WO2015012175A1 - Médicament de diagnostic et médicament thérapeutique pour le cancer du poumon à petites cellules - Google Patents

Médicament de diagnostic et médicament thérapeutique pour le cancer du poumon à petites cellules Download PDF

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WO2015012175A1
WO2015012175A1 PCT/JP2014/068937 JP2014068937W WO2015012175A1 WO 2015012175 A1 WO2015012175 A1 WO 2015012175A1 JP 2014068937 W JP2014068937 W JP 2014068937W WO 2015012175 A1 WO2015012175 A1 WO 2015012175A1
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nsr100
mir
small cell
lung cancer
cell lung
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正仁 下條
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学校法人関西医科大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3023—Lung
    • 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
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity

Definitions

  • the present invention relates to a method for detecting small cell lung cancer using the expression level of nSR100 gene as an index, a diagnostic agent used in the method, and a therapeutic agent for small cell lung cancer targeting nSR100 gene.
  • Lung cancer is a major cause of cancer-related deaths worldwide, and can be broadly classified into two types: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC).
  • SCLC small cell lung cancer
  • NSCLC non-small cell lung cancer
  • Small cell lung cancer is known to have a high relevance to smoking. For example, nicotine, a component of tobacco smoke, promotes tumor growth and angiogenesis, and inhibits apoptosis of cancer cells It has been reported.
  • small cell lung cancer is highly malignant and rapidly grows and metastasizes, so it is very important to detect it early.
  • small cell lung cancer is difficult to treat effectively because of its high resistance to chemotherapy. Therefore, development of a more effective treatment method is demanded.
  • REST RE 1- s ilencing t ranscription factor gene
  • Non-Patent Document 1 The REST gene normally expresses a protein having nine zinc finger domains, but a splicing variant protein (sREST protein) having only five zinc finger domains is specifically expressed in small cell lung cancer. Therefore, small cell lung cancer can be detected by using the expression level of this sREST protein or sREST mRNA as an index.
  • NSR100 although (n eural-specific SR -related protein of 100 kDa) protein expressed from the gene is known to have the function of controlling the splicing, the expression of the brain, olfactory bulb, and eyes, etc. of the nervous Since it is limited to tissue, it is considered to be involved in splicing specific to nerve tissue (Non-patent Document 2).
  • An object of the present invention is to find a novel small cell lung cancer marker gene and provide a method for detecting small cell lung cancer and a diagnostic agent used in the method. Furthermore, it aims at providing the therapeutic agent of small cell lung cancer.
  • the present inventors have surprisingly found that the nSR100 gene, whose expression is reported to be restricted to neural tissue, is specifically expressed in small cell lung cancer cells. I found it. In connection with this, the present inventors also found that the amount of miSR-suppressed miRNA increased in the blood of SCLC patients. Furthermore, we found an association between nSR100 gene expression and malignancy of small cell lung cancer. As a result of further research based on these findings, the present invention was completed.
  • the present invention includes the following aspects.
  • Item 1 (A) a polynucleotide having at least 15 bases continuous in the base sequence of human nSR100 mRNA and / or a polynucleotide complementary to the polynucleotide; or (b) a base sequence of human nSR100 mRNA or a base sequence complementary thereto. A polynucleotide having at least 15 bases that hybridizes under stringent conditions; A diagnostic agent for small cell lung cancer containing any of the above.
  • Item 2 The diagnostic agent for small cell lung cancer according to Item 1, wherein the polynucleotide is a probe or a primer.
  • a ′ a polynucleotide having at least 15 bases consecutive in the base sequence of miR-4279, miR-4419b, miR-4516, or miR-4635 and / or a polynucleotide complementary to the polynucleotide, or (b ′ A polynucleotide having at least 15 bases that hybridizes under stringent conditions to the base sequences of miR-4279, miR-4419b, miR-4516, and miR-4635, or a base sequence complementary thereto, A diagnostic agent for small cell lung cancer containing any of the above.
  • Item 4. The diagnostic agent for small cell lung cancer according to Item 3, wherein the polynucleotide is a probe or a primer.
  • Item 5 A diagnostic agent for small cell lung cancer, comprising an antibody that recognizes human nSR100 protein.
  • Item 6. (1) a step of measuring the amount of human nSR100 mRNA or a nucleic acid derived therefrom in a sample derived from lung tissue collected from a subject using the diagnostic agent according to item 1 or 2, and (2) the above step (1 ) Is the amount of human nSR100 mRNA or nucleic acid derived from it (sample mRNA expression level) in human nSR100 mRNA or the amount of nucleic acid derived from it in a sample derived from a tissue not containing small cell lung cancer tissue or nerve tissue ( A step for comparison with the expression level of the control mRNA), Have (3) A method for detecting small cell lung cancer, wherein the test mRNA expression level is higher than the control mRNA expression level, and the subject is a determination index that the subject has small cell lung cancer.
  • Item 7. (1 ") The diagnosis according to Item 3 or 4, wherein at least one amount selected from the group consisting of miR-4279, miR-4419b, miR-4516, and miR-4635 in a blood sample collected from a subject is used And (2 ′′) at least one selected from the group consisting of miR-4279, miR-4419b, miR-4516, and miR-4635 measured in the above step (1 ′′).
  • the amount (miRNA amount to be tested) is selected from the group consisting of miR-4279, miR-4419b, miR-4516, and miR-4635 in a blood sample collected from a subject who is not a small cell lung cancer patient (eg, a healthy subject) Comparing with at least one amount (control miRNA amount) Have (3 ′′) A method for detecting small cell lung cancer, wherein the test miRNA amount is higher than the control miRNA amount, and the subject is an indicator for determining that the subject has small cell lung cancer.
  • Item 8. (1 ′) a step of measuring the expression level of human nSR100 protein in a sample derived from lung tissue collected from a subject using the diagnostic agent according to item 3, and (2 ′) measured in the above step (1 ′). Comparing the expression level of human nSR100 protein (test protein expression level) with the expression level of human nSR100 protein (control protein expression level) in a sample derived from a tissue not containing small cell lung cancer tissue or nerve tissue, Have (3 ′) A method for detecting small cell lung cancer, wherein the test protein expression level is higher than the control protein expression level, and the determination index is that the subject has small cell lung cancer.
  • Item 9 A therapeutic agent for small cell lung cancer, comprising a nucleic acid that suppresses human nSR100 mRNA expression, human nSR100 protein expression, or human nSR100 protein function.
  • Item 10 The therapeutic agent for small cell lung cancer according to Item 9, wherein the nucleic acid is siRNA that specifically suppresses the expression of human nSR100 mRNA.
  • Item 11 The therapeutic agent for small cell lung cancer according to Item 9, wherein the nucleic acid is at least one selected from the group consisting of miR-4279, miR-4419b, miR-4516, and miR-4635.
  • Item 12 A therapeutic agent for small cell lung cancer, comprising an antibody that recognizes human nSR100 protein.
  • nSR100 gene new small cell lung cancer marker gene
  • a diagnostic agent used in the method Furthermore, a therapeutic agent for small cell lung cancer can also be provided.
  • small cell lung cancer can be detected easily and efficiently by using the expression product amount of nSR100 gene or the amount of miSR suppression of nSR100 gene as an index.
  • nSR100 protein regulates the generation of sREST mRNA (or protein), a known small cell lung cancer marker, increasing the expression level of nSR100 mRNA (or protein) in small cell lung cancer cells and suppressing nSR100 gene
  • the increase in the blood level of miRNA is expected to occur prior to the increase in the expression level of sREST mRNA (or protein). Therefore, small cell lung cancer can be detected earlier by using the nSR100 gene expression level or the nSR100 gene suppression miRNA level as an index.
  • the malignancy of small cell lung cancer (such as tumor-forming ability in tissues other than the lung) can also be evaluated.
  • REST genes I to VI
  • REST protein and sREST protein The structures of REST genes (I to VI) and the expression products (REST protein and sREST protein) are shown. Each box from I to VI indicates the exons constituting the REST gene.
  • White bars indicate the structure of the REST protein or sREST protein, and the gray box in the bar indicates the zinc finger domain.
  • the sREST protein is a protein having five zinc finger domains on the N-terminal side, which is generated when a stop codon appears between exon V and exon VI due to frame shift.
  • A shows the result of expression analysis of nSR100 protein in SCLC cells
  • B shows the result of expression analysis of nSR100 protein, REST protein, and sREST protein in SCLC cells.
  • a and B show the abbreviations of the cell names used for expression analysis. Shown on the right side of B is the name of the detected protein.
  • A shows the expression analysis results of nSR100 protein in SCLC cells (N417) adherently cultured in suspension culture or extracellular matrix
  • B shows SCLC cells adherently cultured in suspension culture or extracellular matrix.
  • the expression analysis results of sREST mRNA (upper), REST mRNA (lower left), and nSR100 mRNA (lower right) are shown.
