる。あるいは、正常動物における発現量を予め同定しておき、例えば、その平均値＋2SD（平均値＋標準偏差の2倍）をカットオフ値として規定し、被験動物より採取した試料中のCt-SLCO1B3遺伝子の発現量又はCt-SLCO1B3の量が、当該カットオフ値を超えた場合に、該被験動物は、癌の悪性度が高いか、将来悪性化するリスクが高いと判定することもできる。 The expression level of Ct-SLCO1B3 in the sample can be measured by the amount of mRNA of Ct-SLCO1B3 or the amount of Ct-SLCO1B3 protein, and these are used in the above screening method using the expression level of the gene or the protein as an index. It can be measured by the same method as described.
As a result of the above measurement, the expression level of Ct-SLCO1B3 gene or the amount of Ct-SLCO1B3 protein in the sample collected from the test animal is equal to the expression level of Ct-SLCO1B3 gene or Ct-SLCO1B3 protein in the sample collected from the normal animal. If the amount is significantly higher than the amount, the subject animal can be determined to have developed cancer or have a higher risk of developing in the future. Alternatively, the expression level in a normal animal is identified in advance, for example, the average value + 2SD (average value + twice the standard deviation) is defined as a cut-off value, and the Ct-SLCO1B3 gene in a sample collected from the test animal When the expression level of or the amount of Ct-SLCO1B3 exceeds the cut-off value, the test animal can also be determined to have a high cancer malignancy or a high risk of becoming malignant in the future.
６．癌の予防又は治療方法
　本発明は、本発明の核酸を用いた、癌の予防又は治療方法（本明細書中、本発明の予防又は治療方法とも称する）を提供する。本発明の予防又は治療方法に関連する各用語の定義及び態様は、上記に記載したものと同一である。
　一態様として、本発明の予防又は治療方法は、予防又は治療を必要とする動物に、予防又は治療上有効量の本発明の核酸又は本発明の医薬組成物を投与する工程を含む。予防又は治療を必要とする動物としては、癌に罹患しているか又は癌の罹患の可能性の高い動物が挙げられ、好ましくは、癌に罹患しているか又は癌の罹患の可能性の高いヒトである。また、本発明の予防又は治療方法は、本発明の試験方法により「癌の悪性度が高いか、将来悪性化するリスクが高い」と判定された動物、特に該試験方法により「癌の悪性度が高いか、将来悪性化するリスクが高い」と診断されたヒトに対して用いることができる。
　本発明の核酸又は本発明の医薬組成物の好ましい投与方法の一例としては、癌の可能性が疑われる組織、癌に罹患している組織、癌の悪性度が高いか将来悪性化するリスクが高い組織への局所投与が挙げられる。例えば、本発明の試験方法により癌の悪性度が高いか、将来悪性化するリスクが高いと判断された動物（例、ヒト）の予防又は治療を行う場合、本発明の試験方法工程（a）にて当該被験動物より採取した試料が由来する組織（例、肺、肝臓、膵臓、食道等）に、本発明の核酸又は本発明の医薬組成物を局所投与することすることができる。本発明の試験方法と本発明の予防又は治療方法を組み合わせることにより、効果的な予防又は治療を行うことが可能となる。 6). Method for Preventing or Treating Cancer The present invention provides a method for preventing or treating cancer (hereinafter also referred to as the method for preventing or treating the present invention) using the nucleic acid of the present invention. The definitions and embodiments of each term relating to the prevention or treatment method of the present invention are the same as those described above.
In one aspect, the preventive or therapeutic method of the present invention includes a step of administering a prophylactically or therapeutically effective amount of the nucleic acid of the present invention or the pharmaceutical composition of the present invention to an animal in need of prevention or treatment. Examples of animals that require prevention or treatment include animals that have cancer or are highly likely to have cancer, preferably humans who have cancer or are highly likely to have cancer. It is. In addition, the prevention or treatment method of the present invention can be applied to animals determined to be “high cancer malignancy or high risk of malignancy in the future” by the test method of the present invention. Can be used for humans who are diagnosed as having a high risk of malignancy in the future.
Examples of preferable administration methods of the nucleic acid of the present invention or the pharmaceutical composition of the present invention include tissues suspected of having cancer, tissues suffering from cancer, risk of cancer having high malignancy or becoming malignant in the future. Local administration to high tissues is mentioned. For example, when the prevention or treatment of an animal (eg, human) determined to have a high malignancy of cancer or a high risk of malignancy in the future by the test method of the present invention, the test method step (a) of the present invention The nucleic acid of the present invention or the pharmaceutical composition of the present invention can be locally administered to a tissue (eg, lung, liver, pancreas, esophagus, etc.) from which the sample collected from the test animal is derived. By combining the test method of the present invention and the prevention or treatment method of the present invention, effective prevention or treatment can be performed.
　以下に本発明に用いる培地組成物の分析例、試験例を実施例として具体的に述べることで、本発明を更に詳しく説明するが、本発明はこれらによって限定されるものではない。 Hereinafter, the present invention will be described in more detail by specifically describing analysis examples and test examples of the medium composition used in the present invention as examples, but the present invention is not limited thereto.
実施例１：　肺癌臨床検体を用いた解析によるSLCO1B3高発現肺癌症例の同定
方法：
（１）　組織からのRNA抽出
　術後臨床検体をQIAZOL（QIAGEN）700μLと破砕用φ5mmジルコニアビーズ1個が入ったサンプルチューブに入れ、Micro smash MS-100（TOMY）を用いて破砕し、ホモジネートを得た。破砕条件としては、4800rpm、30秒を2回繰り返し、途中1分間の氷上放置とした。組織ホモジネートにクロロホルム140μLを添加して混和後、12,000gで15分間遠心し、得られた水層をRNA抽出に用いた。全自動核酸抽出装置QIA Qube（QIAGEN）を使用し、miRNeasy Mini kit（QIAGEN）を用いてtotal　RNAを抽出精製した。
（２）　RNA濃度測定と純度確認
　RNAの濃度測定には極微量分光光度計Nano Dropを使用した。また純度の確認にはRNA 1μLをExperion RNA StdSens Analysis chip（BIO-RAD）に添加し、全自動チップ電気泳動システムExperion Automated Electrophoresis station（BIO-RAD）にて解析した。なおRNA純度は、RQI（RNA Quality Indicator）を指標とした。RQIは、RNAの完全性を1（分解度が極めて高い）から10（分解していない）の間の数値で表している。以後のmicroarray解析にはRQIが7より大きい値のRNAを用いた。
（３）　Exon array解析
　非小細胞肺癌術後検体（#1－4）の癌部及び非癌部より得られたtotal RNA 100ngを用いて、Ambion WT Expression Kit（Applied Biosystems）によりcDNA合成を行った。得られたcDNA 5.5μgをGeneChip WT Terminal Labeling Kit（Affymetrix）により断片化、ビオチン化した。ビオチン化されたcDNAをもとにHybridization Cocktailを作成後、GeneChip Human Exon 1.0 ST Array（Affymetrix）に注入し、45℃、60rpmで17時間hybridizationを行った。Hybridization終了後、Fluidics Station（Affymetrix）を使用してGeneChip Hybridization Wash and Stain Kit（Affymetrix）によりarrayの自動洗浄・染色を行い、GeneChip Scanner 3000によりスキャンした。データ解析は、Gene Spring 12.1を用いた。
結果：
　結果を図1に示す。#2、3の癌部検体においてSLCO1B3の高発現が認められた。しかしながらそれらの検体のexon2のシグナル強度のみ他の検体と同程度の低いシグナル強度を示した。 Example 1: Identification of lung cancer cases with high expression of SLCO1B3 by analysis using lung cancer clinical specimens Method:
(1) RNA extraction from tissue Post-operative clinical specimens are placed in a sample tube containing 700 μL of QIAZOL (QIAGEN) and one φ5 mm zirconia bead for disruption, and disrupted using Micro smash MS-100 (TOMY). Obtained. As crushing conditions, 4800 rpm, 30 seconds was repeated twice, and left on ice for 1 minute. After adding 140 μL of chloroform to the tissue homogenate and mixing, the mixture was centrifuged at 12,000 g for 15 minutes, and the resulting aqueous layer was used for RNA extraction. Total RNA was extracted and purified using miRNeasy Mini kit (QIAGEN) using a fully automatic nucleic acid extraction device QIA Qube (QIAGEN).
(2) RNA concentration measurement and purity confirmation An ultra-trace spectrophotometer Nano Drop was used for RNA concentration measurement. To confirm the purity, 1 μL of RNA was added to an Experion RNA StdSens Analysis chip (BIO-RAD) and analyzed by a fully automatic chip electrophoresis system Experion Automated Electrophoresis station (BIO-RAD). RNA purity was determined using RQI (RNA Quality Indicator) as an index. RQI represents RNA integrity as a number between 1 (very high degradation) and 10 (not degraded). For subsequent microarray analysis, RNA with RQI greater than 7 was used.
(3) Exon array analysis cDNA synthesis using Ambion WT Expression Kit (Applied Biosystems) using 100 ng of total RNA obtained from cancerous and non-cancerous post-operative specimens of non-small cell lung cancer (# 1-4) It was. The obtained cDNA (5.5 μg) was fragmented and biotinylated using GeneChip WT Terminal Labeling Kit (Affymetrix). A Hybridization Cocktail was created based on biotinylated cDNA, then injected into GeneChip Human Exon 1.0 ST Array (Affymetrix), and subjected to hybridization at 45 ° C. and 60 rpm for 17 hours. After completion of hybridization, the array was automatically washed and stained with GeneChip Hybridization Wash and Stain Kit (Affymetrix) using Fluidics Station (Affymetrix), and scanned with GeneChip Scanner 3000. For the data analysis, Gene Spring 12.1 was used.
result:
The results are shown in Figure 1. High expression of SLCO1B3 was observed in the cancer specimens # 2 and # 3. However, only the signal intensity of exon2 of those specimens showed a signal intensity as low as other specimens.
実施例２：　肺癌細胞株を用いた解析によるSLCO1B3高発現肺癌症例の同定
方法：
（１）　肺癌細胞株からのRNA抽出と濃度測定、純度確認、exon array解析
　非小細胞肺癌細胞株A549、EBC-1、HLC-1、LC-1F、LC-2/ad、LK-2、NCI-H1650、NCI-H1975、NCI-H2228、PC-14、II-18、RERF-LC-AI、RERF-LC-KJ、Sq-1のペレットに対しQIAZOL（QIAGEN）を700μL添加し、さらにクロロホルム140μLを添加して混和後、実施例１－（１）と同様の方法にてtotal RNAを抽出精製した。濃度測定、純度確認は実施例１－（２）とexon arrayは実施例１－（３）と同様の方法で行った。
結果：
　結果を図２に示す。非小細胞肺癌細胞株でも、肺癌臨床検体と同様にSLCO1B3高発現細胞の存在が確認できた。さらにこれら高発現が認められた細胞においてもSLCO1B3のexon2のシグナル強度は低いことが分かった。 Example 2: Identification of lung cancer cases with high expression of SLCO1B3 by analysis using lung cancer cell lines Method:
(1) RNA extraction and concentration measurement from lung cancer cell lines, purity confirmation, exon array analysis Non-small cell lung cancer cell lines A549, EBC-1, HLC-1, LC-1F, LC-2 / ad, LK-2, Add 700 μL of QIAZOL (QIAGEN) to the pellets of NCI-H1650, NCI-H1975, NCI-H2228, PC-14, II-18, RERF-LC-AI, RERF-LC-KJ, Sq-1, and then chloroform After adding 140 μL and mixing, total RNA was extracted and purified in the same manner as in Example 1- (1). Concentration measurement and purity confirmation were performed in the same manner as Example 1- (2) and exon array in the same manner as in Example 1- (3).
result:
The results are shown in FIG. In non-small cell lung cancer cell lines, the presence of SLCO1B3 high-expressing cells was confirmed as in the case of lung cancer clinical specimens. Furthermore, the signal intensity of exon2 of SLCO1B3 was found to be low even in cells in which these high expression levels were observed.
