WO2021132309A1 - Pharmaceutical composition - Google Patents

Pharmaceutical composition Download PDF

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WO2021132309A1
WO2021132309A1 PCT/JP2020/048114 JP2020048114W WO2021132309A1 WO 2021132309 A1 WO2021132309 A1 WO 2021132309A1 JP 2020048114 W JP2020048114 W JP 2020048114W WO 2021132309 A1 WO2021132309 A1 WO 2021132309A1
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mir
gene
cancer
tumor
cells
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PCT/JP2020/048114
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French (fr)
Japanese (ja)
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稲澤　譲治
泰行 玄
井上　純
智輝 村松
祐希 ▲高▼川
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国立大学法人 東京医科歯科大学
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Publication of WO2021132309A1 publication Critical patent/WO2021132309A1/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
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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

Definitions

  • the present invention relates to a pharmaceutical composition.
  • Cancer is the leading cause of death in Japan, and according to the statistics of the National Cancer Center (2012), the probability of getting cancer in a lifetime is high at 63% for Japanese men and 47% for women. According to Ministry of Health, Labor and Welfare statistics (2015), cancer accounts for 32% of Japanese deaths, 24% of women, and about one-third of the total population dies of cancer. Cancer deaths continue to increase, with 11.4 million people worldwide expected to die from cancer annually in 2030.
  • WO 2019/107487 focuses on the relationship between bromodomain protein (BRD4) and cancer, and contains specific microRNAs and pharmaceutical compositions used in the treatment of tumors. Proposed.
  • An object of the present invention is to provide a pharmaceutical composition that suppresses the expression of a bromodomain protein (BRD4) gene and can be used for treating tumors.
  • BBD4 bromodomain protein
  • the first aspect encodes at least one microRNA selected from the group consisting of miR-1293, miR-876-3p, miR-4438, miR-6751, miR-634 and miR-92a-2-5p.
  • it contains a polynucleotide having the same base sequence as the transcript of a gene or its processing product, or a base sequence in which 1 to 5 bases are substituted, deleted or added in the base sequence of the transcript or its processing product.
  • the second aspect encodes at least one microRNA selected from the group consisting of miR-1293, miR-876-3p, miR-4438, miR-6751, miR-634 and miR-92a-2-5p.
  • BRD4 gene-related disease containing a polynucleotide having the same base sequence as the transcript of a gene or its processing product, or a base sequence in which 1 to 5 bases are substituted, deleted or added in the base sequence of the transcript or its processing product. It is a pharmaceutical composition used for the treatment of.
  • the third aspect encodes at least one microRNA selected from the group consisting of miR-1293, miR-876-3p, miR-4438, miR-6751, miR-634 and miR-92a-2-5p.
  • a method of treating a tumor including administering the dose to the subject.
  • the fourth aspect encodes at least one microRNA selected from the group consisting of miR-1293, miR-876-3p, miR-4438, miR-6751, miR-634 and miR-92a-2-5p.
  • a method of treating a BRD4 gene-related disorder which comprises administering an amount to a subject.
  • the present invention it is possible to provide a pharmaceutical composition that suppresses the expression of the BRD4 gene and can be used for treating tumors.
  • FIG. 5 is a diagram showing the effect on cell proliferation when knockdown of APEX1 gene, PRA1 gene, and POLD4 gene is performed on colorectal cancer cell line using siRNA.
  • microRNA includes a polynucleotide (oligonucleotide) composed of a ribonucleotide, a polynucleotide (oligonucleotide) composed of a ribonucleotide and a modified nucleotide, and a polynucleotide (oligonucleotide) composed of a modified nucleotide. included.
  • the microRNA may be single-stranded or double-stranded.
  • the pharmaceutical composition is at least one microRNA selected from the group consisting of miR-1293, miR-876-3p, miR-4438, miR-6751, miR-634 and miR-92a-2-5p.
  • a polynucleotide having the same base sequence as a transcript of a gene encoding (hereinafter, also referred to as a specific microRNA) or a processing product thereof, or a polynucleotide having the same base sequence as the transcript thereof or a processing product thereof contains 1 to 5 bases.
  • the pharmaceutical composition may be an agent that suppresses the expression of the BRD4 gene.
  • the pharmaceutical composition is at least one micro selected from the group consisting of miR-1293, miR-876-3p, miR-4438, miR-6751, miR-634 and miR-92a-2-5p.
  • the tumor to be treated may be a tumor expressing the BRD4 gene.
  • the pharmaceutical composition encodes at least one microRNA selected from the group consisting of miR-1293, miR-876-3p, miR-4438, miR-6751 and miR-92a-2-5p.
  • BRD4 is a protein having two bromodomains that bind to acetylated modified lysine and a region that binds to p-TEFb (positive transcription evolution factor b) on the C-terminal side. Acetylation modification of histones is positively correlated with transcriptional activity, and p-TEFb activates transcription by inactivating a protein that suppresses transcriptional RNA polymerase II.
  • BRD4 is said to have the function of mobilizing p-TEFb and promoting transcription by binding to the lysine of acetylated modified histones.
  • abnormal activation of BRD4 gene expression is thought to contribute to tumor cell proliferation. Therefore, suppressing the expression of the BRD4 gene may be effective in the treatment of cancer.
  • MicroRNAs are endogenous, suppressing gene expression by interfering with their translation or stabilization through binding of the target transcript (mRNA) to the coding (CDS) region or the 3'untranslated region (3'UTR). It is a small non-coding RNA.
  • mRNA target transcript
  • CDS coding
  • 3'UTR 3'untranslated region
  • miR-1293, miR-876-3p, miR-4438, miR-6751, miR-634 and miR-92a-2-5p are excellent for tumor cells in which expression of BRD4 gene is observed by suppressing expression of BRD4 gene.
  • at least miR-876-3p suppresses the expression of the RSK1 gene and has an excellent growth-suppressing effect on tumor cells in which the expression of the RSK1 gene is observed.
  • at least miR-1293 has an excellent growth inhibitory effect on tumor cells in which expression of at least one selected from the group consisting of APEX1, RPA1 and POLD4 is observed.
  • the specific microRNA By suppressing the expression of not only the BRD4 gene but also other tumor-related genes, the specific microRNA can exhibit a more excellent growth-suppressing effect on tumor cells. Furthermore, at least miR-1293 has an excellent growth inhibitory effect on tumor cells having a pathogenic mutation in the BRCA1 / 2 gene or in a BRCAness state.
  • the polynucleotide contained in the pharmaceutical composition encodes a microRNA selected from the group consisting of miR-1293, miR-876-3p, miR-4438, miR-6751, miR-634 and miR-92a-2-5p. It may have the same base sequence as the transcript of the gene or its processing product.
  • the primary transcript of the gene encoding the microRNA is called the early transcript (pri-miRNA) and generally has a stem-loop hairpin structure.
  • Pri-miRNAs are converted to mature miRNA precursors (pre-miRNAs) with a stem-loop structure by an RNase III-like enzyme called Drosha.
  • transcripts of genes encoding microRNAs or processing products thereof include tri-miRNAs, pre-miRNAs and mature miRNAs.
  • double-stranded mature miRNA there are three embodiments: when only one strand exerts a desired effect, when each strand exerts a desired effect, and when a double-stranded state exerts a desired effect.
  • the miRNA-derived polynucleotides herein may be either single-stranded or double-stranded, preferably double-stranded.