  • the left 2 lanes show the results when the primary antibody (anti-nSR100 antibody) was not reacted
  • the right 2 lanes show the results when the primary antibody (anti-nSR100 antibody) was reacted
  • w / o represents the expression analysis result in SCLC cells cultured in suspension
  • w represents the expression analysis result in SCLC cells adhered and cultured on the extracellular matrix.
  • the lower part of each graph shows the type of cell used for expression analysis. + ECL indicates that cells cultured on the extracellular matrix were used
  • (plate) indicates that cells that had adhered to the culture dish were used, and (medium) did not adhere to the culture dish and was in the medium. It shows that the cells that were floating were used.
  • each graph of B shows the relative expression level when the expression level of GAPDH mRNA is 1.
  • A shows the expression analysis results of REST mRNA (upper), sREST mRNA (middle), and nSR100 mRNA (lower) in NCI-N417 cells in which nSR100 gene is suppressed
  • B shows the results of expression analysis of these proteins.
  • the lower part of each graph of A and the upper part of B show the types of cells used for expression analysis.
  • siRNA (-) indicates that NCI-N417 cells treated with RNAi using non-specific siRNA were used, and siRNA (+) represents NCI-N417 cells treated with RNAi using siRNA against the nSR100 gene. Indicates that it was used.
  • Shown on the right side of B is the name of the detected protein.
  • the vertical axis of each graph of A shows the relative expression level when the expression level of GAPDH mRNA is 1.
  • A shows the photograph of the mouse
  • B shows the expression analysis result of nSR100 (TM) mRNA, sREST (TM) mRNA and REST in the formed tumor.
  • TM nSR100
  • sREST TM
  • REST in the formed tumor.
  • I to III are photographs of mice 60 days after injection
  • IV is a photograph of mice 46 days after injection.
  • the lower part of each graph shows the type of sample injected.
  • N417 is a suspension culture NCI-N417 cell sample (pattern I)
  • N417 + ECL is an adhesion culture NCI-N417 cell sample (pattern III)
  • the vertical axis of each graph of B shows the relative expression level when the expression level of GAPDH mRNA is 1.
  • A shows the results when NCI-N417 cells are used
  • B shows the results when NCI-H82 cells are used
  • C shows the results when NCI-H1650 cells are used.
  • siRNA-A indicates siRNA targeting target sequence 1 (SEQ ID NO: 10)
  • siRNA-B indicates siRNA targeting target sequence 2 (SEQ ID NO: 11)
  • siRNA-C indicates target SiRNA targeted to sequence 3 (SEQ ID NO: 12)
  • siRNA-D represents siRNA targeted to target sequence 4 (SEQ ID NO: 13)
  • siRNA mix represents an equimolar mixture of siRNA-A to siRNA-D Show.
  • the vertical axis represents the blood concentration of each miRNA. “Control” on the horizontal axis indicates data on healthy subjects, “SCLC” indicates data on SCLC patients, “NSCLC” indicates data on NSCLC patients, and “gastric cancer” indicates data on gastric cancer patients.
  • the vertical axis represents the blood concentration of miR-4516.
  • ⁇ Normal '' on the horizontal axis shows data for healthy subjects
  • ⁇ SCLC '' shows data for SCLC patients
  • ⁇ NSCLC '' shows data for NSCLC patients
  • ⁇ gastric '' shows data for gastric cancer patients
  • prostate indicates data for prostate cancer patients
  • kidney indicates data for kidney cancer patients.
  • nucleotide sequences nucleotide sequences
  • nucleic acids in this specification are defined by IUPAC-IUB (IUPAc-IUB communication on Biological Nomenclature, Eur. J. Biochem., 138; 9 (1984)).
  • IUPAC-IUB IUPAc-IUB communication on Biological Nomenclature, Eur. J. Biochem., 138; 9 (1984)
  • “Guidelines for the preparation of specifications including base sequences or amino acid sequences” (edited by the Patent Office) and common symbols in the field.
  • gene refers to a regulatory region, a coding region, an exon, and an intron without distinction unless otherwise specified.
  • DNA includes double-stranded DNA including human genomic DNA, single-stranded DNA including cDNA and synthetic DNA (sense strand), and 1 having a sequence complementary to the sense strand. Both double-stranded DNA (antisense strand) and fragments thereof are included.
  • RNA includes not only single-stranded RNA but also single-stranded RNA having a sequence complementary thereto, and further double-stranded RNA composed thereof, unless otherwise specified. Used for purposes. The RNA includes synthetic RNA such as total RNA, mRNA, rRNA, siRNA.
  • nucleotide “oligonucleotide”, and “polynucleotide” are synonymous with nucleic acid and include both DNA and RNA. These may be double-stranded or single-stranded, and in the case of “nucleotide” having a certain sequence (or “oligonucleotide”, “polynucleotide”), it is complementary to this unless otherwise specified. “Nucleotide” (or “oligonucleotide” and “polynucleotide”) having a typical sequence is also intended to be inclusive. Furthermore, when the “nucleotide” (or “oligonucleotide” and “polynucleotide”) is RNA, the base symbol “T” shown in the sequence listing shall be read as “U”.
  • human nSR100 mRNA is not particularly limited as long as it is an nSR100 mRNA expressed in cells constituting the human body.
  • human nSR100 mRNA include mRNA consisting of the base sequence shown in SEQ ID NO: 1, but as long as the above, 90% or more identity to the base sequence shown in SEQ ID NO: 1, preferably 95% or more It may be mRNA consisting of a base sequence having identity, more preferably 98% or more, and even more preferably 99% or more.
  • miR-4279 “miR-4419b”, “miR-4516”, and “miR-4635” are particularly limited as long as they are the miRNAs expressed in the cells constituting the human body. Not. These miRNAs mean precursor miRNA and / or mature miRNA, but preferably mean mature miRNA.
  • miR-4279 includes, for example, mRNA consisting of the base sequence shown in SEQ ID NO: 14
  • miR-4419b includes, for example, mRNA consisting of the base sequence shown in SEQ ID NO: 15
  • miR-4516 includes, for example, SEQ ID NO: MRNA having the base sequence shown in FIG.
  • miR-4635 includes, for example, the mRNA having the base sequence shown in SEQ ID NO: 17, but as long as the above, the base sequences shown in these SEQ ID NOs. Alternatively, it may be an mRNA comprising a nucleotide sequence having 90% or more identity, preferably 95% or more identity, more preferably 98% or more identity, and even more preferably 99% or more identity.
  • the “human nSR100 protein” is not particularly limited as long as it is an nSR100 protein expressed in cells constituting the human body.
  • the human nSR protein include a protein consisting of the amino acid sequence shown in SEQ ID NO: 2, but 90% or more identity, preferably 95% or more to the amino acid sequence shown in SEQ ID NO: 2 as far as described above. It may be a protein comprising an amino acid sequence having identity, more preferably 98% or more, and even more preferably 99% or more.
  • nucleic acid suppressing the expression of human nSR100 mRNA and the “nucleic acid suppressing the expression of human nSR100 protein” in the present invention include not only siRNA against human nSR100 mRNA but also miRNA, antisense poly (oligonucleotide), ribozyme And decoy are included.
  • nucleic acid that suppresses the function of human nSR100 protein in the present invention means a nucleic acid that has an action of suppressing the function of human nSR100 protein, and includes aptamers for human nSR100 protein.
  • the “antibody” referred to in the present invention includes one of the above antibodies having antigen-binding properties, such as a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single chain antibody, or a Fab fragment or a fragment generated by a Fab expression library. Parts are included.
  • the “diagnostic agent” refers to one that is directly or indirectly used for diagnosing the presence or absence of small cell lung cancer or the extent thereof.
  • these (poly) (oligo) nucleotides and antibodies can be used as probes for detecting the above-described nSR100 mRNA (or protein) expressed in lung tissue in vivo and in vitro, and (poly) (oligo) ) Nucleotides can be effectively used as primers for amplifying the gene expressed in vivo.
  • the present invention provides (a) a polynucleotide having at least 15 bases continuous in the base sequence of human nSR100 mRNA and / or a polynucleotide complementary to the polynucleotide, or (b) A polynucleotide having at least 15 bases that hybridizes under stringent conditions to the base sequence of human nSR100 mRNA or a base sequence complementary thereto,
  • the present invention relates to a diagnostic agent for small cell lung cancer containing any of the above.
  • the diagnostic agent of the present invention is a primer for specifically recognizing and amplifying human nSR100nmRNA or a nucleic acid derived therefrom (cDNA or the like), or a human nSR100 mRNA or nucleic acid (cDNA or the like derived therefrom) specifically. It can be used as a probe for detecting automatically.