実施例３：　ヒト正常組織におけるSLCO1B3の発現
方法：
　Human Multiple Tissue cDNA Panel（CLONTECH）をMilli-Q水にて10倍希釈した溶液をtemplateとして使用した。下に示す組成にてPCR反応液を作成し、同じく下に示すPCR条件にて反応を行った。なお、GAPDHはAmpliTaq Gold（Applied biosystems）、SLCO1B3 full lengthはKOD-plus-（TOYOBO）にてPCR反応を行った。 Example 3: Expression of SLCO1B3 in normal human tissues Method:
A solution obtained by diluting Human Multiple Tissue cDNA Panel (CLONTECH) 10 times with Milli-Q water was used as a template. A PCR reaction solution was prepared with the composition shown below, and the reaction was performed under the PCR conditions shown below. GAPDH was subjected to PCR reaction with AmpliTaq Gold (Applied biosystems), and SLCO1B3 full length was subjected to PCR reaction with KOD-plus- (TOYOBO).
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
・primer
　SLCO1B3 fulllength F:ATGGACCAACATCAACATTTGAATAAAAC（配列番号：33）
　SLCO1B3 fulllength R:TTAGTTGGCAGCAGCATTGTCTTG（配列番号：34）
　GAPDH F:CCATCACCATCTTCCAGGAG（配列番号：35）
　GAPDH R:AATGAGCCCCAGCCTTCTCC（配列番号：36）
結果：
　結果を図3に示す。調べた正常組織においてSLCO1B3は肝臓にのみ検出された。 ・ Primer
SLCO1B3 fulllength F: ATGGACCAACATCAACATTTGAATAAAAC (SEQ ID NO: 33)
SLCO1B3 fulllength R: TTAGTTGGCAGCAGCATTGTCTTG (SEQ ID NO: 34)
GAPDH F: CCATCACCATCTTCCAGGAG (SEQ ID NO: 35)
GAPDH R: AATGAGCCCCAGCCTTCTCC (SEQ ID NO: 36)
result:
The results are shown in Figure 3. In normal tissues examined, SLCO1B3 was detected only in the liver.
実施例：４　Ct-SLCO1B3、Lt-SLCO1B3のsplicing form
方法：
　肺癌組織におけるSLCO1B3の5’側の配列を確認するためSMARTer RACE cDNA増幅キット（Clontech）を用いて5’-RACEを行い、塩基配列を決定した。
結果：
　結果を図４に示す。肺癌に発現するSLCO1B3はexon2を使用しておらず、代わりにintronをexonとして使用していることが分かった。この解析中にThakkarらが、癌においては肝臓で発現する正常型のSLCO1B3（肝臓型SLCO1B3：Lt-SLCO1B3）と異なり、exon1、2を使用せずintronをexon1*として使用する癌型SLCO1B3（Ct-SLCO1B3）が存在することを報告した。我々が塩基配列を決定したものもCt-SLCO1B3と同じものであった。 Example: 4 Ct-SLCO1B3, Lt-SLCO1B3 splicing form
Method:
In order to confirm the 5'-side sequence of SLCO1B3 in lung cancer tissue, 5'-RACE was performed using SMARTer RACE cDNA amplification kit (Clontech), and the nucleotide sequence was determined.
result:
The results are shown in FIG. SLCO1B3 expressed in lung cancer did not use exon2, but instead used intron as exon. During this analysis, Thakkar et al. Differed from normal SLCO1B3 (liver-type SLCO1B3: Lt-SLCO1B3) that is expressed in the liver in cancer. Cancer type SLCO1B3 (Ct -SLCO1B3) was reported. What we determined the base sequence was also the same as Ct-SLCO1B3.
実施例５：　正常肝臓組織と非小細胞肺癌組織におけるSLCO1B3の免疫組織化学染色
方法：
　正常肝臓組織及び非小細胞肺癌組織におけるSLCO1B3の発現を抗SLCO1B3抗体を用いた免疫組織化学的解析により検討した。なお使用抗体はAnti-SLCO1B3 antibody（Sigma-Aldrich、HPA004943）である。
結果：
　結果を図５に示す。肝臓ではSLCO1B3が知られているように細胞膜に発現していた。一方、非小細胞肺癌組織においては、膜局在は認められず、細胞質に局在していた。 Example 5: Immunohistochemical staining of SLCO1B3 in normal liver tissue and non-small cell lung cancer tissue Method:
The expression of SLCO1B3 in normal liver tissue and non-small cell lung cancer tissue was examined by immunohistochemical analysis using anti-SLCO1B3 antibody. The antibody used is Anti-SLCO1B3 antibody (Sigma-Aldrich, HPA004943).
result:
The results are shown in FIG. In the liver, SLCO1B3 was expressed in the cell membrane as is known. On the other hand, in non-small cell lung cancer tissues, membrane localization was not observed, but it was localized in the cytoplasm.
実施例６：　非小細胞肺癌組織におけるCt-SLCO1B3発現の定量的解析
方法：
　臨床検体から抽出したtotal RNA 500ngを用いて、PrimeScript RT reagent Kit（TaKaRa）にてcDNA合成を行い、Milli-Q水にて10倍希釈してからreal-time PCRに用いた。あらかじめ10μMに希釈したprimer及びSsoAdvanced SYBR Green Supermix（BIO-RAD）又はTHUNDERBIRD SYBR qPCR Mix（TOYOBO）をMilli-Q水にて希釈したものにcDNAを加えて20μLとし、CFX96 Real-Time System（BIO-RAD）にてreal-time PCRを行った。 Example 6: Quantitative analysis of Ct-SLCO1B3 expression in non-small cell lung cancer tissues Method:
Using 500 ng of total RNA extracted from clinical samples, cDNA was synthesized with PrimeScript RT reagent Kit (TaKaRa), diluted 10-fold with Milli-Q water, and used for real-time PCR. Primer and SsoAdvanced SYBR Green Supermix (BIO-RAD) or THUNDERBIRD SYBR qPCR Mix (TOYOBO) diluted to 10 μM in advance is diluted with Milli-Q water to make 20 μL, and CFX96 Real-Time System (BIO- RAD) performed real-time PCR.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
・primer
　SLCO1B3 exon1*-3 F:GCTTGGCTTGGGCTCAGA（配列番号：30）
　SLCO1B3 exon1*-3 R:CCAGCAAGAGAAGAGGATATGTCA（配列番号：31）
GAPDHは実施例３と同じ
結果：
　結果を図6に示す。Ct-SLCO1B3は腺癌の55%、扁平上皮癌の72%で検出された。一方その発現は、EGF受容体の変異の有無に関わらず約60%で認められた。 ・ Primer
SLCO1B3 exon1 * -3 F: GCTTGGCTTGGGCTCAGA (SEQ ID NO: 30)
SLCO1B3 exon1 * -3 R: CCAGCAAGAGAAGAGGATATGTCA (SEQ ID NO: 31)
GAPDH has the same results as Example 3:
The results are shown in FIG. Ct-SLCO1B3 was detected in 55% of adenocarcinoma and 72% of squamous cell carcinoma. On the other hand, its expression was observed in about 60% regardless of the presence or absence of mutations in the EGF receptor.
実施例７：　A549細胞におけるCt-SLCO1B3 siRNAのノックダウン効率
方法：
（１）　細胞培養
　500mLのDMEM（和光純薬化学工業）及びRPMI1640（和光純薬化学工業）に対し56℃　30分間のインキュベートにより補体を非働化したFCS（Life Technologies）を55mL、PBSにて希釈した100mg/mLカナマイシン硫酸塩を500μL滅菌後添加した。細胞培養は37℃、5%CO2にて行った。
（２）　siRNAトランスフェクション
　リバーストランスフェクション法を用いた。まず、LipofectamineRNAiMAX（Life Technologies）、Control siRNA （コスモバイオ）及びCt-SLCO1B3　siRNA（GeneDesign）、Opti-MEM（Life Technologies）のトランスフェクション複合体を作成し、siRNA終濃度が10nMとなるようA549細胞懸濁液に添加した。
　使用したsiRNAの配列を下記に示す。
Ct-SLCO1B3 siRNA　#1：
　ACCUGACAGUGGCAAUGUAtt（配列番号：37）
　UACAUUGCCACUGUCAGGUtt（配列番号：38）
Ct-SLCO1B3 siRNA　#2：
　CUGACAGUGGCAAUGUAUGtt（配列番号：39）
　CAUACAUUGCCACUGUCAGtt（配列番号：40）
Ct-SLCO1B3 siRNA　#3：
　UGGCCACGUUACUGAAUCUtt（配列番号：41）
　AGAUUCAGUAACGUGGCCAtt（配列番号：42）
Ct-SLCO1B3 siRNA　#4：
　ACGUUACUGAAUCUACAUGtt（配列番号：43）
　CAUGUAGAUUCAGUAACGUtt（配列番号：44）
　上記のsiRNAはGeneDesignより合成依頼した。
（３）　cDNA合成
　siRNAトランスフェクション後2日目の細胞を回収し、Trizol（Life Technologies）又はillusta RNAspin Mini（GE Healthcare）を用いてtotal RNAを抽出した後、Prime Script RT-PCR Kit（Perfect Real Time）（TaKaRa）を用いてcDNAを合成した。cDNA合成はtotal RNA 500ngを用いて行い、得られたcDNA溶液をMilli-Q水にて10倍希釈してreal-time PCRに使用した。
（４）　Real-time PCR
　検量線用サンプル、測定サンプル、あらかじめ10μMに希釈した各プライマーを用意し、SYBR Premix Ex Taq（TaKaRa）又はTHUNDERBIRD SYBR qPCR Mix(TOYOBO)と混合し、10μLの反応溶液を調製した。これをキャピラリーに分注し、Light Cyclerクイックシステム350S（Roche）にてreal-time PCRを行った。PCR条件を以下に示す。なお、primerは実施例６のSLCO1B3 exon1*-3及び実施例３のGAPDH primer setを用いた。 Example 7: Knockdown efficiency of Ct-SLCO1B3 siRNA in A549 cells Method:
(1) Cell culture FCS (Life Technologies) deactivated by incubation for 30 minutes at 56 ° C in 500 mL of DMEM (Wako Pure Chemical Industries) and RPMI1640 (Wako Pure Chemical Industries) in 55 mL of PBS Diluted 100 mg / mL kanamycin sulfate was added after sterilization of 500 μL. Cell culture was performed at 37 ° C. and 5% CO 2 .
(2) siRNA transfection The reverse transfection method was used. First, a transfection complex of LipofectamineRNAiMAX (Life Technologies), Control siRNA (Cosmo Bio), Ct-SLCO1B3 siRNA (GeneDesign), and Opti-MEM (Life Technologies) was prepared, and A549 cell suspension was prepared so that the final siRNA concentration was 10 nM. Added to the suspension.
The sequence of the siRNA used is shown below.