  • the polynucleotide contained in the pharmaceutical composition is a microRNA selected from the group consisting of miR-1293, miR-876-3p, miR-4438, miR-6751, miR-634 and miR-92a-2-5p. It may have the same base sequence as a mutant that retains the function of the transcript of the encoding gene or its processing product.
  • the mutant may have a base sequence in which 1 to 5 bases are substituted, deleted or added in the base sequence of the transcript of the gene encoding microRNA or the processing product thereof.
  • the number of bases substituted, deleted or added is preferably 1 to 3, and may be 1 or 2.
  • the mutant may have, for example, 80% or more, preferably 85% or more, 90% or more, or 95% or more sequence homology with respect to the original base sequence. Sequence homology is calculated using, for example, BLAST.
  • miR-1293 may be hsa-miR-1293 and may have the base sequence of SEQ ID NO: 1.
  • miR-876-3p may be hsa-miR-876-3p and may have the nucleotide sequence of SEQ ID NO: 2.
  • miR-4438 may be hsa-miR-4438 and may have the nucleotide sequence of SEQ ID NO: 3.
  • miR-6751 may be hsa-miR-6751, hsa-miR-6751-5p, and may have the nucleotide sequence of SEQ ID NO: 4.
  • miR-634 may be hsa-miR-634 and may have the nucleotide sequence of SEQ ID NO: 5.
  • miR-92a-2-5p may be hsa-miR-92a-2-5p and may have the base sequence of SEQ ID NO: 6.
  • the pharmaceutical composition may contain the polynucleotide as a single strand or may contain the polynucleotide as a double strand.
  • the polynucleotide When the polynucleotide is contained as a double strand, it may have at least a partially double strand, and at least one of the ends of the double strand may have a single strand portion. May have at least one pair of mismatched base pairs or base deficiencies.
  • the double-stranded portion has a single-stranded portion at the end, it is preferable to have the single-stranded portion at least on the 3'side.
  • the chain length of the single-stranded portion is, for example, 2 residues or more and 20 residues or less, preferably 2 residues or more and 12 residues or less, or 2 residues or more and 5 residues or less.
  • the mismatched base pairs may be arranged continuously or discontinuously, and the total number of mismatched base pairs is, for example, 10 bases. It may be pair or less, preferably 6 base pairs or less, or 4 base pairs or less, and may be, for example, 1 base pair or more, 2 base pairs or more, or 3 base pairs or more.
  • complementary base pairs include thermodynamically stable non-Watson click type wobble base pairs (eg, GU).
  • the double-stranded polynucleotide contained in the pharmaceutical composition may have the base sequence of any of the following (1) to (6) or the base sequence of a mutant thereof.
  • the pharmaceutical composition may contain at least one selected from the group consisting of double-stranded polynucleotides having the base sequence of any of the following (1) to (6) or the base sequence of a variant thereof. ..
  • the first double-stranded polynucleotide is derived from the gene encoding hsa-miR-1293.
  • the first double-stranded polynucleotide consists of a single-stranded polynucleotide having the nucleotide sequence "UGGGUGGUCUGGAGAUUGUGGC” (SEQ ID NO: 1) and a single-stranded polynucleotide having the nucleotide sequence "GCACAAAUCUCCGGACCUUA” (SEQ ID NO: 7). It is a chain polynucleotide.
  • SEQ ID NO: 1 is the base sequence of hsa-miR-1293 (mature miRNA) and can form the complementary structure shown below.
  • the RNA on the 5'side having the nucleotide sequence of SEQ ID NO: 1 is positioned as the guide strand
  • the RNA on the 3'side having the nucleotide sequence of SEQ ID NO: 7 is positioned as the passenger strand.
  • the second double-stranded polynucleotide is derived from the gene encoding hsa-miR-876-3p.
  • the second double-stranded polynucleotide is a substantially complementary 2 consisting of a single-stranded polynucleotide having the nucleotide sequence "UGGUGUUUAGAAAGUAAUUCA" (SEQ ID NO: 2) and a single-stranded polynucleotide having the nucleotide sequence "UGGAUUUCUUGUAUCCA” (SEQ ID NO: 8). It is a main chain polynucleotide.
  • SEQ ID NO: 2 is the base sequence of hsa-miR-876-3p (mature miRNA) and can form a substantially complementary structure shown below.
  • the RNA on the 3'side having the nucleotide sequence of SEQ ID NO: 2 is positioned as the guide strand
  • the RNA on the 5'side having the nucleotide sequence of SEQ ID NO: 8 is positioned as the passenger strand.
  • substantially complementary means a portion in which a part of the double-stranded polynucleotide is a single strand and / or a portion in which a part of the base pair is not bonded by a hydrogen bond, as shown in the following formula. It is in a state of constituting a double-stranded polynucleotide as a whole while containing (mismatch).
  • the third double-stranded polynucleotide is derived from the gene encoding hsa-miR-4438.
  • the third double-stranded polynucleotide is a complementary two-stranded polynucleotide consisting of the single-stranded polynucleotide of the base sequence "CAAGGCUUAGAAAAGACAGU” (SEQ ID NO: 3) and the single-stranded polynucleotide of the base sequence "ACUGUCUUUCUAAGCCUGUG” (SEQ ID NO: 9). It is a chain polynucleotide.
  • SEQ ID NO: 3 is the base sequence of hsa-miR-4438 (mature miRNA) and can form the complementary structure shown below.
  • the RNA on the 3'side having the nucleotide sequence of SEQ ID NO: 3 is positioned as the guide strand
  • the RNA on the 5'side having the nucleotide sequence of SEQ ID NO: 9 is positioned as the passenger strand.
  • the fourth double-stranded polynucleotide is derived from the gene encoding hsa-miR-6751-5p.
  • the fourth double-stranded polynucleotide is a substantially complementary 2 consisting of a single-stranded polynucleotide having the nucleotide sequence "UGGGGGGUGAGGUGUGGUGUCUGUGG” (SEQ ID NO: 4) and a single-stranded polynucleotide having the nucleotide sequence "ACUGAGCCUCUCUCUCUCCAG" (SEQ ID NO: 10). It is a main chain polynucleotide.
  • SEQ ID NO: 4 is the base sequence of hsa-miR-6751-5p (mature miRNA), which can form a substantially complementary structure shown below.
  • the RNA on the 5'side having the nucleotide sequence of SEQ ID NO: 4 is positioned as the guide strand
  • the RNA on the 3'side having the nucleotide sequence of SEQ ID NO: 10 is positioned as the passenger strand.
  • the fifth double-stranded polynucleotide is derived from the gene encoding hsa-miR-634.
  • the fifth double-stranded polynucleotide is a substantially complementary 2 consisting of a single-stranded polynucleotide having the nucleotide sequence "AACCAGCACCCCAAACUUGGAC” (SEQ ID NO: 5) and a single-stranded polynucleotide having the nucleotide sequence "AUCGAGGGUUGGGGGCUUGGU” (SEQ ID NO: 11). It is a main chain polynucleotide.
  • SEQ ID NO: 5 is the base sequence of hsa-miR-634 (mature miRNA) and can form a substantially complementary structure as shown below.
  • the RNA on the 3'side having the nucleotide sequence of SEQ ID NO: 5 is positioned as the guide strand
  • the RNA on the 5'side having the nucleotide sequence of SEQ ID NO: 11 is positioned as the passenger strand.
  • the sixth double-stranded polynucleotide is derived from the gene encoding hsa-miR-92a-2.