  • the diagnostic agent of the present invention is a polynucleotide comprising the base sequence (full-length sequence) of human nSR100 mRNA as long as it selectively recognizes (specifically) human nSR100 mRNA or a nucleic acid derived from it (cDNA or the like). It may be a polynucleotide comprising its complementary sequence. Moreover, the polynucleotide which consists of a partial sequence of the said full length sequence or its complementary sequence may be sufficient. In this case, examples of the partial sequence include a polynucleotide having at least 15 consecutive base lengths arbitrarily selected from the base sequence of the full-length sequence or its complementary sequence.
  • nSR100nmRNA can be specifically detected in, for example, Northern blotting, and human nSR100 mRNA or its origin in RT-PCR.
  • nucleic acid cDNA, etc.
  • cDNA nucleic acid
  • amplification product is derived from human nSR100 mRNA. That's fine.
  • the diagnostic agent of the present invention is characterized by comprising a polynucleotide having at least 15 bases continuous in the base sequence of human nSR100 mRNA and / or a polynucleotide complementary to the polynucleotide.
  • the complementary polynucleotide (complementary strand, reverse strand) is a full-length sequence of a polynucleotide consisting of the base sequence of human nSR100 mRNA, or a partial sequence thereof having a base sequence of 15 bases in length at least in the base sequence (Hereinafter, these are also referred to as “positive strands” for the sake of convenience), it means a polynucleotide having a base-complementary relationship based on a base pair relationship such as A: T and G: C. .
  • such a complementary strand is not limited to the case where it forms a completely complementary sequence with the target positive strand base sequence, but has a complementary relationship that allows it to hybridize with the target positive strand under stringent conditions.
  • stringent conditions here combine complexes or probes as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego CA). It can be determined based on the melting temperature (Tm) of the nucleic acid. For example, as washing conditions after hybridization, conditions of about “1 ⁇ SSC, 0.1% SDS, 37 ° C.” can be mentioned.
  • the complementary strand is preferably one that maintains a hybridized state with the target positive strand even when washed under such conditions.
  • the more stringent hybridization conditions are about “0.5 ⁇ SSC, 0.1% SDS, 42 ° C.”
  • the more severe hybridization conditions are “0.1 ⁇ SSC, 0.1% SDS, 65 ° C.”.
  • a complementary strand a strand consisting of a base sequence that is completely complementary to the base sequence of the target positive strand, and at least 90%, preferably 95%, more preferably Examples thereof include a chain composed of a base sequence having 98% or more identity, more preferably 99% or more identity.
  • the diagnostic agent of the present invention can be designed using, for example, the vector NTI (manufactured by Infomax) based on the base sequence of human nSR100 mRNA shown in SEQ ID NO: 1, for example.
  • a primer or probe candidate sequence obtained by applying the nucleotide sequence of the human nSR100 mRNA to the vector NTI software, or a sequence partially including the sequence can be used as a primer or a probe.
  • the diagnostic agent of the present invention has a length of at least 15 consecutive bases as described above, specifically, the length can be appropriately selected and set according to the use of the diagnostic agent. it can.
  • the diagnostic agent of the present invention can be used as a primer or probe according to a conventional method in a known method for specifically detecting a specific gene, such as Northern blotting, RT-PCR, in situ hybridization, etc. .
  • a specific gene such as Northern blotting, RT-PCR, in situ hybridization, etc. .
  • the lung tissue of the subject and its surrounding tissues, or a part of the lung tissue is collected with a biopsy, etc., and total RNA prepared according to a conventional method is used.
  • a nucleic acid such as cDNA
  • a nucleic acid derived from the RNA may be used.
  • a polynucleotide having at least 15 consecutive nucleotides in the nucleotide sequence of human nSR100SR mRNA and / or a polynucleotide complementary thereto examples thereof include those having a base length of usually 15 bases to 100 bases, preferably 15 bases to 50 bases, more preferably 15 bases to 35 bases.
  • a detection probe those having a base length of usually 15 bases to the entire sequence, preferably 15 bases to 1000 bases, more preferably 100 bases to 1000 bases can be exemplified.
  • the diagnostic agent (probe or primer) of the present invention includes a suitable label for detecting human nSR100 mRNA or nucleic acid (cDNA, etc.) derived therefrom, such as fluorescent dye, enzyme, protein, radioisotope, chemiluminescent substance And those with biotin added thereto.
  • a suitable label for detecting human nSR100 mRNA or nucleic acid (cDNA, etc.) derived therefrom such as fluorescent dye, enzyme, protein, radioisotope, chemiluminescent substance And those with biotin added thereto.
  • fluorescent dye used in the present invention those generally labeled with nucleotides and used for detection and quantification of nucleic acids can be suitably used.
  • HEX 4, 7, 2 ′, 4 ′, 5 ′, 7 '-hexachloro-6-carboxylfluorescein (green fluorescent dye), fluorescein, NED (trade name, Applied Biosystems, yellow fluorescent dye), or 6-FAM (trade name, Applied Biosystems, yellow) Green fluorescent dye), rhodamine or a derivative thereof (for example, tetramethylrhodamine (TMR)), but is not limited thereto.
  • TMR tetramethylrhodamine
  • a method for labeling a nucleotide with a fluorescent dye an appropriate one of known labeling methods can be used [see Nature Biotechnology, 14, 303-308 (1996)].
  • Commercially available fluorescent labeling kits can also be used (for example, oligonucleotide ECL 3'-oligo labeling system manufactured by Amersham Pharmacia).
  • the probe oligo or polynucleotide
  • the diagnostic agent of the present invention is a probe in which the probe (oligo or polynucleotide) is immobilized (for example, a DNA chip, cDNA microarray, oligo DNA array, membrane filter, etc. on which the probe is immobilized. )).
  • the solid phase used for immobilization is not particularly limited as long as it can immobilize oligos or polynucleotides. Examples thereof include glass plates, nylon membranes, microbeads, silicon chips, capillaries or other substrates. be able to.
  • the immobilization of the oligo or polynucleotide to the solid phase is a method of placing a pre-synthesized oligo or polynucleotide on the solid phase, or a method of synthesizing the target oligo or polynucleotide on the solid phase. Also good.
  • the immobilization method is well known in the art depending on the type of immobilization probe, for example, using a commercially available spotter (such as Amersham) in the case of a DNA microarray [for example, photolithographic technique (Affymetrix) In-situ synthesis of oligonucleotides using inkjet technology (Rosetta Inpharmatics).
  • a commercially available spotter such as Amersham
  • a DNA microarray for example, photolithographic technique (Affymetrix) In-situ synthesis of oligonucleotides using inkjet technology (Rosetta Inpharmatics).
  • the probe formed by hybridization with labeled DNA or RNA prepared based on RNA collected from a biological tissue.
  • the presence or expression level (expression level) of human nSR100 mRNA in lung tissue by detecting a complex of the DNA and the labeled DNA or RNA using the labeled DNA or RNA label as an index it can.
  • the above-mentioned DNA chip or the like only needs to contain one or more kinds of the diagnostic agent (probe) of the present invention that can bind to human nSR100 mRNA or a nucleic acid derived therefrom (cDNA or the like).
  • the diagnostic agent (probe) of the present invention that can bind to human nSR100 mRNA or a nucleic acid derived therefrom (cDNA or the like).
  • the diagnostic agent of the present invention is useful for detecting small cell lung cancer in a subject.
  • the diagnosis of small cell lung cancer using the diagnostic agent comprises the amount of human nSR100n mRNA or nucleic acid derived therefrom (test mRNA expression level) in a sample derived from a subject's lung tissue, and the small cell lung cancer tissue and This can be done by determining the difference in the amount of human nSR100 mRNA or nucleic acid derived therefrom (control mRNA expression level) in a tissue-derived sample that does not contain nerve tissue.
  • test mRNA expression level is increased by 50% or more compared to the control mRNA expression level, preferably increased by 100% or more, more preferably increased by 200% or more, the subject has small cell lung cancer. Is likely to be.
  • the present invention is (a ′) at least 15 bases continuous in the base sequence of miR-4279, miR-4419b, miR-4516, or miR-4635 (nSR100 gene-suppressed miRNA) And / or a polynucleotide complementary to the polynucleotide, or (b ′) a string to the base sequence of miR-4279, miR-4419b, miR-4516, and miR-4635 or a base sequence complementary thereto A polynucleotide having at least 15 bases that hybridizes under mild conditions;
  • the present invention relates to a diagnostic agent for small cell lung cancer containing any of the above.
  • the diagnostic agent of the present invention is a primer for specifically recognizing and amplifying nSR100 gene-suppressed miRNA or a nucleic acid derived from it (cDNA or the like), or a nSR100 gene-suppressed miRNA or a nucleic acid derived from it (cDNA or the like).