Ct-SLCO1B3 siRNA # 1:
ACCUGACAGUGGCAAUGUAtt (SEQ ID NO: 37)
UACAUUGCCACUGUCAGGUtt (SEQ ID NO: 38)
Ct-SLCO1B3 siRNA # 2:
CUGACAGUGGCAAUGUAUGtt (SEQ ID NO: 39)
CAUACAUUGCCACUGUCAGtt (SEQ ID NO: 40)
Ct-SLCO1B3 siRNA # 3:
UGGCCACGUUACUGAAUCUtt (SEQ ID NO: 41)
AGAUUCAGUAACGUGGCCAtt (SEQ ID NO: 42)
Ct-SLCO1B3 siRNA # 4:
ACGUUACUGAAUCUACAUGtt (SEQ ID NO: 43)
CAUGUAGAUUCAGUAACGUtt (SEQ ID NO: 44)
The above siRNA was requested for synthesis from GeneDesign.
(3) cDNA synthesis Cells on the second day after transfection of siRNA were collected, total RNA was extracted using Trizol (Life Technologies) or illusta RNAspin Mini (GE Healthcare), and then Prime Script RT-PCR Kit (Perfect Real Time) (TaKaRa) was used to synthesize cDNA. cDNA synthesis was performed using 500 ng of total RNA, and the obtained cDNA solution was diluted 10-fold with Milli-Q water and used for real-time PCR.
(4) Real-time PCR
A calibration curve sample, a measurement sample, and each primer previously diluted to 10 μM were prepared and mixed with SYBR Premix Ex Taq (TaKaRa) or THUNDERBIRD SYBR qPCR Mix (TOYOBO) to prepare a 10 μL reaction solution. This was dispensed into capillaries, and real-time PCR was performed using the Light Cycler Quick System 350S (Roche). PCR conditions are shown below. As the primer, SLCO1B3 exon1 * -3 of Example 6 and GAPDH primer set of Example 3 were used.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
結果：
　結果を図７に示す。使用したCt-SLCO1B3 siRNAの中で＃３、＃４が約50%以上のノックダウン効果を示した。 result:
The results are shown in FIG. Among the Ct-SLCO1B3 siRNAs used, # 3 and # 4 showed a knockdown effect of about 50% or more.
実施例８：　A549細胞の足場依存性増殖に対するCt-SLCO1B3　siRNAの効果
方法：
　siRNAトランスフェクション後2日目の細胞を回収し、1000 cells/wellとなるよう96 well plateへ播種した。その後24時間ごとに20mM HEPES（pH7.4）に溶解したWST-1（DOJINDO）溶液と1-methoxy PMS（DOJINDO）溶液を9：1で混合したものを10μL/wellずつ添加し、1時間30分後に450nmで吸光度を測定した。対照波長として630nmを使用した。
結果：
　結果を図8に示す。Ct-SLCO1B3 siRNA（図8では#4を使用）は、A549細胞の足場依存性増殖に影響を示さなかった。 Example 8: Effect of Ct-SLCO1B3 siRNA on anchorage-dependent growth of A549 cells Method:
Cells on the second day after siRNA transfection were collected and seeded on a 96 well plate at 1000 cells / well. After that, every 24 hours, WST-1 (DOJINDO) solution dissolved in 20 mM HEPES (pH 7.4) and 1-methoxy PMS (DOJINDO) solution mixed at a ratio of 9: 1 was added at 10 μL / well for 1 hour 30 Absorbance was measured after 450 minutes at 450 nm. A reference wavelength of 630 nm was used.
result:
The results are shown in FIG. Ct-SLCO1B3 siRNA (using # 4 in FIG. 8) had no effect on anchorage-dependent growth of A549 cells.
実施例９：　A549細胞の足場非依存性増殖に対するCt-SLCO1B3　siRNAの効果
方法：
　FCeM-D（日産化学工業株式会社）に対して10% FCS及びカナマイシンを加えたものを培地として用い、細胞低吸着加工が施されたHydro Cell plate（株式会社セルシード）にて培養を行った。siRNAトランスフェクション後2日目の細胞を500 cells/wellとなるよう96well plateへ再播種し、48時間培養後、実施例８と同様にWST-1試薬を加え、吸光度を測定した。
結果：
　結果を図9に示す。Ct-SLCO1B3 siRNA（図9では#4を使用）は、A549細胞の足場非依存性増殖を抑制した。 Example 9: Effect of Ct-SLCO1B3 siRNA on anchorage-independent growth of A549 cells Method:
Using FCeM-D (Nissan Chemical Co., Ltd.) supplemented with 10% FCS and kanamycin as a culture medium, the cells were cultured on a Hydro Cell plate (Cell Seed Co., Ltd.) that had been subjected to low cell adsorption processing. The cells on day 2 after siRNA transfection were replated on a 96-well plate at 500 cells / well, cultured for 48 hours, WST-1 reagent was added in the same manner as in Example 8, and the absorbance was measured.
result:
The results are shown in FIG. Ct-SLCO1B3 siRNA (using # 4 in FIG. 9) suppressed anchorage-independent growth of A549 cells.
実施例１０：　A549細胞の遊走能に対するCt-SLCO1B3 siRNAの効果
方法：
　siRNAトランスフェクション後2日目の細胞を回収し、24 well plateがコンフルエントとなるよう播種した。24時間後にピペットチップの先で増殖細胞の一部を削り、その0、6、12、24、36、48時間後に細胞の遊走面積をImage Jを用いて定量評価した。細胞の撮影にはFSX100（Olympus）を使用した。
結果：
　結果を図10に示す。Ct-SLCO1B3 siRNA（図10では#4を使用）は、A549細胞の遊走能を抑制した。 Example 10: Effect of Ct-SLCO1B3 siRNA on the migration ability of A549 cells Method:
Cells on day 2 after siRNA transfection were collected and seeded so that the 24-well plate was confluent. After 24 hours, a portion of the proliferating cells was scraped with the tip of a pipette tip, and 0, 6, 12, 24, 36, and 48 hours later, the cell migration area was quantitatively evaluated using Image J. FSX100 (Olympus) was used for cell photography.
result:
The results are shown in FIG. Ct-SLCO1B3 siRNA (using # 4 in FIG. 10) suppressed the migration ability of A549 cells.
実施例１１：　A549細胞の浸潤能に対するCt-SLCO1B3 siRNAの効果
方法：
　BD Matrigel Basement Membrane Matrix High Concentration（BD Biosciences）を無血清DMEMにて40倍希釈し、CIM-Plate16（Roche）へコートした。siRNAトランスフェクション後2日目の細胞を回収し、4×104 cells/wellとなるよう希釈してCIM-Plateへ播種し、1時間常温にて放置後xCELLigence（Roche）にセットし測定を開始した。
結果：
　結果を図11に示す。コントロールsiRNAトランスフェクションではA549は顕著に浸潤したのに対し、Ct-SLCO1B3　siRNA（図11では#4を使用）はその浸潤作用を顕著に抑制した。 Example 11: Effect of Ct-SLCO1B3 siRNA on invasive ability of A549 cells Method:
BD Matrigel Basement Membrane Matrix High Concentration (BD Biosciences) was diluted 40-fold with serum-free DMEM and coated onto CIM-Plate 16 (Roche). Collect cells on day 2 after siRNA transfection, dilute to 4 × 10 4 cells / well, seed on CIM-Plate, leave at room temperature for 1 hour, set to xCELLigence (Roche) and start measurement did.
result:
The results are shown in FIG. In control siRNA transfection, A549 was significantly infiltrated, whereas Ct-SLCO1B3 siRNA (# 4 was used in FIG. 11) remarkably suppressed its invasion effect.
実施例１２：　Ct-SLCO1B3ノックダウンA549細胞における細胞接着分子発現の免疫細胞染色
方法：
　トランスフェクション48時間後の細胞をカバーガラスを入れた12 well plateに5×104 cells/wellずつ再播種し、さらに48時間37℃でインキュベートした。その後、－20℃ methanol（Wako 137-01823）を培地の代わりに500μL／wellずつ加え、15分間－20℃で固定を行った。PBS-T（0.1% TWEEN20／PBS）で10分3回washし、Blocking Buffer（組成0.3%TWEEN20（Wako167-11515）／5%牛血清アルブミン（SIGMA A3912-1009 Lot#LBF7177V）／PBS）で1時間ブロッキングを行った。抗体希釈Buffer（組成0.3%TWEEN20／1%牛血清アルブミン／PBS）で1000倍希釈したoccludin抗体（SIGMA HPA005933）及びE-cadherin 23E10抗体（Cell Signaling #8834）を添加して4℃で一晩インキュベートした。翌日、PBS-Tで10分　3回washを行い、1000倍希釈したAlexa Fluor 488 goat anti-rabbit IgG（Invitrogen）及びRhodamine Red goat anti-rabbit IgG（Invitrogen）を添加して暗所・室温で1時間インキュベートし、PBS-Tによるwashを行った。Dapi Fluoromount-G（Southen Biotech 0100-20）を用いて封入した後に、システム生物顕微鏡BX51（OLIMPUS）を用いて観察した。
結果：
　結果を図12に示す。Ct-SLCO1B3 siRNAのトランスフェクションにより、occludinやE-cadherinの発現上昇が認められた。これらの上昇がCt-SLCO1B3の細胞増殖抑制作用に関わっていることが推測された。 Example 12: Immune cell staining of cell adhesion molecule expression in Ct-SLCO1B3 knockdown A549 cells Method:
Cells 48 hours after transfection were replated at 5 × 10 4 cells / well in a 12-well plate with a cover glass, and incubated at 37 ° C. for a further 48 hours. Thereafter, −20 ° C. methanol (Wako 137-01823) was added in an amount of 500 μL / well instead of the medium, and fixation was performed at −20 ° C. for 15 minutes. Wash 10 times 3 times with PBS-T (0.1% TWEEN20 / PBS) and 1 with Blocking Buffer (composition 0.3% TWEEN20 (Wako167-11515) / 5% bovine serum albumin (SIGMA A3912-1009 Lot # LBF7177V) / PBS) Time blocking was performed. Add occludin antibody (SIGMA HPA005933) and E-cadherin 23E10 antibody (Cell Signaling # 8834) diluted 1000 times with antibody dilution buffer (composition 0.3% TWEEN20 / 1% bovine serum albumin / PBS) and incubate overnight at 4 ° C did. The next day, wash with PBS-T for 10 minutes 3 times, add 1000-fold diluted Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen) and Rhodamine Red goat anti-rabbit IgG (Invitrogen) and add 1 in the dark at room temperature. Incubated for a time and washed with PBS-T. After encapsulating with Dapi Fluoromount-G (Southen Biotech 0100-20), observation was performed using system biological microscope BX51 (OLIMPUS).
result:
The results are shown in FIG. Ct-SLCO1B3 siRNA transfection showed increased expression of occludin and E-cadherin. It was speculated that these increases were related to the cell growth inhibitory action of Ct-SLCO1B3.