  • the sixth double-stranded polynucleotide is a substantially complementary 2 consisting of a single-stranded polynucleotide having the nucleotide sequence "GGGUGGGAUUUGUGCAUUAC” (SEQ ID NO: 6) and a single-stranded polynucleotide having the nucleotide sequence "UAUUGCACUUGUCCGCGCCUGU" (SEQ ID NO: 12). It is a main chain polynucleotide.
  • SEQ ID NO: 6 is the base sequence of hsa-miR-92a-2 (mature miRNA) and can form a substantially complementary structure as shown below.
  • the RNA on the 5'side having the nucleotide sequence of SEQ ID NO: 6 is positioned as the guide strand
  • the RNA on the 3'side having the nucleotide sequence of SEQ ID NO: 12 is positioned as the passenger strand.
  • the nucleotide constituting the polynucleotide may contain at least one ribonucleotide, and may contain at least one of the corresponding deoxyribonucleotide and modified nucleotide in place of the ribonucleotide.
  • Modified nucleotides include nucleotides with a modified phosphate moiety, nucleotides with a modified sugar moiety, nucleotides with a modified base moiety, and the like.
  • the modified nucleotide may be one in which any of the phosphoric acid moiety, the sugar moiety and the base moiety is modified, and may be a combination of two or more kinds of modifications.
  • JP-A-10-304889, WO 2005/021570, JP-A-10-195098, JP-A-2002-521310, WO 2007/143315, International Publication No. Publication No. 2008/043753, International Publication No. 2008/029619, International Publication No. 2008/049085, etc. can be referred to.
  • Non-crosslinked modification and crosslinked modification are known as modifications of the sugar moiety.
  • Non-crosslinking type modification include modification of the 2'-hydroxyl group such as fluorination (F) at the 2'position, O-methylation, and MOE formation, and morpholinonucleation.
  • examples of the crosslinked modification include LNA (2', 4'-BNA) conversion, ENA conversion and the like.
  • the polynucleotide according to this embodiment can be synthesized by using a known method for synthesizing a polynucleotide or the like.
  • Examples of the method for synthesizing a polynucleotide include a phosphoromidide method and an improved method thereof, an H-phosphonate method and an improved method thereof, and an enzyme synthesis method (in vitro transcription method). It is also possible to apply the nucleic acid of a commercially available product or the nucleic acid of which is manufactured by consignment.
  • the pharmaceutical composition may further contain other antitumor compounds as an active ingredient in addition to the polynucleotide.
  • Other antitumor compounds include, for example, antimetabolites, molecular targeted drugs, alkylating agents, plant alkaloids, anticancer antibiotics, platinum preparations, hormonal agents, biological response regulators, immune checkpoints. Inhibitors and the like can be mentioned.
  • the pharmaceutical composition may be prepared in the form of a pharmaceutical composition by containing a suitable pharmaceutical pharmaceutical carrier together with the nucleic acid (polynucleotide) as the active ingredient described above.
  • a suitable pharmaceutical pharmaceutical carrier can be selected according to the mode of use, and excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, surfactants, diluents and the like can be used. Can be used. It is also possible to prepare a desiccant that can be made liquid at the time of use by adding an appropriate carrier to the nucleic acid as the active ingredient.
  • the pharmaceutical composition may contain a polynucleotide as an active ingredient in a pharmaceutically acceptable carrier in a state of being encapsulated or bound.
  • a pharmaceutically acceptable carrier in a state of being encapsulated or bound.
  • drug delivery systems such as high molecular weight micelles, nanoparticles composed of cyclodextrin-containing polymers, stable nucleic acid lipid particles, and multifunctional envelope-type nanostructures can be utilized to further improve the effects of pharmaceutical compositions. It is possible.
  • the form of the pharmaceutical composition is not particularly limited as long as it can effectively contain the nucleic acid as the active ingredient, and is a solid agent such as a tablet, a powder, a granule, or a pill, or an ointment. It may be an agent or a pill agent. It is also preferable to use an injection form such as a liquid, a suspension, or an emulsion. At present, topical administration of pharmaceutical compositions is effective. Dosage forms suitable for this dosage form are usually in the form of injections, ointments, or haps.
  • the active ingredient is directly injected into the tumor by injection from outside the body such as intravenous, subcutaneous, intradermal, or intramuscular injection, and further, the active ingredient is injected into the body using an endoscope. It is performed by directly injecting the active ingredient into a tumor (intradigestive tract, uterus, bladder, etc.). Topical administration using an ointment or a happing agent is carried out in such a manner that the active ingredient is directly infiltrated against skin cancer.
  • the dose of the nucleic acid as the active ingredient to the human body is, for example, 0.01 ⁇ g or more and 1000 mg or less per adult per day. This administration may be performed once a day, or twice or more and 5 times or less, and may be performed every day or every few days.
  • the pharmaceutical composition is used in the treatment of tumors.
  • the treatment of the tumor may be any treatment performed on the tumor, and examples thereof include treatment of the tumor, improvement, suppression of progression (prevention of deterioration), prevention, alleviation of symptoms caused by the tumor, and the like. ..
  • the pharmaceutical composition contains a polynucleotide that suppresses the expression of the BRD4 gene
  • the tumor to be treated preferably contains a tumor cell expressing the BRD4 gene.
  • Tumors to be treated include esophageal cancer, lung cancer, oral cancer, gastric cancer, colon cancer, uterine cancer, skin cancer, brain tumor, neuroblastoma, glioblastoma, breast cancer, pancreatic cancer, and ovary.
  • the tumors to be treated are preferably colon cancer, pancreatic cancer, oral cancer, lung cancer, esophageal cancer, gastric cancer, uterine cancer, skin cancer, hematological tumor, brain tumor, neuroblastoma, collagen bud. It may be at least one selected from the group consisting of tumor, breast cancer, ovarian cancer, prostate cancer, bladder cancer, esophageal cancer, liver cancer and renal cancer.
  • topical administration that is, a tumor in which the pharmaceutical composition can be brought into direct contact with the tumor is suitable as a treatment target.
  • the pharmaceutical composition may be used in the treatment of tumors expressing the RSK1 gene. It is particularly suitable when the pharmaceutical composition contains, for example, a polynucleotide derived from a gene encoding miR-876-3p as an active ingredient.
  • RSK1 is a member of the RSK (ribosomal S6 kinase) family and is a growth factor-controlled serine threonine kinase.
  • RSK1 is involved in mitogen-activated kinase (MAPK) cascade activation and stimulates cell proliferation and differentiation.
  • MAPK mitogen-activated kinase
  • RSK1 activated by the MAPK cascade is known to activate S6 kinase, and as a result, the mTOR pathway is activated to promote cell proliferation and survival. Inhibitors of this mTOR pathway have already been clinically applied in the treatment of cancer.
  • the pharmaceutical composition may be used in the treatment of tumors expressing at least one gene selected from the group consisting of APEX1, RPA1 and POLD4. It is particularly suitable when the pharmaceutical composition contains, for example, a polynucleotide derived from a gene encoding miR-1293 as an active ingredient.
  • APEX1, RPA1 and POLD4 are all genes involved in DNA repair. It is considered that replication stress is applied to cancer due to the accumulation of gene mutations and structural abnormalities, and PARP inhibitors are clinically applied as therapeutic agents that inhibit the DNA repair pathway.