  • cDNA or the like a primer for specifically recognizing and amplifying nSR100 gene-suppressed miRNA or a nucleic acid derived from it
  • cDNA or the like a nSR100 gene-suppressed miRNA or a nucleic acid derived from it
  • the diagnostic agent of the present invention can selectively (specifically) recognize nSR100 gene-suppressing miRNA or a nucleic acid derived from it (cDNA or the like)
  • the diagnostic agent of nSR100 gene-suppressing miRNA is a polynucleotide comprising the base sequence (full-length sequence) of nSR100 gene-suppressing miRNA It may be a nucleotide or a polynucleotide consisting of its complementary sequence.
  • the polynucleotide which consists of a partial sequence of the said full length sequence or its complementary sequence may be sufficient.
  • examples of the partial sequence include a polynucleotide having at least 15 consecutive base lengths arbitrarily selected from the base sequence of the full-length sequence or its complementary sequence.
  • nSR100 gene-suppressed miRNA can be specifically detected, and in RT-PCR method, nSR100 gene-suppressed miRNA or it can be detected.
  • cDNA or the like the derived nucleic acid
  • the detected product or amplified product is derived from nSR100 gene-suppressed miRNA. Anything is acceptable.
  • the diagnostic agent of the present invention is characterized by comprising a polynucleotide having at least 15 bases continuous in the base sequence of the nSR100 gene-suppressing miRNA and / or a polynucleotide complementary to the polynucleotide.
  • the complementary polynucleotide (complementary strand, reverse strand) is the full-length sequence of a polynucleotide consisting of the base sequence of the nSR100 gene-suppressing miRNA, or a portion thereof having a base sequence of at least 15 bases in length in the base sequence
  • such a complementary strand is not limited to the case where it forms a completely complementary sequence with the target positive strand base sequence, but has a complementary relationship that allows it to hybridize with the target positive strand under stringent conditions.
  • stringent conditions here combine complexes or probes as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego CA). It can be determined based on the melting temperature (Tm) of the nucleic acid. For example, as washing conditions after hybridization, conditions of about “1 ⁇ SSC, 0.1% SDS, 37 ° C.” can be mentioned.
  • the complementary strand is preferably one that maintains a hybridized state with the target positive strand even when washed under such conditions.
  • the more stringent hybridization conditions are about “0.5 ⁇ SSC, 0.1% SDS, 42 ° C.”
  • the more severe hybridization conditions are “0.1 ⁇ SSC, 0.1% SDS, 65 ° C.”.
  • a complementary strand a strand consisting of a base sequence that is completely complementary to the base sequence of the target positive strand, and at least 90%, preferably 95%, more preferably Examples thereof include a chain composed of a base sequence having 98% or more identity, more preferably 99% or more identity.
  • the diagnostic agent of the present invention can be designed using known program software based on the base sequence of the nSR100 gene-suppressed miRNA shown in SEQ ID NO: 14, 15, 16, or 17, for example.
  • the diagnostic agent of the present invention has a length of at least 15 consecutive bases as described above, specifically, the length can be appropriately selected and set according to the use of the diagnostic agent. it can.
  • the diagnostic agent of the present invention can be used as a primer or probe according to a conventional method in a known method for specifically detecting a specific gene, such as Northern blotting, RT-PCR, in situ hybridization, etc. . By using this, it is possible to evaluate the amount of nSR100 gene-suppressed miRNA in the blood sample of the subject.
  • RNA samples to be measured As a sample to be measured, depending on the type of detection method to be used, a blood sample of a subject may be collected by blood collection or the like, and total RNA prepared from the blood sample according to a conventional method may be used, or derived from the RNA. Nucleic acids (cDNA, etc.) may be used.
  • a polynucleotide having at least 15 consecutive nucleotides in the base sequence of the nSR100 gene-suppressing miRNA and / or a complementary polynucleotide thereto examples of nucleotides generally have a length of 15 to 21 bases, preferably 16 to 21 bases.
  • a polynucleotide having at least 15 consecutive bases in the base sequence of nSR100 gene-suppressing miRNA and / or a polynucleotide complementary thereto is usually 15 to 21 bases, preferably 16 bases to One having 21 bases can be exemplified.
  • the diagnostic agent (probe or primer) of the present invention includes an appropriate label for detecting nSR100 gene-suppressed miRNA or a nucleic acid (cDNA, etc.) derived therefrom, such as fluorescent dye, enzyme, protein, radioisotope, chemiluminescence Includes substances, biotin and the like added.
  • an appropriate label for detecting nSR100 gene-suppressed miRNA or a nucleic acid (cDNA, etc.) derived therefrom such as fluorescent dye, enzyme, protein, radioisotope, chemiluminescence Includes substances, biotin and the like added.
  • fluorescent dye used in the present invention those generally labeled with nucleotides and used for detection and quantification of nucleic acids can be suitably used.
  • HEX 4, 7, 2 ′, 4 ′, 5 ′, 7 '-hexachloro-6-carboxylfluorescein (green fluorescent dye), fluorescein, NED (trade name, Applied Biosystems, yellow fluorescent dye), or 6-FAM (trade name, Applied Biosystems, yellow) Green fluorescent dye), rhodamine or a derivative thereof (for example, tetramethylrhodamine (TMR)), but is not limited thereto.
  • TMR tetramethylrhodamine
  • a method for labeling a nucleotide with a fluorescent dye an appropriate one of known labeling methods can be used [see Nature Biotechnology, 14, 303-308 (1996)].
  • Commercially available fluorescent labeling kits can also be used (for example, oligonucleotide ECL 3'-oligo labeling system manufactured by Amersham Pharmacia).
  • the probe oligo or polynucleotide
  • the diagnostic agent of the present invention is a probe in which the probe (oligo or polynucleotide) is immobilized (for example, a DNA chip, cDNA microarray, oligo DNA array, membrane filter, etc. on which the probe is immobilized. )).
  • the solid phase used for immobilization is not particularly limited as long as it can immobilize oligos or polynucleotides. Examples thereof include glass plates, nylon membranes, microbeads, silicon chips, capillaries or other substrates. be able to.
  • the immobilization of the oligo or polynucleotide to the solid phase is a method of placing a pre-synthesized oligo or polynucleotide on the solid phase, or a method of synthesizing the target oligo or polynucleotide on the solid phase. Also good.
  • the immobilization method is well known in the art depending on the type of immobilization probe, for example, using a commercially available spotter (such as Amersham) in the case of a DNA microarray [for example, photolithographic technique (Affymetrix) In-situ synthesis of oligonucleotides using inkjet technology (Rosetta Inpharmatics).
  • a commercially available spotter such as Amersham
  • a DNA microarray for example, photolithographic technique (Affymetrix) In-situ synthesis of oligonucleotides using inkjet technology (Rosetta Inpharmatics).
  • the probe formed by hybridization with labeled DNA or RNA prepared based on RNA collected from a biological tissue.
  • the amount of nSR100 gene-suppressed miRNA in the blood can be evaluated by detecting the complex of the labeled DNA or RNA with the label of the labeled DNA or RNA as an index.
  • the above-mentioned DNA chip or the like only needs to contain one or more kinds of the diagnostic agent (probe) of the present invention that can bind to the nSR100 gene-suppressing miRNA or a nucleic acid derived therefrom (cDNA or the like).
  • the diagnostic agent (probe) of the present invention that can bind to the nSR100 gene-suppressing miRNA or a nucleic acid derived therefrom (cDNA or the like).
  • the diagnostic agent of the present invention is useful for detecting small cell lung cancer in a subject.
  • the diagnosis of small cell lung cancer using the diagnostic agent is performed by measuring the amount of nSR100 gene-suppressed miRNA or nucleic acid derived therefrom (amount of test miRNA) in a subject's blood sample and a subject who is not a patient with small cell lung cancer (for example, it can be carried out by determining the difference in the amount of nSR100 gene-suppressed miRNA or nucleic acid derived therefrom (control miRNA amount) in a blood sample collected from a healthy person. In this case, specifically, if the test miRNA level is increased by 50% or more, preferably 100% or more, more preferably 200% or more, compared to the control miRNA level, the subject has small cell lung cancer. The possibility increases.
  • the present invention provides an antibody capable of specifically recognizing human nSR100 protein as a diagnostic agent.
  • Specific examples of the antibody include an antibody capable of specifically recognizing a protein consisting of the amino acid sequence represented by SEQ ID NO: 2.
  • the present invention is based on the knowledge that human nSR100 protein expression is specifically increased in lung tissue of patients with small cell lung cancer, and by detecting the presence or absence of human nSR100 protein expression and the degree thereof in a subject. This is based on the idea that whether or not the subject has small cell lung cancer and the degree thereof can be specifically detected.
  • the above-mentioned antibody is a tool (diagnostic agent) that can diagnose whether or not the subject has small cell lung cancer by detecting the presence or absence or the extent of the increased expression of the human nSR100 protein in the subject. ).
  • the form of the antibody of the present invention is not particularly limited, and may be a polyclonal antibody using human nSR100 protein as an immunizing antigen or a monoclonal antibody thereof. Furthermore, an antibody having antigen-binding ability to a polypeptide consisting of at least continuous amino acids of the human nSR100 protein, usually 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids, is also included in the antibodies of the present invention. .