実施例１３：　Ct-SLCO1B3ノックダウンA549細胞における細胞接着分子、上皮間葉転換制御分子の発現解析
方法：
　一次抗体はE-cadherin、snail、slug（以上Cell Signaling Technology）、occludin、β－actin（以上SIGMA-ALDRICH）を1000倍希釈して用いた。
　細胞をPBSで2回washし、Lysis buffer（10mM Tris、150mM NaCl、1% sodium deoxycholate、0.1% SDS、1% Triton X-100）となるようMilli-Q水に溶解し、使用直前にprotease inhibitorを0.1%量加えて細胞溶解液を回収した。氷上で30分放置後、15,000 rpmにて30分遠心し、上清を回収した。この上清についてDC Protein Assay Reagent（Bio-Rad）を用いLowry法にてタンパク定量を行った。各サンプル間のタンパク濃度をそろえたのち、上清と等量の2×sample buffer（0.5M Tris-HCl（pH6.8）2.5mL、10% SDS 4mL、2－メルカプトエタノール 0.5mL、スクロース 1mLをMilli-Q水で溶解して10mLとした）を加え、37℃で30分加熱しWestern blot用細胞溶解液を得た。
　アクリルアミドゲルを作成し、SDS-PAGE running buffer（Tris base 15.1g、glycine 72.0g、SDS 5.0gをMilli-Q水に溶解して1000mLとした。使用時にMilli-Q水で5倍希釈して用いた）を用いて各lysateの電気泳動を行った。電気泳動終了後のゲルはtransfer buffer（Tris base 30.3g、glycine 144gをMilli-Q水に溶解して1000mLとした。使用時にこのbuffer 20mLに対し、メタノール 40mL、Milli-Q水 140mLを加えて用いた。）に浸し、15分程シェーカーにてなじませた。またImmobilon-P Transfer Membrane（Millipore）はメタノールに浸したのちtransfer bufferに浸し、20分程シェーカーにてなじませた。その後サブマリン式にて25V、60分のtransferを行った。転写終了後のメンブレンはTBS-T（1M Tris-HCl（pH7.4）10mL、5M NaCl 30mL、Tween 20 1mLをMilli-Q水に溶解して1000mLとした。）にて数回washした後、TBS-Tにて3%となるよう希釈したskim milk（森永）にて20分程度のブロッキングを行った。TBS-Tで数回wash後、Can Get Signal Immuno Reaction Enhancer Solution Solution 1 for primary antibody（TOYOBO）にて希釈した一次抗体で一晩4℃にてインキュベートした。翌日TBS-Tで15分×4回washを行った後、Can Get Signal Immuno Reaction Enhancer Solution Solution 2 for secondary antibody（TOYOBO）にて希釈したHRP標識抗rabbit IgG抗体又はHRP標識抗mouseIgG抗体（いずれもCell Signaling Technology）で1時間常温にてインキュベートした。さらにTBS-Tで15分×4回washを行った後、ECL Prime Western Blotting Detection System（GE Healthcare）を滴下して５分間暗所で放置し、ImageQuant LAS 4000mini（GE Healthcare）にて発光を検出した。
結果：
　結果を図１３に示す。Ct-SLCO1B3のノックダウンにより、上皮間葉転換に関わる転写因子であるsnail、slugの発現低下と、それに起因することが推測される接着分子E-cadherin、occludinの発現上昇が認められた。 Example 13: Expression analysis of cell adhesion molecule and epithelial-mesenchymal transition regulatory molecule in Ct-SLCO1B3 knockdown A549 cells Method:
As primary antibodies, E-cadherin, snail, slug (hereinafter Cell Signaling Technology), occludin, and β-actin (hereinafter SIGMA-ALDRICH) were diluted 1000 times.
The cells were washed twice with PBS, dissolved in Milli-Q water to become Lysis buffer (10 mM Tris, 150 mM NaCl, 1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100), and immediately before use, a protease inhibitor Was added in an amount of 0.1% to recover the cell lysate. After standing on ice for 30 minutes, the supernatant was collected by centrifugation at 15,000 rpm for 30 minutes. The supernatant was subjected to protein quantification using DC Protein Assay Reagent (Bio-Rad) by the Lowry method. After aligning the protein concentration between each sample, add 2 × sample buffer (0.5M Tris-HCl (pH6.8) 2.5mL, 10% SDS 4mL, 2-mercaptoethanol 0.5mL, sucrose 1mL) in the same volume as the supernatant. The resultant was dissolved in Milli-Q water to make 10 mL), and heated at 37 ° C. for 30 minutes to obtain a cell lysate for Western blotting.
Prepare an acrylamide gel and dissolve SDS-PAGE running buffer (Tris base 15.1 g, glycine 72.0 g, SDS 5.0 g in Milli-Q water to 1000 mL. When used, dilute 5 times with Milli-Q water. Each lysate was subjected to electrophoresis. The gel after electrophoresis was transferred buffer (Tris base 30.3g, glycine 144g dissolved in Milli-Q water to 1000mL. When used, add 40mL methanol and 140mL Milli-Q water to this buffer 20mL. And soaked in a shaker for about 15 minutes. Immobilon-P Transfer Membrane (Millipore) was soaked in methanol, soaked in transfer buffer, and blended on a shaker for about 20 minutes. Thereafter, a transfer of 25 V and 60 minutes was performed by a submarine method. After completion of the transfer, the membrane was washed several times with TBS-T (1 M Tris-HCl (pH 7.4) 10 mL, 5 M NaCl 30 mL, Tween 20 1 mL dissolved in Milli-Q water to 1000 mL), Blocking was performed for about 20 minutes with skim milk (Morinaga) diluted to 3% with TBS-T. After washing several times with TBS-T, incubation was performed overnight at 4 ° C. with a primary antibody diluted with Can Get Signal Immuno Reaction Enhancer Solution 1 for primary antibody (TOYOBO). The following day, after washing with TBS-T for 15 minutes x 4 times, HRP-labeled anti-rabbit IgG antibody or HRP-labeled anti-mouse IgG antibody diluted with Can Get Signal Immuno Reaction Enhancer Solution Solution 2 for secondary antibody (TOYOBO) (both Cell Signaling Technology) for 1 hour at room temperature. After washing with TBS-T for 15 minutes x 4 times, ECL Prime Western Blotting Detection System (GE Healthcare) was dropped and left in the dark for 5 minutes, and luminescence was detected with ImageQuant LAS 4000mini (GE Healthcare). did.
result:
The results are shown in FIG. Knockdown of Ct-SLCO1B3 showed a decrease in the expression of snail and slug, transcription factors involved in epithelial-mesenchymal transition, and an increase in the expression of adhesion molecules E-cadherin and occludin, which are presumed to be caused by them.
実施例１４：　Ct-SLCO1B3ノックダウンA549細胞におけるmatrix metalloprotease 9（MMP9）の発現解析
方法：
（１）　real-time PCR
　方法は実施例７と同様。
・primer
　MMP9　F :ACCTCGAACTTTGACAGCGACA（配列番号45）
　MMP9　R :GATGCCATTCACGTCGTCCTTA（配列番号46） Example 14: Expression analysis of matrix metalloprotease 9 (MMP9) in Ct-SLCO1B3 knockdown A549 cells Method:
(1) real-time PCR
The method is the same as in Example 7.
・ Primer
MMP9 F: ACCTCGAACTTTGACAGCGACA (SEQ ID NO: 45)
MMP9 R: GATGCCATTCACGTCGTCCTTA (SEQ ID NO: 46)
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
（２）　Western blot
　一次抗体はMMP-9（Cell Signaling Technology）を1000倍希釈して用いた。操作は実施例１３と同様。
（３）　gelatin zymography
　siRNAトランスフェクション48時間後に無血清DMEMで24時間37℃でインキュベートした後、回収した上清500μLをCentrifugal Filter Unit Anicom Ultra-0.5mL、50Kに通してタンパク質を濃縮した。また、サンプルごとの細胞数をカウントした。濃縮した上清は等量の6×Sample Buffer（Milli-Q 1.6mL、625mM Tris 2.4mL、Glycerol 2.4mL、10%SDS、4.8mL BPB（bromo phenol blue）微量）を添加し常温で数分放置した。0.1% Gelatin（Sigma）／10%アクリルアミドゲルを作成し、サンプルを細胞数がそろうようにアプライし、Western Blotと同様の方法で電気泳動した。この時、上清した時の細胞数で上清のサンプルのアプライ量を規定した。ゲルを30分間　Zymogram Renaturating Buffer（life technology）に浸透させた後、30分間　Zymogram Developing Buffer（life technology）に浸透させた。その後、さらにZymogram Developing Bufferを取り換えた後、37℃で1晩インキュベートさせた。ゲルをCBB染色液（組成　methanol 250mL／acetic acid 50mL／MQ 200mL／Coomassie Brilliant Blue（CBB）微量）に浸して約１時間振とうさせた。その後、脱色液（組成　methanol 25mL／acetic acid 37.5mL／MQ 437.5mL）に浸し、約半日振とうさせ、ImageQuant LAS 4000によるバンドの撮影を行いImage Jにて定量した。
結果：
　結果を図14に示す。Ct-SLCO1B3のノックダウンにより、浸潤に機能するMMP9のmRNA、タンパク質発現抑制が認められた。またその酵素活性の低下もzymographyにより確認した。 (2) Western blot
The primary antibody used was MMP-9 (Cell Signaling Technology) diluted 1000 times. The operation is the same as in Example 13.
(3) gelatin zymography
After 48 hours of siRNA transfection, serum-free DMEM was incubated for 24 hours at 37 ° C., and then 500 μL of the collected supernatant was passed through Centrifugal Filter Unit Anicom Ultra-0.5 mL, 50K to concentrate the protein. The number of cells for each sample was counted. Concentrated supernatant is added with equal volume of 6 × Sample Buffer (Milli-Q 1.6mL, 625mM Tris 2.4mL, Glycerol 2.4mL, 10% SDS, 4.8mL BPB (bromophenol blue)) and left at room temperature for several minutes did. A 0.1% Gelatin (Sigma) / 10% acrylamide gel was prepared, and the sample was applied so that the number of cells was the same, and electrophoresed in the same manner as Western Blot. At this time, the applied amount of the supernatant sample was defined by the number of cells at the time of supernatant. The gel was infiltrated with Zymogram Renaturating Buffer (life technology) for 30 minutes and then infiltrated with Zymogram Developing Buffer (life technology) for 30 minutes. Thereafter, the Zymogram Developing Buffer was further replaced, followed by overnight incubation at 37 ° C. The gel was soaked in a CBB staining solution (composition methanol 250 mL / acetic acid 50 mL / MQ 200 mL / Coomassie Brilliant Blue (CBB)) and shaken for about 1 hour. After that, the sample was immersed in a decolorizing solution (composition methanol 25 mL / acetic acid 37.5 mL / MQ 437.5 mL), shaken for about half a day, a band was photographed with ImageQuant LAS 4000, and quantified with Image J.
result:
The results are shown in FIG. By knocking down Ct-SLCO1B3, mRNA and protein expression of MMP9 functioning in invasion was suppressed. The decrease in enzyme activity was also confirmed by zymography.