  • the pharmaceutical composition when aiming for "coexistence with cancer", it can be an indication target for pharmaceutical compositions. That is, the pharmaceutical composition is also used when aiming to prevent malignant transformation of cancer and complete natural life by suppressing the expression of genes such as RSK1, APEX1, RPA1, and POLD4, as well as the expression of BRD4 gene in cancer. Things are considered suitable.
  • the above gene expression can be quantified according to a conventional method.
  • a method for quantifying the expression of BRD4 gene, RSK1 gene, APEX1 gene, RPA1 gene, POLD4 gene and the like Northern blotting method, Western blotting method, in situ hybridization method (ISH method), quantitative RT-PCR method and the like.
  • Examples thereof include (including real-time PCR method), RNase protection assay, in situ transcription method, expression array analysis method, immunohistochemical staining method, and modifications of these methods.
  • genes such as BRD4, RSK1, APEX1, RPA1, and POLD4 in the tumor to be treated may be overexpressed.
  • the presence or absence of overexpression of a predetermined gene can be specified as follows. Appropriate cutoff values are set comprehensively or individually for each tumor based on the normal expression level of each gene. By applying the above-mentioned gene expression quantification method to a tumor to be treated and applying the cutoff value to the expression quantification value of a predetermined gene in the tumor, an excess of the predetermined gene is applied. The presence or absence of expression can be specified. Cancers in which such overexpression of a predetermined gene is observed can be specified as an appropriate application target of the pharmaceutical composition.
  • the pharmaceutical composition may be used in the treatment of tumors having a pathogenic mutation in the BRCA1 / 2 gene. It is particularly suitable when the pharmaceutical composition contains, for example, a polynucleotide derived from a gene encoding miR-1293 as an active ingredient.
  • the BRCA1 / 2 gene is a gene that plays an important role in homologous recombination repair at the time of DNA damage, and BRCA1 and BRCA2 are known. Examples of pathogenic mutations in the BRCA1 / 2 gene include frameshift mutations, nonsense mutations, and methylation. Further, the tumor to be treated may be a so-called BRCAness. BRCAness is a DNA damage repair mechanism similar to the pathological mutation of the BRCA1 / 2 gene, which causes homologous recombination dysfunction due to modulation of other factors even if no pathological mutation is found in the BRCA1 / 2 gene itself. The state in which the abnormality of is caused.
  • PAPR poly (ADP ribose) polymerase
  • PAPR poly (ADP ribose) polymerase
  • a pharmaceutical composition containing a polynucleotide derived from a gene encoding miR-1293 is also expected to be effective against tumors that have become resistant to PAPR inhibitors.
  • the target of treatment of the pharmaceutical composition is not limited to the tumor, but may be a disease exhibiting the above-mentioned genetic characteristics.
  • Diseases to be treated based on genetic characteristics include, for example, acute myelogenous leukemia (AML), mixed lineage leukemia (MLL), and barkit lymphoma as diseases related to the BRD4 gene.
  • lymphoma the most common brain tumors in childhood, medulloblastoma, MYCN-amplified neuroblastoma, castration-resistant prostate cancer (CRPC), Ewing's sarcoma, colorectal cancer, lymphoma, leukemia, multiple myeloma, solid tumor (eg, cancer of the pancreas and prostate) ), Breast cancer, non-small cell lung carcinoma (NSCLC), myelodysplastic syndromes (MDS), myeloproliferative neoplasms, foliar tumors that cannot be classified (Phyllodes) tumor), diffuse large B-cell lymphoma (DLBCL), triple negative breast cancer (TNBC), pancreatic ductal adenocarcinoma, polymorphic glioma Cancer (glioblastoma multiforme), type 2 diabetes (Type 2 diabetes mellitus), ischemic heart diseases (for example, Int. J. Mol. Sci. 2016, 17, _1849; Cold Spring
  • a tumor treatment method is a method of treating a tumor, which comprises administering an effective amount of the pharmaceutical composition to a subject.
  • the tumor to be treated is as described above, and is, for example, a tumor expressing at least one gene selected from the group consisting of BRD4, RSK1, APEX1, RPA1 and POLD4. Further, the tumor may have a pathological mutation in the BRCA1 / 2 gene or is in a BRCAness state.
  • the details of the pharmaceutical composition and the administration method are as described above.
  • the subject of treatment is, for example, a mammal, which includes a human.
  • the target of treatment may be a non-human animal or a human.
  • the present invention also includes, as another aspect, the use of the polynucleotide in the production of a pharmaceutical composition used in the treatment of a tumor, the use of the polynucleotide in the treatment of a tumor, and the polynucleotide used in the treatment of a tumor. To do.
  • MicroRNA and siRNA A synthetic product of double-stranded microRNA using hsa-miR-1293 (mature miRNA) as a guide strand (SEQ ID NO: 1) was obtained from Ambion (Ambion, Inc .: USA) (trade name: MC13698).
  • the microRNA is a double-stranded RNA having the same base sequence as the following natural type, and contains a modified nucleotide.
  • this double-stranded RNA will be referred to as "miR-1293" unless otherwise specified.
  • a synthetic product of double-stranded microRNA using hsa-miR-876-3p (mature miRNA) as a guide strand (SEQ ID NO: 2) was obtained from Ambion (trade name: MC12886).
  • the microRNA is a double-stranded RNA having the same base sequence as the following natural type, and contains a modified nucleotide.
  • this double-stranded RNA will be referred to as "miR-876-3p" unless otherwise specified.
  • a synthetic product of double-stranded microRNA using hsa-miR-4438 (mature miRNA) as a guide strand (SEQ ID NO: 3) was obtained from Ambion (trade name: MC22634).
  • the microRNA is a double-stranded RNA having the same base sequence as the following natural type, and contains a modified nucleotide.
  • this double-stranded RNA will be referred to as "miR-4438" unless otherwise specified.
  • a synthetic product of double-stranded microRNA using hsa-miR-6751-5p (mature miRNA) as a guide strand (SEQ ID NO: 4) was obtained from Ambion (trade name: MC27194).
  • the microRNA is a double-stranded RNA having the same base sequence as the following natural type, and contains a modified nucleotide.
  • this double-stranded RNA will be referred to as "miR-6751" unless otherwise specified.
  • microRNA A synthetic product of double-stranded microRNA using hsa-miR-634 (mature miRNA) as a guide strand (SEQ ID NO: 5) was obtained from Ambion (trade name: MC11538).
  • the microRNA is a double-stranded RNA having the same base sequence as the following natural type, and contains a modified nucleotide.
  • this double-stranded RNA will be referred to as "miR-634" unless otherwise specified.
  • a synthetic product of double-stranded microRNA using hsa-miR-92a-2 (mature miRNA) as a guide strand (SEQ ID NO: 6) was obtained from Ambion (trade name: MC12524).
  • the microRNA is a double-stranded RNA having the same base sequence as the following natural type, and contains a modified nucleotide.
  • this double-stranded RNA will be referred to as "miR-92a-2" unless otherwise specified.
  • Control miRNA negative control # 1; miR-NC (Ambion) was used as the control microRNA.
  • RSK1 siGENOME SMARTpool: M-003025-04
  • APEX1 siGENOME SMARTpool: M-01237-01
  • BRD4 siGENOME SMARTpool: M-004937-02-0005
  • RPA1 siGENOME49ol
  • the siRNAs for POLD4 siGENOME SMARTpool: M-014013-1) were obtained from Dermacon.