  • the antibodies of the present invention can also be produced according to these conventional methods (Current protocol in Molecular Biology, Chapter 11.12 to 11.13 (2000)).
  • an oligopeptide having a partial amino acid sequence of the human nSR100 protein using a human nSR100 protein expressed and purified in Escherichia coli or the like according to a conventional method or according to a conventional method Can be synthesized to immunize non-human animals such as rabbits, and obtained from the sera of the immunized animals according to a conventional method.
  • a human nSR100 protein expressed and purified in Escherichia coli or the like according to a conventional method, or a non-human animal such as a mouse is immunized with an oligopeptide having a partial amino acid sequence of the protein, and the obtained spleen cells and It can be obtained from hybridoma cells prepared by cell fusion with myeloma cells (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.4-11.11).
  • the human nSR100 protein used as an immunizing antigen for antibody production is based on the gene sequence information (for example, SEQ ID NO: 1) provided by the present invention, DNA cloning, construction of each plasmid, transfection into a host, It can be obtained by culturing the transformant and recovering the protein from the culture. These operations are based on methods known to those skilled in the art or methods described in the literature (Molecular Cloning, T.Maniatis et al., CSH Laboratory (1983), DNA Cloning, DM. Glover, IRL PRESS (1985)). Can be done.
  • a recombinant DNA capable of expressing a gene encoding human nSR100 protein in a desired host cell is prepared, introduced into the host cell, transformed, and the transformant is cultured.
  • a protein as an immunizing antigen for producing the antibody of the present invention can be obtained.
  • the partial peptide of human nSR100 protein can also be produced by a general chemical synthesis method (peptide synthesis) according to the amino acid sequence information (SEQ ID NO: 2) provided by the present invention.
  • the antibody of the present invention may be prepared using an oligopeptide having a partial amino acid sequence of human nSR100 protein.
  • the oligo (poly) peptide used for the production of such an antibody does not need to have a functional biological activity, but preferably has an immunogenic property similar to that of human nSR100 protein.
  • An oligo (poly) peptide preferably having this immunogenic property and consisting of at least 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids in the amino acid sequence of human nSR100 protein can be exemplified.
  • Such an antibody against an oligo (poly) peptide can also be produced by enhancing the immunological reaction using various adjuvants depending on the host.
  • adjuvants include, but are not limited to, Freund's adjuvant, mineral gels such as aluminum hydroxide, and surfaces such as lysolecithin, pluronic polyol, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol.
  • Active substances, human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum are included.
  • the antibody of the present invention has the property of specifically binding to human nSR100 protein, human nSR100 protein expressed in the lung tissue of a subject can be specifically detected by using the antibody. That is, the antibody is useful as a probe for detecting the presence and level of human nSR100 protein in a sample derived from lung tissue of a subject.
  • the lung tissue of the subject and its surrounding tissues, a part of the lung tissue is collected with a biopsy, etc., and using a tissue extract or protein prepared according to a conventional method, for example, immunoblotting, ELISA, etc.
  • a tissue extract or protein prepared according to a conventional method for example, immunoblotting, ELISA, etc.
  • human nSR100 protein can be detected by using the antibody of the present invention as a probe according to a conventional method.
  • the amount of human nSR100 protein (test protein expression level) in the sample derived from the lung tissue of the subject and the amount of human nSR100 protein in the sample derived from the tissue not including small cell lung cancer tissue and nerve tissue (control) can be done by determining the difference in protein expression level. In this case, specifically, if the test protein expression level is increased by 50% or more, preferably by 100% or more, more preferably by 200% or more compared to the control protein expression level, the test subject has small cell lung cancer. Is likely to be.
  • the present invention also provides a diagnostic kit for use in the detection and diagnosis of small cell lung cancer comprising the above diagnostic agent.
  • the kit contains at least one oligo or polynucleotide (which may be labeled or immobilized on a solid phase) or the antibody used as the probe or primer.
  • the kit includes other reagents and instruments necessary for carrying out the method of the present invention, such as a hybridization reagent, a probe label, a label detection agent, and a buffer as necessary. May be included as appropriate.
  • Detection method (diagnosis method) of small cell lung cancer includes the following steps (I) to (III): (I) a step of measuring the amount of human nSR100 mRNA or a nucleic acid derived therefrom in a sample derived from lung tissue collected from a subject, or the amount of human nSR100 protein, (II) The amount of human nSR100 mRNA or nucleic acid derived from it measured in the above step (I) or the amount of human nSR100 protein (test mRNA / protein expression level) does not include small cell lung cancer tissue and nerve tissue Steps in comparison with the amount of human nSR100 mRNA or nucleic acid derived from it in a tissue-derived sample, or the amount of human nSR100 protein (control mRNA / protein expression level), and (III) the test compared to the control mRNA / protein expression level A step in which a high mRNA / protein expression level is used as an indicator for determining
  • test mRNA / protein expression level is higher than the control mRNA / protein expression level obtained by such a method, the subject can be judged to have small cell lung cancer.
  • sample used here examples include a sample prepared from lung tissue collected from a subject. Specifically, an RNA-containing sample prepared from the tissue, a sample containing a nucleic acid (cDNA or the like) further prepared therefrom, or a sample containing a protein prepared from the tissue can be mentioned. A sample containing such RNA, nucleic acid or protein can be prepared from a lung tissue of a subject using a biopsy or the like, and prepared therefrom according to a conventional method.
  • RNA-containing sample prepared from the tissue a sample containing a nucleic acid (cDNA or the like) further prepared therefrom, or a sample containing a protein prepared from the tissue can be mentioned.
  • a sample containing such RNA, nucleic acid or protein can be prepared from a lung tissue of a subject using a biopsy or the like, and prepared therefrom according to a conventional method.
  • test mRNA / protein expression level is higher than the control mRNA / protein expression level. Specifically, for example, the test mRNA / protein expression level is increased by 50% or more, preferably by 100% or more, more preferably by 200% or more compared to the control mRNA / protein expression level. be able to.
  • One embodiment of the detection method of the present invention includes the following steps (I ′) to (III ′): (I ′) an amount of at least one selected from the group consisting of miR-4279, miR-4419b, miR-4516, and miR-4635 (nSR100 gene-suppressed miRNA) in a blood sample collected from a subject, A step of measuring using the diagnostic agent according to 3 or 4, (II ′) at least one amount selected from the group consisting of miR-4279, miR-4419b, miR-4516, and miR-4635 (amount of test miRNA) measured in the above step (1 ′′), At least one amount selected from the group consisting of miR-4279, miR-4419b, miR-4516, and miR-4635 in a blood sample collected from a subject who is not a small cell lung cancer patient (eg, a healthy subject) (control miRNA And (III ′) a step in which the test miRNA amount is higher than the control
  • the subject can be judged to have small cell lung cancer.
  • RNA-containing sample prepared from blood or a sample containing a nucleic acid (cDNA or the like) further prepared therefrom can be mentioned.
  • a sample containing such RNA or nucleic acid can be prepared from a subject's blood by blood sampling or the like and prepared therefrom according to a conventional method.
  • test miRNA amount being higher than the control miRNA amount.
  • the amount of test miRNA can be increased by 50% or more, preferably 100% or more, more preferably 200% or more compared to the control miRNA amount.
  • the detection method of the present invention is specifically performed as follows according to the type of measurement object.
  • the detection method is carried out by measuring the amount of human nSR100 mRNA or nucleic acid derived therefrom in the sample. Specifically, Northern blot method, RT-PCR method, DNA chip analysis method, in situ hybridization analysis method, etc., using the diagnostic agent (polynucleotide) of the present invention containing the aforementioned polynucleotide as a primer or probe It can implement by performing the well-known method of these.
  • the presence or amount of human nSR100 mRNA or a nucleic acid derived from it in a sample can be measured by using the diagnostic agent of the present invention as a probe.
  • the diagnostic agent of the invention complementary strand radioisotopes (such as 32 P, 33 P: RI) or a fluorescent substance labeled with a, it, of the subject and transferred to a nylon membrane or the like according to a conventional method
  • a signal derived from the diagnostic agent label such as a duplex between the diagnostic agent and the subject sample mRNA
  • a signal derived from the diagnostic agent label such as a duplex between the diagnostic agent and the subject sample mRNA
  • a radiation detector BAS-1800II, manufactured by Fuji Film
  • a fluorescence detector a fluorescence detector
  • a diagnostic agent is labeled according to the protocol, hybridized with mRNA derived from the subject's sample, and then a signal derived from the label of the diagnostic agent.
  • a method of detecting and measuring with a multi-bioimager STORM860 can also be used.
  • the presence or amount of human nSR100 mRNA or a nucleic acid derived from it in a sample can be measured by using the above-described diagnostic agent of the present invention as a primer.