実施例１５：　Ct-SLCO1B3が発現していない肺癌細胞株NCI-H23を用いたCt-SLCO1B3、Lt-SLCO1B3高発現細胞株の樹立
方法：
（１）　Ct-SLCO1B3、Lt-SLCO1B3高発現用plasmid vectorの作製
　KOD-plus（TOYOBO）を用いてCt-SLCO1B3及びLt-SLCO1B3全長のPCR反応を行った。KOD-plus-PCR反応液組成及びLt-SLCO1B3 primerは実施例３参照。PCR条件及びCt-SLCO1B3全長primerは以下の通りである。得られたPCR産物はアガロースゲル電気泳動後Wizard SV Gel and PCR Clean-up System（Promega）にて切り出し精製を行った。この産物90ngとPerfectly Blunt Cloning Kit（Millipore）を用いてpT7 Blue vector（Millipore）へligationを行った。これをDH5α（Life Technologies）にトランスフォーメーション後、ampicillin含有LB寒天培地に播種し、37℃で一晩インキュベートした。形成されたコロニーを採取し、ampicillin含有LB液体培地3mLで一晩培養し、得られた大腸菌液からWizard Plus SV Minipreps DNA Purification System（Promega）によりプラスミドを精製し、シーケンスの一致を確認した。
　得られたプラスミド及びpcDNA 3.0をMilli-Q水にて44μLにメスアップし、5μLの10×buffer K、1μL　BamHI（TaKaRa）を加え30℃で16時間酵素処理を行った。その後、Wizard SV Gel and PCR Clean-up Systemにて精製した。これらを再びMilli-Q水にて44μLにメスアップし、5μLの10×buffer tangoと1μLのXbaI（fermentas）を加え37℃で20時間酵素処理した。アガロースゲル電気泳動後Wizard SV Gel and PCR Clean-up Systemにて切り出し精製を行った。得られた産物をligation high（TOYOBO）を用いて20時間ligation反応を行った後、DH5α（Life Technologies）にトランスフォーメーションした。これをampicillin含有LB寒天培地に播種し、37℃で一晩インキュベートした。得られたコロニーについて以下のプロトコルを用いてdirect PCRを行い、PCR産物が出来たコロニーをCt-SLCO1B3、Lt-SLCO1B3の全長が組み込まれたpcDNA3.0として以降の実験に用いた。
・primer
　　　　CMV promoter Forward primer:TGACGCAAATGGGCGGTA（配列番号47）
　　　　SLCO1B3 exon1*-3 Reverse primer:CCAGCAAGAGAAGAGGATATGTCA （配列番号31） Example 15: Establishment of Ct-SLCO1B3 and Lt-SLCO1B3 highly expressing cell lines using lung cancer cell line NCI-H23 in which Ct-SLCO1B3 is not expressed Method:
(1) Preparation of plasmid vectors for high expression of Ct-SLCO1B3 and Lt-SLCO1B3 A PCR reaction of full length Ct-SLCO1B3 and Lt-SLCO1B3 was performed using KOD-plus (TOYOBO). See Example 3 for KOD-plus-PCR reaction solution composition and Lt-SLCO1B3 primer. PCR conditions and Ct-SLCO1B3 full length primer are as follows. The obtained PCR product was subjected to agarose gel electrophoresis and then excised and purified by Wizard SV Gel and PCR Clean-up System (Promega). Using this product 90 ng and Perfectly Blunt Cloning Kit (Millipore), ligation was performed to pT7 Blue vector (Millipore). This was transformed into DH5α (Life Technologies) and then inoculated on an ampicillin-containing LB agar medium and incubated overnight at 37 ° C. The formed colonies were picked and cultured overnight in 3 mL of ampicillin-containing LB liquid medium, and the plasmid was purified from the resulting Escherichia coli solution by Wizard Plus SV Minipreps DNA Purification System (Promega) to confirm sequence matching.
The obtained plasmid and pcDNA 3.0 were made up to 44 μL with Milli-Q water, 5 μL of 10 × buffer K and 1 μL BamHI (TaKaRa) were added, and enzyme treatment was performed at 30 ° C. for 16 hours. Then, it refine | purified in Wizard SV Gel and PCR Clean-up System. These were again made up to 44 μL with Milli-Q water, 5 μL of 10 × buffer tango and 1 μL of XbaI (fermentas) were added, and enzyme treatment was performed at 37 ° C. for 20 hours. After agarose gel electrophoresis, it was excised and purified by Wizard SV Gel and PCR Clean-up System. The obtained product was subjected to ligation reaction for 20 hours using ligation high (TOYOBO), and then transformed into DH5α (Life Technologies). This was seeded on ampicillin-containing LB agar medium and incubated overnight at 37 ° C. The obtained colonies were subjected to direct PCR using the following protocol, and the colonies from which PCR products were produced were used in subsequent experiments as pcDNA3.0 into which the full lengths of Ct-SLCO1B3 and Lt-SLCO1B3 were incorporated.
・ Primer
CMV promoter Forward primer: TGACGCAAATGGGCGGTA (SEQ ID NO: 47)
SLCO1B3 exon1 * -3 Reverse primer: CCAGCAAGAGAAGAGGATATGTCA (SEQ ID NO: 31)
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
（２）　Ct-SLCO1B3、Lt-SLCO1B3安定高発現NCI-H23の作製
　NCI-H23細胞を24 well plateへ5×104  cells/wellとなるよう播種した。翌日Ct-SLCO1B3、Lt-SLCO1B3導入pcDNA3.0 vectorを500ng、Lipofectamine 2000（Life Technologies）2μLをOpti-MEM培地内で混合してtransfectionを行い、5時間後に培地を交換した。さらに翌日から終濃度1.0μg／mLのG418（WAKO）を添加し、7日間セレクションを行った。transfectionを行っていないNCI-H23細胞がすべて死滅したことを確認した後、各実験に細胞を用いた。
（３）　real-time PCR
　方法は実施例７と同じであり、使用したprimerは以下の通りである。
　　　　SLCO1B3 exon8-9 F :CCATACCATTTTTTTTCTTGCCGA（配列番号48）
　　　　SLCO1B3 exon8-9 R :ACAGGGGATTGGTAAGGATGC（配列番号49）
（４）　western blot
　一次抗体はSLCO1B3（abcam）を500倍希釈にて用いた。操作は実施例１３と同様である。
結果：
　Lt-SLCO1B3とCt-SLCO1B3安定高発現細胞NCI-H23を樹立することができた。ウエスタンブロット及びRT―PCRの結果を図15に示す。 (2) Preparation of Ct-SLCO1B3 and Lt-SLCO1B3 stably expressing NCI-H23 NCI-H23 cells were seeded on a 24-well plate at 5 × 10 4 cells / well. The next day, 500 ng of Ct-SLCO1B3 and Lt-SLCO1B3-introduced pcDNA3.0 vector and 2 μL of Lipofectamine 2000 (Life Technologies) were mixed in Opti-MEM medium, and transfection was performed. After 5 hours, the medium was changed. Furthermore, G418 (WAKO) with a final concentration of 1.0 μg / mL was added from the next day, and selection was performed for 7 days. After confirming that all non-transfected NCI-H23 cells were killed, the cells were used for each experiment.
(3) real-time PCR
The method is the same as in Example 7, and the primers used are as follows.
SLCO1B3 exon8-9 F: CCATACCATTTTTTTTCTTGCCGA (SEQ ID NO: 48)
SLCO1B3 exon8-9 R: ACAGGGGATTGGTAAGGATGC (SEQ ID NO: 49)
(4) western blot
As the primary antibody, SLCO1B3 (abcam) was used at a 500-fold dilution. The operation is the same as in Example 13.
result:
Lt-SLCO1B3 and Ct-SLCO1B3 stable high expression cells NCI-H23 could be established. The results of Western blot and RT-PCR are shown in FIG.
実施例１６：　Ct-SLCO1B3、Lt-SLCO1B3高発現NCI-H23細胞株の足場非依存性増殖
方法：
　Ct-SLCO1B3及びLt-SLCO1B3高発現NCI-H23細胞を回収し、6000 cells／90μLとなるようFCeM－R培地（日産化学工業株式会社）に希釈しnon coating 96 well plate（IWAKI）へ90μLずつ播種した。その後24、72、120、168時間後にWST-1溶液と1-methoxy PMS溶液（いずれもDOJINDO）を9：1で混合したものを10μL／wellずつ添加し、1時間30分後に450nmで吸光度を測定した。対照波長として630nmを使用した。
結果：
　結果を図16に示す。Ct-SLCO1B3高発現NCI-H23細胞において、足場非依存性増殖促進作用が認められた。 Example 16: Anchorage-independent growth of Ct-SLCO1B3, Lt-SLCO1B3 high expression NCI-H23 cell line Method:
NCI-H23 cells with high expression of Ct-SLCO1B3 and Lt-SLCO1B3 are collected, diluted in FCeM-R medium (Nissan Chemical Industry Co., Ltd.) to 6000 cells / 90 μL, and seeded at 90 μL in a non coating 96 well plate (IWAKI) did. After 24, 72, 120 and 168 hours, add WST-1 solution and 1-methoxy PMS solution (both DOJINDO) 9: 1 at 10 μL / well, and after 1 hour 30 minutes, absorb the absorbance at 450 nm. It was measured. A reference wavelength of 630 nm was used.
result:
The results are shown in FIG. In CCI-SLCO1B3 highly expressing NCI-H23 cells, an anchorage-independent growth promoting action was observed.
実施例１７：　Ct-SLCO1B3、Lt-SLCO1B3高発現NCI-H23細胞株の遊走能
方法：
　細胞を1×105cells／wellとなるよう48 well plateへ播種した。24時間後にチップの先で傷を付け、0、6、12、24、36、48時間後に細胞の遊走面積をImage Jソフトウエアを用いて遊走能を測定した。撮影はFSX100（Olympus）を用いた。
結果：
　結果を図17に示す。Ct-SLCO1B3高発現NCI-H23細胞において、遊走能の亢進が認められた。 Example 17: Ct-SLCO1B3, Lt-SLCO1B3 high expression NCI-H23 cell line migration ability Method:
Cells were seeded in a 48 well plate at 1 × 10 5 cells / well. After 24 hours, the tip of the chip was scratched, and after 0, 6, 12, 24, 36, and 48 hours, the migration area of the cells was measured using Image J software. Photographing was performed using FSX100 (Olympus).
result:
The results are shown in FIG. In Ct-SLCO1B3 highly expressing NCI-H23 cells, enhanced migration ability was observed.
実施例１８：　CRISPR／Cas9システムを利用したCt-SLCO1B3発現抑制A549細胞の作製
方法：
（１）　Ct-SLCO1B3 exon1* guide sequence導入px330の作製
　5μgのpX330-U6-Chimeric_BB-CBh-hSpCas9（Addgene）をBbsI（Thermo Scientific）にて37℃で一晩消化した後、Wizard SV Gel and PCR Clean-up System（Promega）にて精製した。これをAlkaline Phosphatase、Calf Intestinal（CIP）（NEB）にて37℃で一晩脱リン酸化反応を行い、アガロースゲル電気泳動後Wizard SV Gel and PCR Clean-up Systemにて切り出し精製を行った。一方でCt-SLCO1B3 exon1*内の配列を標的とするguide sequence（下表）について、100μMのsence oligo DNA及びantisence oligo DNA 20μLずつをアニールさせた。その後T4 Polynucleotide Kinase（TAKARA）及びATP（TAKARA）を用い37℃で一晩リン酸化反応を行った。得られたリン酸化oligo DNAとBbsIにて消化し脱リン酸化したpx330とligation high（TOYOBO）によるligation反応を行った。得られたplasmidを用いてCompetent Quick DH5α（TOYOBO）に対してtransformationを行い、Wizard Plus SV Minipreps DNA Purification System（Promega）にてplasmidを精製した。 Example 18: Generation of Ct-SLCO1B3 expression-suppressed A549 cells using CRISPR / Cas9 system Method:
(1) Ct-SLCO1B3 exon1 * guide sequence introduced px330 production 5 μg of pX330-U6-Chimeric_BB-CBh-hSpCas9 (Addgene) was digested overnight at 37 ° C with BbsI (Thermo Scientific), then Wizard SV Gel and PCR Purified with Clean-up System (Promega). This was dephosphorylated overnight at 37 ° C. with Alkaline Phosphatase and Calf Intestinal (CIP) (NEB). After agarose gel electrophoresis, it was excised and purified with Wizard SV Gel and PCR Clean-up System. On the other hand, 100 μM sence oligo DNA and 20 μL of antisence oligo DNA were annealed for each guide sequence (the table below) targeting the sequence in Ct-SLCO1B3 exon1 *. Thereafter, phosphorylation was performed overnight at 37 ° C. using T4 Polynucleotide Kinase (TAKARA) and ATP (TAKARA). The resulting phosphorylated oligo DNA and BbsI digested with dephosphorylated px330 and ligation reaction with ligation high (TOYOBO) were performed. The obtained plasmid was transformed into Competent Quick DH5α (TOYOBO), and the plasmid was purified by Wizard Plus SV Minipreps DNA Purification System (Promega).