  • Antibodies As antibodies for Western blot, anti-BRD4 antibody (cell signing), anti-cleave PARP antibody (cell signing), anti-RSK1 antibody (R & D Systems), anti-APEX1 antibody (proteintech), anti-phosphorylation RSK1 antibody (R & D Systems), anti-phosphorylated P70S6K antibody (cell signing), anti-phosphorylated S6 antibody (cell signing), anti-MYC antibody (cell signing), anti-POLD4 antibody (proteintech), anti- ⁇ H2AX antibody (Cell signing) and anti- ⁇ -actin antibody (Sigma) were used.
  • Anti-BRD4 antibody (Atlas antibodies), anti-RSK1 antibody (Atlas antibodies), anti-APEX1 antibody (proteintech), anti-RPA1 antibody (abcam), and anti-POLD4 antibody (MyBioSour) for immunohistochemical staining. ..
  • Example A1 Primary screening of miRNA that suppresses cell proliferation HCT116 +/ + cells and HCT116 ⁇ / ⁇ cells (6000 cells / well) are included in the miRNA library [miRVana miRNA mimic Library V21 (Ambion)]. 10 nmol / L of 2565 types of miRNA or control miRNA (miR-NC) was introduced using Lipofectamine RNAiMAX (Invitrogen) according to the attached procedure manual. Cell viability was evaluated by crystal violet (CV) staining assay 3 days after introduction. The CV staining assay was performed as follows. Cells were washed with PBS and fixed with 10% formaldehyde PBS containing 0.2% CV for 5 minutes.
  • CV staining assay was performed as follows. Cells were washed with PBS and fixed with 10% formaldehyde PBS containing 0.2% CV for 5 minutes.
  • FIG. 1 shows the results of the primary screening in the above HCT116 +/+ and HCT116 ⁇ / ⁇ cells.
  • the ratio of the number of surviving cells when compared with the control miRNA when 2565 kinds of miRNAs are introduced is shown.
  • the horizontal axis shows the result of HCT116 +/ + cells, and the vertical axis shows the result of HCT116 ⁇ / ⁇ cells.
  • the relative number of viable cells when the number of viable cells when transfected with the control miRNA is 1.
  • the ratio to the control was set to less than 0.3 as a standard for the cell proliferation inhibitory effect.
  • 138 types of miRNAs showed a growth inhibitory effect on both HCT116 + / + and HCT116 ⁇ / ⁇ cells.
  • the cell proliferation inhibitory effect was observed in 630 types in HCT116 + / +, 141 types were observed in HCT116 ⁇ / ⁇ , and 138 types of miRNAs were common to both.
  • Table 14 shows the results of the secondary screening.
  • the ratio of the number of surviving cells when compared with the control miRNA when each of the 138 kinds of miRNAs extracted in the primary screening is introduced is shown.
  • the relative number of viable cells when the number of viable cells when transfected with the control miRNA is 1.
  • the ratio with the control was set to less than 0.6.
  • 7 types of miRNAs showing a growth inhibitory effect in all 10 types of cancer cells are shown.
  • Example A2> (Western blot) HCT116 +/ + cells, HCT116 into which miR-NC, miR-876-3p, miR-1293, miR-4438, miR-6751-5p, miR-92a-5p, miR-634, or miR-3140-3p were introduced.
  • Western blots were performed on the samples obtained from ⁇ / ⁇ cells and HOC313 cells to examine the expression level of BRD4. Western blotting was performed as follows. SDS-PAGE was performed on the lysates of all cells, and the protein was transferred to a PVDF membrane (GE Healthcare).
  • the membrane was reacted with the antibody overnight at 4 ° C.
  • the dilution ratio of the primary antibody was anti-BRD4 antibody (1/1000) and anti- ⁇ -actin antibody (1/5000).
  • HRP-binding anti-mouse or anti-rabbit IgG antibody both 1/5000 was exposed to HRP-binding anti-mouse or anti-rabbit IgG antibody (both 1/5000) at room temperature for 1 hour.
  • the bound antibody was visualized using the SuperSignal West Femto Substrate (Thermo Fisher Scientific). The results are shown in Fig. 2-1.
  • miR-876-3p As shown in FIG. 2-1 in these three types of cancer cell lines, miR-876-3p, miR-1293, miR-4438, miR-6751-5p, miR-92a-5p, miR-634, or Suppression of BRD4 gene expression was observed by introducing miR-3140-3p.
  • Example A3> (Luciferase Reporter Assay) The interaction between miRNA and the BRD4 gene was evaluated by a luciferase reporter assay using a construct in which the 3'UTR region of the BRD4 gene was incorporated into a luciferase vector.
  • FIG. 2-2 (A) shows the results of verification of miR-1293 by the luciferase reporter assay.
  • the luciferase reporter plasmid used for the assay is pmiRGlo Dual-Luciferase miRNA Target Expression Vector (Promega).
  • Wt oligonucleotide
  • Mt oligonucleotide
  • the luciferase reporter plasmid was introduced into HOC313 cells using Lipofectamine 2000 (In vitrogen) according to the attached procedural document, and the next day miR-1293 or control miRNA was introduced. Two days later, firefly luciferase activity and sea shiitake mushroom luciferase activity were measured using Dual-Luciferase Reporter Assay System (Promega). Relative luciferase activity was calculated by standardizing firefly luciferase activity by correcting for the corresponding internal standard control, Shiitake mushroom luciferase activity.
  • miR-1293 With the introduction of miR-1293, a decrease in luciferase activity corresponding to the wild-type 3'UTR region was observed at two locations, R1 and R4, in the 3'UTR region of BRD4, and the nucleotide sequence of the guide chain of miR-1293 was observed. Luciferase activity was restored in the vector in which the matching region was mutated. From these results, it was shown that miR-1293 significantly suppressed the expression of the BRD4 gene directly by the action of the BRD4 gene on the two 3'UTR regions of R1 and R4.
  • FIG. 2-2 shows the results of verification by the luciferase reporter assay of miR-876-3p.
  • the assay was performed in the same manner as above except that the sequence of the region was mutated.
  • miR-876-3p significantly suppressed the expression of the BRD4 gene directly by the action of the BRD4 gene on the 3'UTR region.
  • FIG. 2-2 (C) shows the results of verification by the luciferase reporter assay of miR-6751-5p.
  • the assay was performed in the same manner as above except that the sequence of the region was mutated.
  • miR-6751-5p significantly suppressed the expression of the BRD4 gene directly by the action of the BRD4 gene on the 3'UTR region.
  • Figure 2-3 shows the effect on cell proliferation when siRNA against the BRD4 gene is allowed to act on a colon cancer cell line (HCT116 ⁇ / ⁇ cells) and the BRD4 gene is knocked down. For comparison, the effect on cell proliferation when control siRNA (si-NC) is introduced is also shown.
  • the results of Western blotting performed in the same manner as in Example A2 are shown in FIG. 2-3 (B).
  • Target cancer cell lines include colon cancer cell lines (HCT116 + / + cells, HCT116 ⁇ / ⁇ cells), oral cancer cell lines (HOC313 cells, HSC2 cells), and lung cancer cell lines (A549 cells (non-small cell lung cancer)).
  • Cell line)) and esophageal cancer cell line (KYSE150 cells) were used to evaluate the cell growth inhibitory effect of the introduction of miRNA. 10 nmol / L of miR-876-3p, miR-1293 or control miRNA (miR-NC) as a control was introduced into each target cancer cell line using Lipofectamine RNAiMAX according to the procedure manual in the package insert.