  • a pair of primers prepared from the diagnostic agent of the present invention (the above cDNA ( ⁇ )) is prepared so that a cDNA can be prepared from RNA derived from a living tissue of a subject according to a conventional method and the target region can be amplified using this as a template.
  • the method can be exemplified by a method in which a normal strand that binds to the strand (strand) and a reverse strand that binds to the strand +) are hybridized with this and subjected to PCR according to a conventional method, and the resulting amplified double-stranded DNA is detected. .
  • 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 a labeled diagnostic agent is used as a probe to hybridize with the probe 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 with SYBR Green RT-PCR Reagents (Applied Biosystems) and react with ABI PRISM 7700 Sequence Detection System (Applied Biosystems) to detect the reaction product. You can also
  • RNA chip analysis When using DNA chip analysis, prepare a DNA chip to which the above-mentioned diagnostic agent of the present invention is attached as a DNA probe (single-stranded or double-stranded), and extract RNA from biological tissue of a subject by a conventional method.
  • An example is a method in which a double strand of DNA and cRNA formed by hybridization with the prepared cRNA is bound to a labeled probe prepared from the diagnostic agent of the present invention and detected.
  • the detection method is carried out by measuring the amount of human nSR100 protein in the sample. Specifically, it can be carried out by performing a known method such as an immunoblot method using the diagnostic agent (antibody) of the present invention containing the antibody described above.
  • the immunoblot method uses a diagnostic antibody of the present invention as a primary antibody, followed by a labeled antibody (primary antibody) labeled with a radioisotope such as 125 I, a fluorescent substance, an enzyme such as horseradish peroxidase (HRP) as a secondary antibody.
  • a radiometer BAS-1800II: manufactured by Fuji Film Co., Ltd.
  • a fluorescence detector etc., detect the signal derived from the labeling substance such as a radioisotope of the obtained labeled compound or a fluorescent substance. And can be implemented by measuring.
  • the ECL Plus Western Blotting Detection System manufactured by Amersham Pharmacia Biotech
  • the multi-bioimager STORM860 manufactured by Amersham Pharmacia Biotech
  • the detection method is carried out by measuring the amount of nSR100 gene-suppressed miRNA or nucleic acid derived therefrom in the sample. Specifically, Northern blot method, RT-PCR method, DNA chip analysis method, in situ hybridization analysis method, etc., using the diagnostic agent (polynucleotide) of the present invention containing the aforementioned polynucleotide as a primer or probe It can implement by performing the well-known method of these.
  • the presence or amount of the nSR100 gene-suppressed miRNA or nucleic acid derived therefrom can be measured in the sample by using the diagnostic agent of the present invention as a probe.
  • the diagnostic agent of the invention complementary strand radioisotopes (such as 32 P, 33 P: RI) or a fluorescent substance labeled with a, it, of the subject and transferred to a nylon membrane or the like according to a conventional method
  • a signal derived from the diagnostic agent label (RI or a labeling substance such as a fluorescent substance) is used as the duplex between the diagnostic agent and the miRNA derived from the subject sample.
  • Examples thereof include a method of detecting and measuring with a radiation detector (BAS-1800II, manufactured by Fuji Film) or a fluorescence detector.
  • a radiation detector BAS-1800II, manufactured by Fuji Film
  • a fluorescence detector a detector for detecting and measuring with a radiation detector.
  • a diagnostic agent is labeled according to the protocol, hybridized with mRNA derived from the subject's sample, and then a signal derived from the label of the diagnostic agent.
  • a method of detecting and measuring with a multi-bioimager STORM860 manufactured by Amersham Pharmacia Biotech can also be used.
  • the presence or amount of nSR100 gene-suppressed miRNA or a nucleic acid derived therefrom can be measured in the sample by using the above diagnostic agent of the present invention as a primer.
  • a pair of primers prepared from the diagnostic agent of the present invention (the above cDNA ( ⁇ )) is prepared so that a cDNA can be prepared from RNA derived from a living tissue of a subject according to a conventional method and the target region can be amplified using this as a template.
  • the method can be exemplified by a method in which a normal strand that binds to the strand (strand) and a reverse strand that binds to the strand +) are hybridized with this and subjected to PCR according to a conventional method, and the resulting amplified double-stranded DNA is detected. .
  • 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 a labeled diagnostic agent is used as a probe to hybridize with the probe 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 with SYBR Green RT-PCR Reagents (Applied Biosystems) and react with ABI PRISM 7700 Sequence Detection System (Applied Biosystems) to detect the reaction product. You can also
  • RNA chip analysis When using DNA chip analysis, prepare a DNA chip to which the above-mentioned diagnostic agent of the present invention is attached as a DNA probe (single-stranded or double-stranded), and extract RNA from biological tissue of a subject by a conventional method.
  • An example is a method in which a double strand of DNA and cRNA formed by hybridization with the prepared cRNA is bound to a labeled probe prepared from the diagnostic agent of the present invention and detected.
  • the present invention relates to a therapeutic agent or preventive agent for small cell lung cancer, which comprises a nucleic acid that suppresses human nSR100 mRNA expression, human nSR100 protein expression, or human nSR100 protein function, or an antibody that recognizes human nSR100 protein. It relates to therapeutic or prophylactic drugs.
  • the siRNA RNA targeted by the present invention is double-stranded, and is formed by hybridizing a sense strand that is complementary to the target sequence of human nSR100 RNA and an antisense strand that is complementary to the sense strand. ing.
  • the duplex may be a molecule having a closed structure at one end, for example, an siRNA (shRNA) having a hairpin structure.
  • siRNA is not particularly limited as long as it suppresses the expression of human nSR100 mRNA (or protein).
  • siRNA those designed using a known siRNA design program may be used, or commercially available siRNA with a guaranteed suppression function may be used.
  • Preferred target sequences for siRNA include SEQ ID NOs: 10, 11, 12, 13 and the like, preferably among these, SEQ ID NOs: 11, 12, 13 and the like, more preferably SEQ ID NOs: 12, 13 and the like. It is done.
  • nucleic acid that suppresses human nSR100 mRNA (or protein)
  • nucleic acid that suppresses human nSR100 gene
  • examples of such nucleic acid include miRNA (preferably miR-4279, miR-4419b, miR-4516, or miR-4635), antisense poly (oligo) nucleotide, ribozyme, aptamer, and decoy for human nSR100 gene.
  • the antisense poly (oligo) nucleotide has a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of human nSR100 mRNA and hybridizes with human nSR100 mRNA.
  • the antisense polynucleotide may be any antisense poly (oligo) nucleotide as long as it has the above-described action, and includes antisense RNA, antisense DNA, and the like.
  • Antisense poly (oligo) nucleotides are usually composed of about 10 to 1000 bases, preferably about 15 to 500 bases, and more preferably about 16 to 30 bases.
  • the phosphate residues (phosphates) of each nucleotide constituting the antisense DNA are changed to chemically modified phosphate residues such as phosphorothioate, methylphosphonate and phosphorodithionate. May be substituted.
  • These antisense poly (oligo) nucleotides can be produced using a known DNA synthesizer or the like.
  • those having an action of suppressing the function of human nSR100 protein can also be used as an active ingredient of the therapeutic agent for small cell lung cancer of the present invention.
  • examples of such an action include antibodies against human nSR100 protein, particularly neutralizing antibodies.
  • the neutralizing antibody means an antibody having a property of inhibiting the function or activity originally possessed by the antigen by binding to the antigen.
  • the neutralizing antibody against human nSR100 protein refers to an antibody having a property of inhibiting the function or activity of human nSR100 protein by binding to human nSR100 protein.
  • the therapeutic agent or preventive agent for small cell lung cancer of the present invention is an active ingredient (siRNA, antisense poly (oligo) nucleotide) that suppresses the expression of the above human nSR100SR mRNA (or protein) or suppresses the function of human nSR100 protein. , Antibodies, etc.), and optional carriers and additives such as pharmaceutically acceptable carriers and additives.
  • Examples of pharmaceutically acceptable carriers and additives include excipients such as sucrose and starch; binders such as cellulose and methylcellulose; disintegrants such as starch and carboxymethylcellulose; lubricants such as magnesium stearate and aerosil Agents: Fragrances such as citric acid and menthol; Preservatives such as sodium benzoate and sodium bisulfite; Stabilizers such as citric acid and sodium citrate; Suspending agents such as methylcellulose and polyvinylpyrrolide; Surfactants and the like Dispersants; diluents such as water and saline; base waxes and the like, but are not limited thereto.
  • excipients such as sucrose and starch
  • binders such as cellulose and methylcellulose
  • disintegrants such as starch and carboxymethylcellulose
  • lubricants such as magnesium stearate and aerosil Agents: Fragrances such as citric acid and menthol; Preservatives such as sodium benzoate and sodium bisul
  • Preparations suitable for oral administration include liquids, capsules, sachets, tablets, suspensions, emulsions and the like.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which include antioxidants Further, a buffer solution, an antibacterial agent, an isotonic agent and the like may be contained.