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
（２）　puromycin耐性vectorの作製
　px330には細胞へのtransfection後のselectionに使用できる薬剤耐性遺伝子が存在しない。そこでウミシイタケルシフェラーゼベクター　pGL4.84［hRlucCP／Puro］（Promega）よりBglII（Thermo Scientific）-BamHI（TAKARA）でルシフェラーゼ遺伝子部分（hRlucCP）を抜き出し、改変puromycin耐性vectorを作製した。
　2μgのpGL4.84［hRlucCP/Puro］をBglII及びBamH Iにて消化後、アガロースゲル電気泳動を行いWizard SV Gel and PCR Clean-up System（Promega）にて切り出し精製を行った。得られた産物をligation high（TOYOBO）を用いてligation反応を行った。得られたplasmidを用いてCompetent Quick DH5α（TOYOBO）に対してtransformationを行い、Wizard Plus SV Minipreps DNA Purification System（Promega）にてplasmidを精製した。
（３）　Ct-SLCO1B3 knock out A549の作製
　A549細胞を24 well plateへ2×104 cells/wellとなるよう播種した。翌日Ct-SLCO1B3 exon1* guide sequence導入px330 vectorを500ng、puromycin耐性vectorを50ng、Lipofectamine 2000（Life Technologies）を1μLをOpti-MEM（Life Technologies）内で混合し、co-transfectionを行い、6時間後に培地を交換した。さらに翌日から終濃度1.0μg／mLのpuromycinを添加し、3日間セレクションを行った。transfectionを行っていないA549細胞がすべて死滅したことを確認した後、限界希釈法によりクローニングを行った。
（４）　Western blot
　一次抗体はSLCO1B3（abcam）を500倍希釈にて用いた。操作は実施例１３と同様である。
結果：
　いずれのコンストラクトでも顕著なCt-SLCO1B3の発現抑制株が樹立できた。ウエスタンブロットの結果を図18に示す。 (2) Production of puromycin resistance vector px330 has no drug resistance gene that can be used for selection after transfection into cells. Therefore, the luciferase gene part (hRlucCP) was extracted from the Renilla luciferase vector pGL4.84 [hRlucCP / Puro] (Promega) with BglII (Thermo Scientific) -BamHI (TAKARA) to prepare a modified puromycin resistant vector.
2 μg of pGL4.84 [hRlucCP / Puro] was digested with BglII and BamHI, then subjected to agarose gel electrophoresis, and excised and purified with Wizard SV Gel and PCR Clean-up System (Promega). The resulting product was subjected to a ligation reaction using ligation high (TOYOBO). The obtained plasmid was transformed into Competent Quick DH5α (TOYOBO), and the plasmid was purified by Wizard Plus SV Minipreps DNA Purification System (Promega).
(3) Preparation of Ct-SLCO1B3 knockout A549 A549 cells were seeded on a 24-well plate at 2 × 10 4 cells / well. Next day Ct-SLCO1B3 exon1 * guide sequence introduced px330 vector 500ng, puromycin resistant vector 50ng, Lipofectamine 2000 (Life Technologies) 1μL in Opti-MEM (Life Technologies), co-transfection, 6 hours later The medium was changed. Further, puromycin with a final concentration of 1.0 μg / mL was added from the next day, and selection was performed for 3 days. After confirming that all A549 cells that had not been transfected were killed, cloning was performed by the limiting dilution method.
(4) Western blot
As the primary antibody, SLCO1B3 (abcam) was used at a 500-fold dilution. The operation is the same as in Example 13.
result:
In any of the constructs, a remarkable Ct-SLCO1B3 expression-suppressed strain was established. The results of Western blotting are shown in FIG.
実施例１９：　Ct-SLCO1B3発現抑制A549細胞の足場非依存性増殖抑制
方法：
（１）　soft agar assay
　DMEM-10%FCS、2×DMEM-20%FCS（粉末状DMEM（Invitrogen）を1／2量のMilli-Q水にて希釈し、フィルター滅菌して用いた）、1.2% agar（BD pharmigen）を用いて、6 well plateに0.4% agarのbottom agarを作成した。さらに0.3% agarのtop agarを作成し、細胞を1000 cells／wellとなるようtop agar中に播種した。37℃、5%CO2で約3週間培養後、0.05%クリスタルバイオレット（ＷＡＫＯ）／50%メタノールを用いて４℃で一晩染色し、翌日にコロニー数を計数した。
（２）　3D培養WST-1 assay
　実施例９と同様にして行った。
結果：
　結果を図19に示す。いずれの細胞も顕著なコロニー形成抑制、3次元培養での増殖抑制作用が認められた。 Example 19: Inhibition of Ct-SLCO1B3 expression Inhibition of anchorage-independent growth of A549 cells Method:
(1) soft agar assay
DMEM-10% FCS, 2 × DMEM-20% FCS (powdered DMEM (Invitrogen) diluted with 1/2 volume of Milli-Q water and used after filter sterilization), 1.2% agar (BD pharmigen) Was used to prepare 0.4% agar bottom agar on a 6 well plate. Furthermore, 0.3% agar top agar was prepared, and cells were seeded in top agar at 1000 cells / well. After culturing at 37 ° C. and 5% CO 2 for about 3 weeks, the cells were stained overnight at 4 ° C. with 0.05% crystal violet (WAKO) / 50% methanol, and the number of colonies was counted the next day.
(2) 3D culture WST-1 assay
The same operation as in Example 9 was performed.
result:
The results are shown in FIG. All of the cells were found to have significant colony formation inhibition and growth inhibition effects in three-dimensional culture.
実施例２０：　Ct-SLCO1B3発現抑制A549細胞のマウス背部皮下移植による腫瘍形成低下
方法：
　15cm dishにて培養したCt-SLCO1B3 knock out A549 clone No.1-1及びcontrol-1に0.025%EDTA/PBSを滴下し5分放置後、スクレーパーを用いて細胞を回収した。PBSによるwashを2回行い、CountessTM Automated Cell Counter（Invitrogen）にて細胞数及び細胞のviabilityを測定した。4×106 cells／50μLとなるようserum free DMEMにて希釈し、等量のBD Matrigel Basement Membrane Matrix High Concentration（BD Biosciences）を加えた。これをインスリン投与用注射筒へとり、100μLずつ雄のBalb/c slc-nu/nuマウス（５週齢、清水実験材料）へ皮下注射を行った。腫瘍径の測定にはノギスを用いた。腫瘍体積の算出式は腫瘍の長径×短径×短径÷2とした。
結果：
　結果を図20に示す。Ct-SLCO1B3発現抑制A549細胞では、コントロール細胞と比べ顕著な腫瘍形成低下が認められた。 Example 20: Ct-SLCO1B3 expression-suppressed A549 cells were subcutaneously transplanted to the back of the mouse to reduce tumor formation Method:
0.025% EDTA / PBS was added dropwise to Ct-SLCO1B3 knockout A549 clone No. 1-1 and control-1 cultured in a 15 cm dish, allowed to stand for 5 minutes, and then cells were collected using a scraper. Washing with PBS was performed twice, and the number of cells and viability of the cells were measured with a Countess ™ Automated Cell Counter (Invitrogen). It diluted with serum free DMEM so that it might become 4 * 10 < 6 > cells / 50microliter, and equal volume BD Matrigel Basement Membrane Matrix High Concentration (BD Biosciences) was added. This was taken into a syringe for insulin administration, and 100 μL each was subcutaneously injected into male Balb / c slc-nu / nu mice (5 weeks old, Shimizu experimental materials). Vernier calipers were used to measure the tumor diameter. The formula for calculating the tumor volume was tumor major axis × minor axis × minor axis ÷ 2.
result:
The results are shown in FIG. Ct-SLCO1B3 expression-suppressed A549 cells showed a marked decrease in tumor formation compared to control cells.
　Ct-SLCO1B3作用機序のまとめ（図21）
　非小細胞肺癌でCt-SLCO1B3の高発現が見られる。その発現は上皮間葉転換に関わるsnail、slugの発現を上昇させる。それらの転写因子はタイトジャンクション形成に関わり、また癌では発現が低下していることが知られているE-cadherinやoccludinの発現抑制に関わることが指示された。一方、snailやslugは浸潤に関わるMMP9の発現を誘導する。またMMP9はE-cadherinやoccludinの発現も抑制する。Ct-SLCO1B3はこれらの機序により非小細胞肺癌の悪性化を惹起していることが考えられた。よってCt-SLCO1B3はそれが発現する非小細胞肺癌の有望な分子標的となり、その発現を抑制する核酸は分子標的治療薬となることが期待される。 Summary of Ct-SLCO1B3 mechanism of action (Figure 21)
High expression of Ct-SLCO1B3 is seen in non-small cell lung cancer. The expression increases the expression of snail and slug involved in epithelial-mesenchymal transition. These transcription factors were involved in tight junction formation and were also involved in the suppression of E-cadherin and occludin expression, which is known to be down-regulated in cancer. On the other hand, snail and slug induce the expression of MMP9 involved in invasion. MMP9 also suppresses the expression of E-cadherin and occludin. Ct-SLCO1B3 is thought to cause malignant transformation of non-small cell lung cancer by these mechanisms. Therefore, Ct-SLCO1B3 is a promising molecular target for the non-small cell lung cancer that it expresses, and nucleic acids that suppress its expression are expected to be molecular target therapeutics.
実施例２１：　Ct-SLCO1B3のexon1*内におけるsiRNA標的候補配列
方法：
　Ct-SLCO1B3に対する最適siRNA配列を決定する目的で表８に示すsiRNA配列を設定し、合成した。 Example 21: siRNA target candidate sequence within exon1 * of Ct-SLCO1B3 Method:
For the purpose of determining the optimal siRNA sequence for Ct-SLCO1B3, the siRNA sequences shown in Table 8 were set and synthesized.
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
実施例２２：　Ct-SLCO1B3 siRNAのノックダウン効率
方法：
　96 well plateにてA549細胞4000 cells/wellとなるようsiRNA transfectionを行い（方法は実施例７と同様）、1日後に培地交換、2日後にCellAmp Direct RNA Prep Kit for RT-PCR（Real Time）（TaKaRa）にてRNAを回収した。このRNA 2μLを用い、PrimeScript RT-PCR Kit（Perfect Real Time）（TaKaRa）にてcDNAを合成しMilli-Q水にて１０倍希釈した。Light Cycler 96（Roche）を用いてduplicateにてreal-time PCRを行った（方法は実施例７と同様）。
　結果を図２２に示す。 Example 22: Knockdown efficiency of Ct-SLCO1B3 siRNA Method:
Perform siRNA transfection in a 96-well plate so that A549 cells become 4000 cells / well (method is the same as in Example 7), medium change after 1 day, CellAmp Direct RNA Prep Kit for RT-PCR (Real Time) after 2 days RNA was recovered with (TaKaRa). Using 2 μL of this RNA, cDNA was synthesized with PrimeScript RT-PCR Kit (Perfect Real Time) (TaKaRa) and diluted 10-fold with Milli-Q water. Real-time PCR was performed in duplicate using Light Cycler 96 (Roche) (the method is the same as in Example 7).