  • the number of viable cells was evaluated 2 days and 4 days after the introduction.
  • the cell proliferation curve is shown in FIG. 3-1.
  • a micrograph of a colorectal cancer cell line (HCT116 ⁇ / ⁇ cells) 72 hours after introduction is shown in FIG. 3-2.
  • the cell proliferation was significantly suppressed by the introduction of miR-876-3p or miR-1293 in any of the cell lines, and a remarkable proliferation inhibitory effect was observed.
  • FIG. 3-2 in the colorectal cancer cell line, a large number of dead cells were confirmed by introducing miR-876-3p or miR-1293, and the number of viable cells was smaller than that of the control.
  • Example A5> (Apoptosis evaluation 1) 10 nmol / L of miR-NC, miR-876-3p or miR-1293 was added to the colorectal cancer cell line (HCT116 + / +, HCT116 ⁇ / ⁇ cells) and oral cancer cell line (HOC313 cells). RNAiMAX was used and introduced into each cancer cell line according to the procedure manual in the package insert. The proportion of apoptotic cells 72 hours after introduction was evaluated by Annexin V / PI staining. The proportion of annexin V / PI positive cells was evaluated using Accuri C6 Flow Cytometry (BD Biosciences). The results are shown in Fig. 3-3.
  • miR-876-3p or miR-1293 increased the proportion of apoptotic cells in each cell as compared with the control (miR-NC).
  • Apoptosis evaluation 2 10 nmol / L of miR-NC, miR-876-3p or miR-1293 to colorectal cancer cell line (HCT116 +/+, HCT116 ⁇ / ⁇ cells) or oral cancer cell line (HOC313 cells), Lipofectamine RNAiMAX was used and introduced into each cancer cell line according to the procedure manual in the package insert.
  • the expression level of cleared PARP which is a marker of apoptosis, was evaluated by Western blotting on cells 72 hours after introduction. Western blotting was performed in the same procedure as described above. The results are shown in Figure 3-4.
  • miR-876-3p or miR-1293 increased the expression level of cleared PARP in each cell as compared with the control (miR-NC). Shown.
  • miR-876-3p and miR-1293 induce apoptosis in various cancer types including intractable cancers and show a remarkable growth inhibitory effect.
  • Example A1 for colon cancer cell line HCT116 ⁇ / ⁇ cells
  • oral cancer cell line HSC2, HOC313 cells
  • lung cancer cell line A549 cells
  • esophageal cancer cell line KYSE150 cells
  • MiR-NC or miR-876-3p was introduced as described. Forty-eight hours after introduction, gene expression array analysis was performed as follows. Gene expression array analysis was performed using an Agilent 4x44K gene expression array (Agilent Technologies, Inc.) according to its operating manual. The data of the gene expression array analysis was analyzed using GeneSpring software (Agilent Technologies).
  • FIG. 4-1 shows the number of genes whose gene expression decreased by more than 1.5 times as a result of the expression array performed on these miR-introduced cells.
  • 1223 genes were common to 4 or more of the 5 types of cancer cell lines. there were.
  • a pathway analysis of 1223 of these common genes using DAVID software revealed that multiple genes involved in the mTOR pathway shown in the table below were significantly suppressed, resulting in significant suppression. It was shown that the mTOR pathway was suppressed.
  • Example A6> (Western blot) MiR-NC or miR-876-3p was introduced into a colon cancer cell line (HCT116 ⁇ / ⁇ cells) or an oral cancer cell line (HOC313 cells, KOSC3 cells) in the manner described in Example A1. Forty-eight hours after introduction, the expression levels of RSK1, phosphorylated RSK1 (p-RSK1), phosphorylated p70S6K (p-p70S6K) and pS6 (p-S6), which are indicators of activation of the mTOR signal pathway, were adjusted in the same manner as above. It was evaluated by performing Western blotting with the expression of ⁇ -actin protein as a positive control. The results are shown in FIG. 4-2.
  • the expression of RSK1 was remarkably suppressed by the introduction of miR-876-3p in HCT116 ⁇ / ⁇ cells, HOC313 cells, and KOSC3 cells, and further, p-p70S6K of the mTOR signal pathway, Suppression of the expression of p-S6 protein was observed, and suppression of RSK1 and mTOR signal pathway was confirmed by the introduction of miR-876-3p.
  • Example A7 (Luciferase Reporter Assay) The interaction between miR-876-3p and the RSK1 gene was evaluated by a luciferase reporter assay using a construct in which the 3'UTR region of the RSK1 gene was incorporated into a luciferase vector. There is one region in the RSK1 gene that is homologous to the nucleotide sequence of the guide strand of miR-876-3p. A luciferase reporter assay was performed in the same manner as above except that the region was mutated. The results are shown in Figure 4-3.
  • miR-876-3p As shown in FIG. 4-3, the introduction of miR-876-3p showed a decrease in luciferase activity corresponding to the wild-type 3'UTR region, and a region matching the base sequence of the guide chain of miR-876-3p. The luciferase activity was restored in the vector mutated in. From these results, it was shown that miR-876-3p directly suppressed the expression of the RSK1 gene by the action of the RSK1 gene on the 3'UTR region.
  • knockdown of at least one of the BRD4 gene and the RSK1 gene suppresses the expression of the knocked down gene.
  • the expression level of cleared PARP which is an index of apoptosis
  • cell proliferation was suppressed by knocking down at least one of the BRD4 gene and the RSK1 gene, and synergistically suppressed when both were knocked down.
  • FIG. 4-5 is a conceptual diagram schematically showing the presumed mechanism by which miR-876-3p suppresses the growth of tumor cells, which is derived from the results of the above examples.
  • Example A8 Examples A1 were described in a colon cancer cell line (HCT116 ⁇ / ⁇ cells), an oral cancer cell line (HSC2, HOC313 cells), a lung cancer cell line (A549 cells), and an esophageal cancer cell line (KYSE150 cells).
  • HCT116 ⁇ / ⁇ cells colon cancer cell line
  • HSC2, HOC313 cells oral cancer cell line
  • A549 cells lung cancer cell line
  • KYSE150 cells esophageal cancer cell line
  • FIG. 5-1 shows the number of genes whose gene expression decreased by more than 1.5 times as a result of the expression array performed on these miR-introduced cells.
  • 1248 genes were common to 4 or more of the 5 types of cancer cell lines. there were.
  • pathway analysis was performed on 1248 of these common genes using DAVID software, multiple pathways involved in DNA repair, that is, Homologous recombination (homologous recombination repair), base excision repair (base excision repair), and Mismatch repair, nail It was shown that excision repair was suppressed.
  • the table below shows each pathway and the genes involved in that pathway.
  • Example A9> (Western blot) MiR-NC or miR-1293 was introduced into a colon cancer cell line (HCT116 ⁇ / ⁇ cells) or an oral cancer cell line (HOC313 cells) into which miR-1293 was introduced, in the manner described in Example A1. .. Forty-eight hours after the introduction, the expression levels of APEX1, RPA1 and POLD4 proteins were evaluated by performing Western blotting in the same manner as above with the expression of ⁇ -actin protein as a positive control. The results are shown in Figure 5-2.
  • Example A10> (Luciferase Reporter Assay) The interaction of miR-1293 with the APEX1, RPA1 or POLD4 gene was evaluated by a luciferase reporter assay using a construct that incorporated the 3'UTR region of the APEX1, RPA1 or POLD4 gene into a luciferase vector. There is one region homologous to the nucleotide sequence of the guide strand of miR-1293 for each of the APEX1, RPA1 and POLD4 genes. A luciferase reporter assay was performed in the same manner as above, except that mutations were placed in each region. The results are shown in Figure 5-3.