  • parenteral administration preparations include aqueous and non-aqueous sterile suspensions, including suspensions, solubilizers, thickeners, stabilizers, preservatives, and the like. It may be included.
  • the dose of the preventive or therapeutic agent of the present invention varies depending on the body weight and age of the subject to be administered, the severity of the disease, etc., and cannot be generally set.
  • the amount of active ingredient per day for an adult examples include several mg to several tens mg / kg body weight, which can be administered once to several times a day.
  • the above active ingredient is encoded by DNA
  • gene therapy can be performed as it is or by incorporating it into a gene therapy vector.
  • the dosage and administration method of the gene therapy composition vary depending on the patient's weight, age, symptoms, etc., and can be appropriately selected by those skilled in the art.
  • Example 1 Expression analysis of nSR100 gene in SCLC cells The expression of nSR100 protein in small cell lung cancer (SCLC) cells was examined by immunoblotting. At the same time, the expression of the REST protein (lower part of FIG. 1) and the known SCLC marker sREST protein (REST protein splicing variant: upper part of FIG. 1) was also examined. Specifically, it was performed as follows.
  • NCI-N417 cells American Type Culture Collection: ATCC
  • NCI-H82 cells ATCC
  • NCI-H1650 cells ATCC
  • RPMI1640 medium containing 10% FBS, 2 mM L-glutamine, 100 U / ml penicillin, and 100 ⁇ g / ml streptomycin
  • Suspension culture was performed at °C. The cultured cells were collected and the protein was extracted according to a conventional method. The obtained protein extract was used as a sample.
  • Anti-nSR100 antibody (Santa Cruz, sc-139291) diluted to 1/500 with TBS-T solution containing 1% skim milk, and then diluted to 1/1000 with TBS-T solution containing 1% skim milk
  • the anti-REST antibody (Santa Cruz, H-290) or anti-REST antibody (Millipore, 07-579) diluted to 1/5000 with a TBS-T solution containing 1% skim milk was reacted at room temperature for 1 hour.
  • the PVDF membrane was washed with TBS-T solution and diluted to 1/15000 or 1/20000 with TBS-T solution containing 1% skim milk (IRDye700 or IRDye800 labeled antibody (Li-COR)) For 1 hour at room temperature. Detection was performed using Odyssey fluorescence detection system (LI-COR).
  • FIG. 2A shows the result of examining only the expression of nSR100 protein
  • FIG. 2B shows the result of examining the expression of REST and sREST in addition to nSR100 protein.
  • FIG. 2A shows a band (near 75 kDa) specifically expressing nSR100 protein was observed in NCI-N417 cells and NCI-H82 cells, which are SCLC cells.
  • FIG. 2B shows that the nSR100 protein and the known SCLC marker sREST protein are expressed specifically in SCLC cells.
  • FIG. 2 suggests that nSR100 gene is useful as an SCLC marker.
  • Example 2 Analysis of the influence of extracellular matrix components on the expression of nSR100 gene and REST gene It is known that when SCLC cells are cultured on the extracellular matrix, the tumor-forming ability of SCLC cells is enhanced (Reference 1: PNAS USA) 87, 6698-6702, 1990). Therefore, it was examined how the expression of the nSR100 gene and the REST gene is affected by the enhancement of the tumorigenicity. Specifically, it was performed as follows.
  • NCI-N417 cells human SCLC cells
  • NCI-H1650 cells human NSCLC cells
  • Hela cells are ⁇ 1.1.
  • Suspension culture was performed for 48 hours under the same conditions as in Preparation of immunoblot sample>.
  • NCI-N417 cells or NCI-H1650 cells were cultured on a culture dish coated with an extracellular matrix component (Millipore, ECL cell attachment matrix) for 48 hours under the same conditions as in the above suspension culture.
  • Adhesive culture was performed. The coating was performed by spreading a diluted solution obtained by diluting the ECL cell attachment matrix with PBS to 20 ⁇ g / ml on a culture dish, incubating at 37 ° C. for 1 hour, and removing excess water.
  • the nucleic acid amplification reaction was performed at a temperature cycle of 95 ° C. for 5 minutes ⁇ [(95 ° C. for 5 seconds ⁇ 60 ° C. for 5 seconds) ⁇ 40 cycles].
  • the primer sets used are shown in Table 1 by detection target mRNA.
  • nSR100 protein The expression analysis results of nSR100 protein are shown in FIG. 3A, and the expression analysis results of nSR100 mRNA, REST mRNA, and sREST mRNA are shown in FIG. 3B. From FIG. 3A, it was observed that the expression level of nSR100 protein was enhanced by culturing SCLC cells on the extracellular matrix. From the lower right of FIG. 3B, it was shown that the tendency shown in FIG. 3A is also observed at the mRNA level. In addition, the same tendency was observed for sREST mRNA from the top of FIG. 3B. From these results, it was shown that the expression behavior in SCLC cells was the same for both nSR100 and sREST, which is a known SCLC marker. That is, it was strongly suggested that the nSR100 gene is useful as an SCLC marker.
  • nSR100 acts as a regulator of alternative splicing in a cell-specific manner in neural tissue (Non-patent Document 2). Therefore, the influence of nSR100 gene suppression on the expression of REST gene splicing variants (sREST and REST) was examined. Specifically, it was performed as follows.
  • NCI-N417 cells human SCLC cells
  • NCI-H1650 cells human NSCLC cells
  • RNAi> NCI-N417 cells in logarithmic growth phase were seeded in a 6-well plate at 1 ⁇ 10 ⁇ 5 cells / well and cultured for 24 hours. Thereafter, siRNA (Dharmacon, SMART-pool siRNAs) or non-specific siRNA (Dharmacon, SMART-pool siRNAs) for the nSR100 gene was transfected using a commercially available transfection reagent (DharmaFECT siRNA Transfection Reagents (Thermo Scientific)). . The culture was further continued, and the cells were collected 48 hours after transfection. In addition, siRNA (Dharmacon, SMART-pool siRNAs) for the nSR100 gene is a mixture of siRNAs targeting each of the four sequences on nSR100 mRNA shown in Table 2.
  • Quantitative real-time PCR was performed in the same manner as in Example 2 from cells not treated with RNAi and cells treated with RNAi.
  • FIG. 4A The expression analysis results of REST mRNA, sREST mRNA, and nSR100 mRNA are shown in FIG. 4A, and the expression analysis results of REST protein, sREST protein, and nSR100 protein are shown in FIG. 4B.
  • 4A and B show that the suppression of the nSR100 gene decreases the expression of sREST mRNA and protein, and increases the expression of REST mRNA and protein. This suggests that in SCLC cells, the enhancement of nSR100 protein controls the splicing of the REST gene, and as a result, the expression of splicing variants sREST mRNA and protein is increased.
  • the enhancement of the known SCLC marker (sREST) in SCLC cells is controlled by the nSR100 protein, which strongly suggests that the nSR100 gene is useful as an SCLC marker.
  • the results of FIG. 4 that the expression of REST mRNA and protein is enhanced by suppression of the nSR100 gene and the finding that apoptosis of SCLC cells can be induced by forced expression of REST protein (Reference 2: Oncogene 22, 5636- 5645, 2003) suggests that suppression of the nSR100 gene can induce apoptosis of SCLC cells, that is, suppression of the nSR100 gene is effective for the treatment of SCLC.
  • Example 4 Relationship between tumor-forming ability of SCLC cells and expression of nSR100 gene and REST gene The relationship between tumor-forming ability of SCLC cells and expression of nSR100 gene and REST gene was examined. Specifically, it was performed as follows.
  • NCI-N417 cells human SCLC cells
  • Example 2 suspension-cultured NCI-N417 cells were also prepared. The culture was performed for 7 days.
  • FIG. 5A A photograph of the mouse after the tumor formation experiment is shown in FIG. 5A, and the expression analysis results of nSR100 mRNA and sREST mRNA in the formed tumor are shown in FIG. 5B.
  • FIG. 5A no significant tumor formation was observed when SCLC cells cultured in suspension were injected (the middle part on the left side of the mouse of I), but SCLC cells cultured on an extracellular matrix or cultured in suspension.
  • SCLC cells cultured in suspension the middle part on the left side of the mouse of I
  • FIG. 5B A photograph of the mouse after the tumor formation experiment was shown in FIG. 5A, and the expression analysis results of nSR100 mRNA and sREST mRNA in the formed tumor are shown in FIG. 5B.
  • FIG. 5A no significant tumor formation was observed when SCLC cells cultured in suspension were injected (the middle part on the left side of the mouse of I), but SCLC cells cultured on an extracellular matrix or cultured in suspension.