The results are shown in FIG.
実施例２３：　Ct-SLCO1B3 siRNAのA549細胞足場依存性増殖に対する作用評価
方法：
　96 well plateにてA549細胞500 cells/wellとなるようsiRNA transfectionを行い（方法は実施例７と同様）、1日後に培地交換、4日後にWST-1試薬を添加し、吸光度を測定した（方法は実施例８と同様）。
結果：
　結果を図23に示す。 Example 23: Evaluation of the effect of Ct-SLCO1B3 siRNA on A549 cell anchorage-dependent proliferation Method:
The siRNA transfection was performed in a 96-well plate to give A549 cells of 500 cells / well (the method was the same as in Example 7), the medium was changed after 1 day, the WST-1 reagent was added after 4 days, and the absorbance was measured ( The method is the same as in Example 8.)
result:
The results are shown in FIG.
実施例２４：　Ct-SLCO1B3 siRNAのA549細胞足場非依存性増殖に対する作用評価Example 24: Evaluation of the effect of Ct-SLCO1B3 siRNA on A549 cell anchorage-independent growth
方法：
　96 well plateにてA549細胞4000 cells/wellとなるよう表8に記載の二本鎖核酸のsiRNA transfectionを行い（方法は実施例７と同様）、1日後に培地交換、2日後に10% FCS／FCeM-D（日産化学工業）にて30倍希釈した溶液をHydro Cell plate（株式会社セルシード）へ再播種した。浮遊条件下で9日間培養後、WST-1試薬を加え、吸光度を測定した（方法は実施例９と同様）。
結果：
　結果を図24に示す。 Method:
Perform siRNA transfection of the double-stranded nucleic acid shown in Table 8 in a 96-well plate so that A549 cells are 4000 cells / well (the method is the same as in Example 7), medium change after 1 day, and 10% FCS after 2 days / FCeM-D (Nissan Chemical Co., Ltd.) 30-fold diluted solution was replated on Hydro Cell plate (Cell Seed Co., Ltd.). After culturing for 9 days under floating conditions, WST-1 reagent was added and the absorbance was measured (the method was the same as in Example 9).
result:
The results are shown in FIG.
実施例２５：　Ct-SLCO1B3 siRNAのCt-SLCO1B3非発現肺癌細胞NCI-H1975の細胞増殖に対する作用評価
方法：
　96 well plateにてA549細胞1500 cells/wellとなるようsiRNA transfectionを行い（方法は実施例７と同様）、1日後に培地交換、4日後にWST-1試薬を添加し、吸光度を測定した（方法は実施例８と同様）。 Example 25: Evaluation of the effect of Ct-SLCO1B3 siRNA on cell proliferation of non-Ct-SLCO1B3-expressing lung cancer cells NCI-H1975 Method:
The siRNA transfection was performed in a 96-well plate so that A549 cells were 1500 cells / well (the method was the same as in Example 7). After 1 day, the medium was changed. The method is the same as in Example 8.)
結果：
　結果を図25に示す。上記実施例22－25の1次評価により、#26、#27、#29、#30、#32、#33と#4がCt-SLCO1B3特異的作用によりCt-SLCO1B3発現肺癌細胞の足場非依存性増殖を抑制することが示された。そこで次にそれらのsiRNAを用いた2次評価へと進めた。 result:
The results are shown in FIG. According to the primary evaluation in Examples 22-25 above, # 26, # 27, # 29, # 30, # 32, # 33 and # 4 are Ct-SLCO1B3-specific action-dependent Ct-SLCO1B3-expressing lung cancer cell anchorage independent It was shown to suppress sexual growth. Therefore, we proceeded to secondary evaluation using these siRNAs.
実施例２６：　Ct-SLCO1B3 siRNAのA549細胞におけるノックダウン効率の2次評価Example 26: Secondary evaluation of knockdown efficiency of Ct-SLCO1B3 siRNA in A549 cells
方法：
　12 well plateにてA549細胞4×104cells/wellとなるようsiRNA transfectionを行い（方法は実施例７と同様）、1日後に培地交換、2日後にTrizol（Life Technologies）にてRNAを回収し、PrimeScript RT-PCR Kit（Perfect Real Time）（TaKaRa）にてcDNAを合成しMQ水にて10倍希釈した。Light Cycler クイックシステム 350S（Roche）を用いてreal-time PCRを行った（方法は実施例７と同様）。
結果：
　結果を図26に示す。 Method:
Perform siRNA transfection in a 12-well plate so that A549 cells become 4 × 10 4 cells / well (the method is the same as in Example 7). After 1 day, the medium is changed, and after 2 days, RNA is collected with Trizol (Life Technologies). Then, cDNA was synthesized with PrimeScript RT-PCR Kit (Perfect Real Time) (TaKaRa) and diluted 10-fold with MQ water. Real-time PCR was performed using the Light Cycler Quick System 350S (Roche) (the method was the same as in Example 7).
result:
The results are shown in FIG.
実施例２７：　Ct-SLCO1B3 siRNAのA549細胞足場依存性増殖に対する作用２次評価
方法：
　6 well plateにてA549細胞1×105cells/wellとなるようsiRNA transfectionを行い（方法は実施例７と同様）、1日後に培地交換、2日後に96 well plateへ500 cells/wellとなるよう再播種し、接着条件下で5日間培養後、WST-1試薬を加え、吸光度を測定した（方法は実施例８と同様）。
結果：
　結果を図27に示す。 Example 27: Secondary evaluation of the effect of Ct-SLCO1B3 siRNA on A549 cell anchorage-dependent proliferation Method:
Perform siRNA transfection on 6-well plate so that A549 cells become 1 × 10 5 cells / well (method is the same as in Example 7), medium change after 1 day, and 500 cells / well to 96-well plate after 2 days Then, after culturing for 5 days under adhesion conditions, WST-1 reagent was added and the absorbance was measured (the method was the same as in Example 8).
result:
The results are shown in FIG.
実施例２８：　Ct-SLCO1B3 siRNAのA549細胞足場非依存性増殖に対する作用2次評価
方法：
　6 well plateにてA549細胞1×105cells/wellとなるようsiRNA transfectionを行い（方法は実施例７と同様）、1日後に培地交換、2日後に10% FCS／FCeM-D（日産化学工業）にて500 cells／wellとなるよう希釈した細胞懸濁液をHydro Cell plate（株式会社セルシード）へ再播種し、浮遊条件下で9日間培養後、WST-1試薬を加え、吸光度を測定した（方法は実施例９と同様）。
結果：
　結果を図28に示す。 Example 28: Secondary evaluation of the effect of Ct-SLCO1B3 siRNA on A549 cell anchorage-independent growth Method:
Perform siRNA transfection in a 6-well plate to achieve A549 cells of 1 × 10 5 cells / well (the method is the same as in Example 7), medium change after 1 day, 10% FCS / FCeM-D (Nissan Chemical) after 2 days The cell suspension diluted to 500 cells / well in (Industry) was re-seeded on a Hydro Cell plate (Cell Seed Co., Ltd.), cultured for 9 days under floating conditions, added with WST-1 reagent, and the absorbance was measured. (The method is the same as in Example 9).
result:
The results are shown in FIG.
実施例２９：　Ct-SLCO1B3に対する核酸医薬標的配列の決定
　実施例26－28の2次評価結果を踏まえて、Ct-SLCO1B3に対する特異的作用を示すsiRNA標的配列を決定した。その配列はACGTTACTGAATCTACATGTTGCAAGAAGAAAAA（配列番号140）を含む領域に集約されることが分かった（図29）。
　二次評価の結果
　#26、27、29、30、32、33と#4はA549細胞に対して顕著なノックダウン効果を示した。足場非依存増殖に対しては#29は抑制作用を示さず、#30は弱い抑制作用を示したが、#26、27、32、33と#4は顕著な抑制作用を示した。一方#27にはA549細胞の足場依存性増殖抑制作用も弱いながら認められた。よって#4、26、32、33がより特異性が高く、効果的なCt-SLCO1B3に対する配列であることが示された。 Example 29: Determination of nucleic acid drug target sequence for Ct-SLCO1B3 Based on the secondary evaluation results of Examples 26-28, an siRNA target sequence exhibiting a specific action on Ct-SLCO1B3 was determined. The sequence was found to be concentrated in the region containing ACGTTACTGAATCTACATGTTGCAAGAAGAAAAA (SEQ ID NO: 140) (FIG. 29).
Results of the secondary evaluation # 26, 27, 29, 30, 32, 33 and # 4 showed a significant knockdown effect on A549 cells. For anchorage-independent growth, # 29 showed no inhibitory action and # 30 showed a weak inhibitory action, while # 26, 27, 32, 33 and # 4 showed a remarkable inhibitory action. On the other hand, # 27 had a weak anchorage-dependent growth inhibitory effect of A549 cells. Therefore, it was shown that # 4, 26, 32, and 33 are more specific and effective sequences for Ct-SLCO1B3.
実施例３０：　食道癌、肝細胞癌又は膵癌におけるCt-SLCO1B3発現の定量的解析
方法：
　実施例６と同様に、食道癌、肝細胞癌又は膵癌の臨床検体から抽出したtotal RNA 500ngを用いてcDNA合成を行い、real-time PCRによりCt-SLCO1B3の発現量を比較した。
　結果を図30に示す。Ct-SLCO1B3は食道癌、肝細胞癌、膵臓癌では有意な差はみられなかったものの、非癌部と比べて癌部で高発現している検体が存在していることがわかった。 Example 30: Quantitative analysis of Ct-SLCO1B3 expression in esophageal cancer, hepatocellular carcinoma or pancreatic cancer Method:
As in Example 6, cDNA synthesis was performed using 500 ng of total RNA extracted from clinical specimens of esophageal cancer, hepatocellular carcinoma or pancreatic cancer, and the expression level of Ct-SLCO1B3 was compared by real-time PCR.
The results are shown in FIG. Although Ct-SLCO1B3 did not show significant differences in esophageal cancer, hepatocellular carcinoma, and pancreatic cancer, it was found that there were samples that were highly expressed in the cancer area compared to the non-cancer area.