  • miR-1293 significantly reduced the luciferase activity corresponding to the wild-type 3'UTR region in all of the APEX1, RPA1 and POLD4 genes, and the guide chain of miR-1293 was observed.
  • the luciferase activity was significantly restored in the vector in which the mutation was added to the region matching the nucleotide sequence of. From these results, it was shown that miR-1293 directly suppresses the expression of APEX1, RPA1 and POLD4 genes by the action of APEX1, RPA1 and POLD4 genes on the 3'UTR region.
  • siRNA When siRNA is allowed to act on a colon cancer cell line (HCT116 ⁇ / ⁇ cells) and both the BRD4 gene, APEX1 gene or BRD4 gene and APEX1 gene are knocked down at the same time (A), the BRD4 gene, RPA1 gene or BRD4 gene Control siRNA of the effect on gene expression and cell proliferation when both the RPA1 gene and the RPA1 gene are knocked down at the same time (B) and when both the BRD4 gene, POLD4 gene or both the BRD4 gene and the POLD4 gene are knocked down at the same time (C) It was evaluated in comparison with the case where (si-NC) was used. Gene expression was evaluated by Western blot in the same manner as above. The results of Western blotting are shown in FIGS. 5-4 (A) to (C). The effect on cell proliferation is shown in FIG. 5-4 (D).
  • NCS neocarzinostatin
  • Blocking was performed with PBS containing 3% bovine serum, and then anti- ⁇ H2AX antibody (1/400 dilution) was reacted at room temperature for 1 hour.
  • the bound antibody was visualized using an Alexa Fluor555 anti-rabbit IgG antibody (1/500 dilution, Life Technologies).
  • the section was counter-stained with VECTASHILD connecting DAPI (Vector Laboratories), and observed and photographed with a fluorescence microscope of Nikon. The results are shown in Figure 5-5.
  • the proportion of ⁇ H2AX-positive cells in three randomly extracted visual fields in fluorescence microscope observation was measured. The results are shown in Figure 5-6.
  • FIGS. 5-5 and 5-6 a large amount of ⁇ H2AX was observed in miR-1293-introduced cells. That is, it is a result showing that the introduction of miR-1293 suppressed DNA repair from DNA damage caused by neocarzinostatin. Further, as shown in FIGS. 5-5 and 5-6, suppression of APEX1, RPA1, POLD4 and BRD4, which are targets of miR-1293, resulted in suppression of DNA repair as in miR-1293. From these results, it was shown that the DNA repair inhibitory effect of miR-1293 was mediated by the inhibition of APEX1, RPA1, POLD4 and BRD4.
  • Example A12 Homologous recombination repair was evaluated for colorectal cancer cell lines (HCT116 ⁇ / ⁇ cells) when the BRD4 gene was knocked down using siRNA and when miR-1293 was introduced. Evaluation of homologous recombination repair was performed using a DR-GFP vector according to a known method (Genes Dev. 2001 Dec 15.15 (24): 3237-42.).
  • the DR-GFP vector is a vector in which a sequence cleaved by endonuclease Scel is incorporated into the sequence of the fluorescent dye GFP, and when the DNA of the vector cleaved by homologous recombination repair is repaired after being cleaved by Scel It is a vector expressing GFP.
  • the DR-GFP vector was introduced into HCT116 ⁇ / ⁇ cells, and cells in which the DR-GFP vector was stably expressed by neomycin were established.
  • neomycin resistance gene is integrated into the DR-GFP vector, and cells expressing the DR-GFP vector can survive by having resistance to neomycin, but conversely, cells expressing the DR-GFP vector do not. It cannot survive because it is not resistant to neomycin.
  • MiR-1293 or miR-NC as a control was introduced into the DR-GFP expressing HCT116 ⁇ / ⁇ cells, and then treated with endonuclease Scel, and 24 hours later, the number of cells developing DR-GFP was flowed. The analysis was performed by cytometry, and the relative number of cells was calculated when the number of cells in the miR-NC administration group was 1.
  • the number of GFP-expressing cells became lower than that of the control due to knockdown of the BRD4 gene or introduction of miR-1293. That is, it was shown that the introduction of miR-1293 suppresses homologous recombination repair.
  • FIG. 5-8 is a conceptual diagram schematically showing the presumed mechanism by which miR-1293, which is derived from the results of the above examples, suppresses the growth of tumor cells.
  • Example B1> In vivo tumor cell growth inhibitory effect by administration of miR-876-3p and miR-1293 7-week-old Balb / c nude mice were purchased from Oriental Bioservice and bred in a sterile condition. 100 ⁇ l of PBS containing 10 ⁇ 10 6 colorectal cancer cell lines (HCT116 ⁇ / ⁇ cells) was subcutaneously injected into the dorsal flank of mice. The schedule of the in vivo test after this subcutaneous injection is shown in FIG. 6-1.
  • a mixture of 1 nmol miR-876-3p, miR-1293 or control miRNA and 200 ⁇ l AteroGene (KOKEN) was administered into the gap between the tumor and the skin a total of 5 times (from HCT116 ⁇ / ⁇ cell injection). After 3, 7, 10, 14, 17 days). Nineteen days after cell administration, mice were euthanized and tumors were removed. The experimental procedure performed on all mice was approved by the Animal Experiment Committee of Tokyo Medical and Dental University.
  • FIG. 6-2 shows the appearance of a typical mouse subcutaneous tumor 19 days after injection of HCT116 ⁇ / ⁇ cells and a photograph of the excised tumor.
  • Figure 6-3 shows the transition of tumor volume. Tumor volume was calculated as (major) x (minor) 2 x 0.5. As shown in FIG. 6-3, tumor volume was significantly reduced by administration of miR-876-3p, miR-1293 compared to administration of control miRNA (miR-NC).
  • the weight of the removed tumor was weighed for each mouse. The results are shown in Figure 6-4. As shown in FIG. 6-4, the tumor weight in the miR-876-3p or miR-1293-administered group was significantly lighter than that in the control miRNA-administered group.
  • Example B2> Expression analysis of miR-876-3p and miR-1293 was performed in the excised tumor tissue using quantitative RT-PCR. Total RNA was separated from the tumor tissue by a standard method using a TRIsure reagent (Bioline). The single-stranded RNA prepared from the total RNA was amplified using a primer specific to miR-876-3p or miR-1293.
  • Real-time RT-PCR for miR-876-3p and miR-1293 includes ABI Prism 7500 Fast Real-time PCR system (Applied Biosystems), Taqman Universal PCR Master Mix (Applied Biosystems), TaqMan MultiMix (Applied Biosystems).
  • Example B3> The expression levels of the BRD4 gene, RSK1 gene, APEX1 gene, RPA1 gene and POLD4 gene in the excised tumor tissue were evaluated by immunostaining. Tumor tissue was fixed with PBS containing 10% formaldehyde, embedded in paraffin, and sliced into 4 ⁇ m thick sections. Tumor sections were immunohistochemically stained for BRD4, RSK1, APEX1, RPA1 and POLD4 using the avidin-biotin-peroxidase method as shown below. Tumor sections embedded in paraffin were deparaffinized with xylene and then rehydrated with ethanol.