  • a mixture of SCLC cells and Matrigel
  • Example 5 The presence and extent of apoptosis due to inhibition of the nSR100 gene was evaluated using the LDH activity specific to apoptosis induced by SCLC cells due to the suppression of the nSR100 gene as an index. Specifically, it was performed as follows.
  • NCI-N417 cells human SCLC cells
  • NCI-H82 cells human SCLC cells
  • NCI-H1650 cells human NSCLC cells
  • siRNA-A siRNA targeting target sequence 1 (SEQ ID NO: 10)
  • siRNA-B siRNA targeting target sequence 2 (SEQ ID NO: 11)
  • siRNA-C target sequence 3
  • siRNA targeting SEQ ID NO: 12 siRNA targeting target sequence 4 (SEQ ID NO: 13)
  • siRNA mix an equimolar mixture of siRNA-A to siRNA-D
  • siRNA camptothecin
  • camptothecin an anticancer agent
  • LDH activity measurement An LDH activity measurement kit (Takara LDH Cytotoxicity Detection kit, MK401) was used according to the method described in the package insert.
  • Example 6 Analysis of miSR levels in the blood of SCLC patients miRNA expression in SCLC cells was analyzed and miRNAs expected to suppress nSR100 gene expression (hsa-miR-4279 (SEQ ID NO: 14), hsa-miR) -4419b (SEQ ID NO: 15), hsa-miR-4516 (SEQ ID NO: 16), and hsa-miR-4635 (SEQ ID NO: 17) expression levels are decreased in SCLC cells compared to normal cells was found. From this, it was considered that miRNAs that suppress the expression of the nSR100 gene are excreted outside the SCLC cells in SCLC patients, and as a result, the expression level of the nSR100 gene in the SCLC cells is increased. If this hypothesis is correct, SCLC diagnosis is possible by measuring the amount of miRNA excreted outside the SCLC cells. Therefore, in order to confirm this hypothesis, the amount of miRNA in the blood of SCLC patients was analyzed. Specifically, it was performed as follows.
  • a reverse transcription reaction kit Qiagen, miScript II RT Kit
  • the nucleic acid amplification reaction was performed at a temperature cycle of 95 ° C. for 15 minutes ⁇ [(94 ° C. for 15 seconds ⁇ 55 ° C. for 30 seconds ⁇ 70 ° C. for 30 seconds) ⁇ 40 cycles].
  • each miRNA hsa-miR-4279 (sequence number 14), hsa-miR-4419b (sequence number 15), hsa-miR-4516 (sequence number 16), and hsa-miR-4635 (sequence)
  • a primer commercially available from Qiagen was used as the detection primer of No. 17)).
  • each black circle represents the data of each subject. From FIG. 7, the amount of miRNA (miR-4279, miR-4419b, miR-4516, and miR-4635) that suppresses the expression of the nSR100 gene was increased in the blood of SCLC patients. This supported the hypothesis that in SCLC patients, miRNAs that suppress nSR100 gene expression were excreted outside the SCLC cells, resulting in increased expression levels of the nSR100 gene in SCLC cells. It was also shown that SCLC diagnosis is possible by measuring the amount of miRNA in the blood.
  • Example 7 Expression analysis of nSR100 gene regulatory factor in blood of various cancer patients From the results of Example 6 (FIG. 7), miR-4516, which had the highest blood volume, was treated with various cancer patients (SCLC, NSCLC, gastric cancer, bladder cancer, The amount in the blood of prostate cancer, kidney cancer, and healthy subjects was measured in the same manner as in Example 6.
  • each bar shows the data of each subject.
  • FIG. 8 shows that the amount of miR-4516 in blood tends to increase in SCLC patients among various cancer patients. From this, it was shown that SCLC diagnosis can be performed with high accuracy by measuring the amount of miR-4516 in the blood.

Abstract

La présente invention concerne un nouveau gène marqueur du cancer du poumon à petites cellules (gène nSR100) ainsi qu'un procédé de détection du cancer du poumon à petites cellules, un médicament de diagnostic du cancer du poumon à petites cellules et un médicament thérapeutique du cancer du poumon à petites cellules utilisant ce gène.
PCT/JP2014/068937 2013-07-22 2014-07-16 Médicament de diagnostic et médicament thérapeutique pour le cancer du poumon à petites cellules WO2015012175A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020027227A1 (fr) 2018-07-31 2020-02-06 国立大学法人大阪大学 Agent thérapeutique contre le cancer du poumon à petites cellules contenant un oligonucléotide
CN111926080A (zh) * 2014-06-18 2020-11-13 东丽株式会社 肺癌的检测试剂盒或装置以及检测方法
WO2022181807A1 (fr) * 2021-02-25 2022-09-01 国立大学法人大阪大学 Oligonucléotide pour induire le saut de n-exons pendant le traitement des précurseurs de l'arnm rest
CN112513062B (zh) * 2018-07-31 2024-05-10 国立大学法人大阪大学 含有寡聚核苷酸的小细胞肺癌治疗药

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009502115A (ja) * 2005-07-27 2009-01-29 オンコセラピー・サイエンス株式会社 小細胞肺癌の診断方法
WO2011076144A1 (fr) * 2009-12-24 2011-06-30 Fudan University Compositions et méthodes de profilage du cancer du poumon par expression de microarn dans le plasma

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009502115A (ja) * 2005-07-27 2009-01-29 オンコセラピー・サイエンス株式会社 小細胞肺癌の診断方法
WO2011076144A1 (fr) * 2009-12-24 2011-06-30 Fudan University Compositions et méthodes de profilage du cancer du poumon par expression de microarn dans le plasma

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
DU L. ET AL.: "MicroRNA expression distinguishes SCLC from NSCLC lung tumor cells and suggests a possible pathological relationship between SCLCs and NSCLCs", J. EXP. CLIN. CANCER RES., vol. 29, no. 75, 2010, pages 1 - 12 *
GURROLA-DIAZ C. ET AL.: "Reduced expression of the neuron restrictive silencer factor permits transcription of glycine receptor alphal subunit in small- cell lung cancer cells", ONCOGENE, vol. 22, no. 36, 2003, pages 5636 - 5645 *
ORTUNO-PINEDA C. ET AL.: "Binding of hnRNP H and U2AF65 to respective G-codes and a poly- uridine tract collaborate in the N50-5'ss selection of the REST N exon in H69 cells", PLOS ONE, vol. 7, no. 7, 2012, pages 1 - 11 *
RAJ B. ET AL.: "Cross-regulation between an alternative splicing activator and a transcription repressor controls neurogenesis", MOL. CELL, vol. 43, no. 5, 2011, pages 843 - 850, XP028279235, DOI: doi:10.1016/j.molcel.2011.08.014 *
SHIMOJO M. ET AL.: "The small cell lung cancer- specific isoform of RE1-silencing transcription factor (REST) is regulated by neural-specific Ser/Arg repeat-related protein of 100 kDa (nSR100", MOL. CANCER RES., vol. 11, no. 10, 2013, pages 1258 - 1268 *
ZHENG D. ET AL.: "Plasma microRNAs as novel biomarkers for early detection of lung cancer", INT. J. CLIN. EXP. PATHOL., vol. 4, no. 6, 2011, pages 575 - 586 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111926080A (zh) * 2014-06-18 2020-11-13 东丽株式会社 肺癌的检测试剂盒或装置以及检测方法
WO2020027227A1 (fr) 2018-07-31 2020-02-06 国立大学法人大阪大学 Agent thérapeutique contre le cancer du poumon à petites cellules contenant un oligonucléotide
CN112513062A (zh) * 2018-07-31 2021-03-16 国立大学法人大阪大学 含有寡聚核苷酸的小细胞肺癌治疗药
JPWO2020027227A1 (ja) * 2018-07-31 2021-08-10 国立大学法人大阪大学 オリゴヌクレオチドを含有する小細胞肺癌治療薬
US20210277398A1 (en) * 2018-07-31 2021-09-09 Osaka University Small cell lung cancer therapeutic agent containing oligonucleotide
EP3831837A4 (fr) * 2018-07-31 2023-06-14 Osaka University Agent thérapeutique contre le cancer du poumon à petites cellules contenant un oligonucléotide
US11866705B2 (en) 2018-07-31 2024-01-09 Osaka Univerity Small cell lung cancer therapeutic agent containing oligonucleotide
JP7473113B2 (ja) 2018-07-31 2024-04-23 国立大学法人大阪大学 オリゴヌクレオチドを含有する小細胞肺癌治療薬
CN112513062B (zh) * 2018-07-31 2024-05-10 国立大学法人大阪大学 含有寡聚核苷酸的小细胞肺癌治疗药
WO2022181807A1 (fr) * 2021-02-25 2022-09-01 国立大学法人大阪大学 Oligonucléotide pour induire le saut de n-exons pendant le traitement des précurseurs de l'arnm rest

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