実施例３１：　Ct-SLCO1B3 siRNA投与による抗腫瘍作用
方法：
　A549細胞を0.05% EDTAを用いてdishから剥がし、リン酸緩衝生理食塩水（PBS）で2回洗浄した。細胞はDMEM (Code No.041-29775、和光純薬工業株式会社）とBD Matrigel Basement Membrane Matrix High Concentration (Cat.No.354248、BD Bioscience)を1:1で希釈し、4×106 cells/mouse/100 μlでBALB/c Slc-nuマウス各群6匹（5週齢、日本SLC）に皮下移植した。Control siRNA とCt-SLCO1B3 siRNA#4をAteloGene Local Use “Quick Gelation” (Product No. #1490、高研株式会社）のプロトコルに準拠して、siRNA の終濃度2 nmol/200μlとなるように調整し、4日毎・計4回腫瘍の周囲に投与した。腫瘍径の測定にはノギスを用いた．腫瘍体積の算出式は腫瘍の長径×短径×短径÷2とした。
Ct-SLCO1B3 siRNA#4：5’-ACGUUACUGAAUCUACAUGtt（配列番号：43）、5’-CAUGUAGAUUCAGUAACGUtt（配列番号：44）
Control siRNA：5’-AUCCGCGCGAUAGUACGUAtt（配列番号：141）、5’-UACGUACUAUCGCGCGGAUtt（配列番号：142）
結果：
　結果を図31に示す。Ct-SLCO1B3 siRNA投与により抗腫瘍作用が認められた。 Example 31: Anti-tumor effect by administration of Ct-SLCO1B3 siRNA Method:
A549 cells were detached from the dish using 0.05% EDTA and washed twice with phosphate buffered saline (PBS). Cells were diluted 1: 1 with DMEM (Code No.041-29775, Wako Pure Chemical Industries, Ltd.) and BD Matrigel Basement Membrane Matrix High Concentration (Cat. No. 354248, BD Bioscience), 4 × 10 6 cells / Mouse / 100 μl was subcutaneously transplanted into 6 BALB / c Slc-nu mice in each group (5 weeks old, Japan SLC). Control siRNA and Ct-SLCO1B3 siRNA # 4 is adjusted to a final siRNA concentration of 2 nmol / 200 μl according to the AteloGene Local Use “Quick Gelation” (Product No. # 1490, Koken) protocol. Every 4 days, a total of 4 doses were administered around the tumor. A caliper was used to measure the tumor diameter. The formula for calculating the tumor volume was tumor major axis × minor axis × minor axis ÷ 2.
Ct-SLCO1B3 siRNA # 4: 5'-ACGUUACUGAAUCUACAUGtt (SEQ ID NO: 43), 5'-CAUGUAGAUUCAGUAACGUtt (SEQ ID NO: 44)
Control siRNA: 5'-AUCCGCGCGAUAGUACGUAtt (SEQ ID NO: 141), 5'-UACGUACUAUCGCGCGGAUtt (SEQ ID NO: 142)
result:
The results are shown in FIG. Ct-SLCO1B3 siRNA administration showed an antitumor effect.
　本発明により、Ct-SLCO1B3の発現抑制活性を有する核酸、該核酸を有効成分とする医薬組成物などが提供される。本発明の核酸及び医薬組成物は、Ct-SLCO1B3の発現を抑制し、Ct-SLCO1B3の発現に特徴づけられる疾患の治療・予防に有用である。Ct-SLCO1B3の癌特異的exon領域内に対する核酸医薬は、Ct-SLCO1B3の発現と機能を抑制できるため、肺癌の増殖を抑制し、抗腫瘍作用を有する。よって本発明による核酸医薬はCt-SLCO1B3を発現する肺癌等の癌腫に対して革新的な核酸医薬となる。肺癌は癌の死亡率トップを占める疾患である。また肺癌以外にCt-SLCO1B3が発現する難治性癌も認められることから、その効果は多大である。 The present invention provides a nucleic acid having Ct-SLCO1B3 expression inhibitory activity, a pharmaceutical composition containing the nucleic acid as an active ingredient, and the like. The nucleic acid and pharmaceutical composition of the present invention suppress Ct-SLCO1B3 expression and are useful for the treatment and prevention of diseases characterized by Ct-SLCO1B3 expression. Since the nucleic acid drug for the cancer-specific exon region of Ct-SLCO1B3 can suppress the expression and function of Ct-SLCO1B3, it suppresses the growth of lung cancer and has an antitumor effect. Therefore, the nucleic acid drug according to the present invention is an innovative nucleic acid drug for carcinomas such as lung cancer that express Ct-SLCO1B3. Lung cancer is the disease with the highest cancer mortality. In addition to lung cancer, refractory cancer in which Ct-SLCO1B3 is expressed is also observed, so the effect is great.
　ここで述べられた特許、特許出願明細書、科学文献を含む全ての刊行物に記載された内容は、ここに引用されたことによって、その全てが明示されたと同程度に本明細書に組み込まれるものである。 The contents of all publications, including patents, patent application specifications, and scientific literature mentioned herein, are hereby incorporated by reference herein to the same extent as if all were expressed. Is.
　本出願は、日本で出願された特願2015-111859（出願日：2015年6月2日）を基礎としており、その内容は本明細書に全て包含されるものである。 This application is based on Japanese Patent Application No. 2015-111859 (filing date: June 2, 2015) filed in Japan, the contents of which are incorporated in full herein.

Claims (13)

  1. 　センス鎖及びアンチセンス鎖から成り、少なくとも11個の塩基対の二重鎖領域を含む二本鎖核酸であって、前記アンチセンス鎖中の、少なくとも17個のヌクレオチドかつ多くとも30個のヌクレオチドの鎖長のオリゴヌクレオチド鎖において、下記（I）～（II）のいずれかに記載のDNAの塩基配列と完全に相補する標的Ct-SLCO1B3（癌型溶質キャリヤー有機アニオントランスポーターファミリーメンバー１Ｂ３）RNA配列と相補的である、Ct-SLCO1B3の発現を減少させる二本鎖核酸：
    （I）配列番号1の配列からなるDNA、
    （II）（I）記載のDNAと実質的に同一の配列を有する核酸。     A double-stranded nucleic acid consisting of a sense strand and an antisense strand, comprising a double-stranded region of at least 11 base pairs, comprising at least 17 nucleotides and at most 30 nucleotides in said antisense strand The target Ct-SLCO1B3 (cancer-type solute carrier organic anion transporter family member 1B3) RNA sequence that is completely complementary to the DNA base sequence described in any of (I) to (II) below in an oligonucleotide chain having a chain length A double-stranded nucleic acid that decreases the expression of Ct-SLCO1B3 that is complementary to:
    (I) DNA comprising the sequence of SEQ ID NO: 1,
    (II) A nucleic acid having substantially the same sequence as the DNA described in (I).
  2. 　標的Ct-SLCO1B3 RNA配列が、配列番号2の配列に含まれる、請求項１記載の二本鎖核酸。 The double-stranded nucleic acid according to claim 1, wherein the target Ct-SLCO1B3 RNA sequence is contained in the sequence of SEQ ID NO: 2.
  3. 　標的Ct-SLCO1B3 RNA配列が、配列番号6～13からなる群より選択される、請求項1又は2に記載の二本鎖核酸。 The double-stranded nucleic acid according to claim 1 or 2, wherein the target Ct-SLCO1B3 RNA sequence is selected from the group consisting of SEQ ID NOs: 6 to 13.
  4. 　前記アンチセンス鎖は、配列番号14～21からなる群より選択される配列を含む、請求項1～3のいずれか一項に記載の二本鎖核酸。 The double-stranded nucleic acid according to any one of claims 1 to 3, wherein the antisense strand comprises a sequence selected from the group consisting of SEQ ID NOs: 14 to 21.
  5. 　前記センス鎖は、配列番号22～29からなる群より選択される配列を含む、請求項1～4のいずれか一項に記載の二本鎖核酸。 The double-stranded nucleic acid according to any one of claims 1 to 4, wherein the sense strand includes a sequence selected from the group consisting of SEQ ID NOs: 22 to 29.
  6. 　表1記載の配列番号14／配列番号22、配列番号15／配列番号23、配列番号16／配列番号24、配列番号17／配列番号25、配列番号18／配列番号26、配列番号19／配列番号27、配列番号20／配列番号28、及び配列番号21／配列番号29から成る群から選択される1対のアンチセンス鎖／センス鎖の配列を含む、請求項1～5のいずれか一項に記載の二本鎖核酸。 SEQ ID NO: 14 / SEQ ID NO: 22, SEQ ID NO: 15 / SEQ ID NO: 23, SEQ ID NO: 16 / SEQ ID NO: 24, SEQ ID NO: 17 / SEQ ID NO: 25, SEQ ID NO: 18 / SEQ ID NO: 26, SEQ ID NO: 19 / SEQ ID NO: 7. A sequence of a pair of antisense strands / sense strands selected from the group consisting of 27, SEQ ID NO: 20 / SEQ ID NO: 28, and SEQ ID NO: 21 / SEQ ID NO: 29, according to any one of claims 1-5 The double-stranded nucleic acid described.
  7. 　請求項1～6のいずれか一項に記載の二本鎖核酸の、アンチセンス鎖のみからなる一本鎖核酸。 A single-stranded nucleic acid consisting only of an antisense strand of the double-stranded nucleic acid according to any one of claims 1 to 6.
  8. 　Ct-SLCO1B3の発現を抑制する核酸を有効成分として含む医薬組成物。 A pharmaceutical composition comprising, as an active ingredient, a nucleic acid that suppresses the expression of Ct-SLCO1B3.
  9. 　請求項1～7のいずれか一項に記載の核酸を有効成分として含む、医薬組成物。 A pharmaceutical composition comprising the nucleic acid according to any one of claims 1 to 7 as an active ingredient.
  10. 　癌の治療又は予防用である、請求項８又は９記載の医薬組成物。 The pharmaceutical composition according to claim 8 or 9, which is used for treatment or prevention of cancer.
  11. 　癌がCt-SLCO1B3を発現する非小細胞肺癌、肝癌、膵癌又は食道癌である、請求項10記載の医薬組成物。 11. The pharmaceutical composition according to claim 10, wherein the cancer is non-small cell lung cancer, liver cancer, pancreatic cancer or esophageal cancer that expresses Ct-SLCO1B3.
  12. 　以下の（1）～（3）の工程を含む、癌の予防及び／又は治療作用を有する物質のスクリーニング方法：
    （1）Ct-SLCO1B3を発現する細胞に、被検物質を接触させる工程、
    （2）前記細胞におけるCt-SLCO1B3の発現量又は機能を測定する工程、及び
    （3）被検物質の非存在下において測定した場合と比較して、Ct-SLCO1B3の発現量又は機能を低下させる化合物を、癌の予防及び／又は治療作用を有する物質の候補として選択する工程。     A screening method for a substance having a preventive and / or therapeutic action for cancer, comprising the following steps (1) to (3):
    (1) a step of contacting a test substance with a cell expressing Ct-SLCO1B3,
    (2) a step of measuring the expression level or function of Ct-SLCO1B3 in the cell, and (3) a decrease in the expression level or function of Ct-SLCO1B3 as compared to the case of measurement in the absence of the test substance. A step of selecting a compound as a candidate for a substance having a preventive and / or therapeutic action for cancer.
  13. 　（1）及び（2）の工程を含む、被験動物より採取した試料中のCt-SLCO1B3の発現量を測定することを特徴とする癌の悪性度又は悪性化リスクを試験する方法：
    （1）被験動物より採取した試料中の、Ct-SLCO1B3の発現量を測定する工程、
    （2）正常動物由来の試料において測定した場合と比較して、前記発現量が上昇している被験動物を、癌の悪性度が高いか、又は将来悪性化するリスクが高いと判定する工程。     A method for testing the malignancy or risk of malignancy of cancer, comprising measuring the expression level of Ct-SLCO1B3 in a sample collected from a test animal, comprising the steps of (1) and (2):
    (1) a step of measuring the expression level of Ct-SLCO1B3 in a sample collected from a test animal,
    (2) A step of determining that a test animal having an increased expression level has a high cancer malignancy or a high risk of becoming malignant in the future, as compared with a case of measuring in a sample derived from a normal animal.
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