  • the antigen was activated by boiling in 10 mM citrate buffer (pH 6.0) and treated with methanol containing 0.3% hydrogen peroxide to inactivate the endogenous peroxidase. Then, for the section, anti-BRD4 antibody (1/500 dilution), anti-RSK1 antibody (1/100 dilution), anti-APEX1 antibody (1/200 dilution), RPA1 antibody (1/100 dilution) or anti-POLD4 antibody (1). / 100 dilution) was used to react overnight at 4 ° C. The bound antibody was visualized using diaminobenzidine (VECTASTAIN-EluteABCkit: Vector Laboratory). The sections were then counterstained with hematoxylin.
  • VECTASTAIN-EluteABCkit Vector Laboratory
  • FIG. 6-6 The results of immunostaining using the anti-BRD4 antibody are shown in FIG. 6-6.
  • the results of immunostaining using the anti-RSK1 antibody are shown in FIG. 6-7.
  • the results of immunostaining using an anti-APEX1 antibody, an RPA1 antibody or an anti-POLD4 antibody are shown in FIG. 6-8.
  • the administration of miR-876-3p suppressed the expression of the BRD4 gene and the RSK1 gene and suppressed the tumor growth in the in vivo tumor tissue. It was. That is, the pharmaceutical composition containing miR-876-3p is expected to have an effect on various tumors resulting from the expression of at least one selected from the group consisting of BRD4 and RSK1.
  • miR-1293 suppressed the expression of the BRD4 gene, APEX1 gene, RPA1 gene, and POLD4 gene in the in vivo tumor tissue, and suppressed tumor growth. It was shown that That is, the pharmaceutical composition containing miR-1293 is expected to have an effect on various tumors resulting from the expression of at least one selected from the group consisting of BRD4, APEX1, RPA1, and POLD4.
  • miRNAs can inhibit the expression of multiple targets by directly binding to the coding region or 3'UTR region of each gene. This indicates that a single miRNA can target multiple pathways involved in cancer activation.
  • hsa-miR-34a is known to suppress tumor growth via a plurality of targets such as CCND1 gene, CDK6 gene, MYC gene, c-MET gene, and NOTCH gene.
  • miR-876-3p can target the BRD4 and RSK1 genes.
  • miR-1293 can target the BRD4 gene, APEX1 gene, RPA1 gene and POLD4 gene. The introduction of these miRNAs was found to have the effect of suppressing the growth of tumors derived from HCT116 ⁇ / ⁇ cells, which are colon cancer cell lines formed subcutaneously in nude mice.
  • the present embodiment provides a pharmaceutical composition containing a polynucleotide derived from miR-876-3p and which is found to be effective against a tumor in which the BRD4 gene and the RSK1 gene are activated. Further, the present invention provides a pharmaceutical composition containing a polynucleotide derived from miR-1293 and having an effect on a tumor in which the BRD4 gene, APEX1 gene, RPA1 gene and POLD4 gene are activated.
  • Cell lines include breast cancer 14 strains, colon cancer 10 strains, uterine body cancer 5 strains, esophageal cancer 43 strains, gastric cancer 4 strains, liver cancer 2 strains, lung cancer 3 strains, neuroblastoma 1 strain, oral cancer Twenty-two strains, ovarian cancer 22 strains, pancreatic cancer 1 strain, prostate cancer 4 strains, sarcoma 8 strains, thyroid cancer 12 strains, and urinary tract cancer 1 strain were prepared.
  • the table below exemplifies cell lines having a pathological mutation in BRCA1 or BRCA2 (hereinafter, may be abbreviated as BRCA1 / 2) in the prepared cell lines.
  • the pathological mutations of BRCA1 / 2 were intended to be frameshift mutations, nonsense mutations, and methylation.
  • 8,000 cells were seeded in 1 well of 96 well plate and cultured in a medium suitable for each cell line in a 37 ° C., 5% CO 2 environment or an environment free of CO 2. After 24 hours, miR-Negative Control (miR-NC; 20 ⁇ M) or miR-1293 (2.5, 5, 10, or 20 ⁇ M) was mixed with Lipofectamine RNAiMAX (Thermo Fisher scientific) and transfected.
  • miR-NC miR-Negative Control
  • miR-1293 2.5, 5, 10, or 20 ⁇ M
  • the cells were stained with a crystal violet solution (10% formalin, 0.1% crystal violet). Then, it was washed with water, completely dried, and then dissolved in a 2% SDS solution. The lysate was measured with an absorptiometer using a wavelength of 560 nm, and the obtained value was taken as the amount of viable cells. Based on miR-NC, the cell viability at each concentration of miR-1293 was calculated and graphed. A part of the result is shown in FIG. 7-1.
  • the cell line whose cell viability decreased to 0.5 or less at a concentration of less than 20 nM was designated as a miR-1293 high-sensitivity strain, and the miR-1293 sensitivity in the BRCA1 / 2 pathological mutation group (+) and the non-mutation group (-) was determined. evaluated. The results are shown in Figure 7-2.
  • the present embodiment provides a pharmaceutical composition comprising a polynucleotide derived from miR-1293 and found to be effective against tumors having a pathogenic variant in BRCA1 / 2 or in a state of BRCAness. .. Furthermore, it provides a pharmaceutical composition that is expected to be effective against tumors that are resistant to PAPR inhibitors.

Abstract

Provided is a pharmaceutical composition that suppresses the expression of a bromodomain protein (BRD4) gene and that can be used for treating a tumor. This pharmaceutical composition used for treating a tumor includes a polynucleotide having a base sequence identical to a transcription product of a gene encoding at least one micro-RNA selected from the group consisting of miR-1293, miR-876-3p, miR-4438, miR-6751, miR-634, and miR-92a-2-5p or to a processing product of the transcription product, or having a base sequence obtained by substituting, deleting, or adding 1-5 bases in said base sequence.

Description

医薬組成物Pharmaceutical composition
　本発明は、医薬組成物に関する。 The present invention relates to a pharmaceutical composition.
　日本人の死因の第一位はがんであり、国立がんセンターの統計（２０１２年）によると生涯においてがんに罹患する確率は日本人男性６３％、女性４７％と高い。また厚生労働省の統計（２０１５年）によると、日本人の死亡原因に占めるがんの割合は、男性３２％、女性２４％、全人口の約３分の１ががんで亡くなっている。がんによる死亡は増加し続け、２０３０年には世界で年間１１４０万人ががんで死亡すると予測されている。 Cancer is the leading cause of death in Japan, and according to the statistics of the National Cancer Center (2012), the probability of getting cancer in a lifetime is high at 63% for Japanese men and 47% for women. According to Ministry of Health, Labor and Welfare statistics (2015), cancer accounts for 32% of Japanese deaths, 24% of women, and about one-third of the total population dies of cancer. Cancer deaths continue to increase, with 11.4 million people worldwide expected to die from cancer annually in 2030.
　２００３年に終了したヒトゲノム計画により、遺伝子レベル、タンパク質分子レベルでがんを理解し、それを診断及び治療に応用しようとする動きは加速し、その結果、がんの診断及び治療の進歩は目覚ましい。特に、治療面では、従来の抗がん剤とは異なる機序として、がん細胞において活性化している遺伝子やタンパク質を標的としたいわゆる分子標的治療薬の進歩、開発も進んでいる。診断時にがんで活性化している分子を同定することでこのような分子標的治療薬が臨床応用されている。 The Human Genome Project, completed in 2003, has accelerated the move to understand cancer at the genetic and protein molecular levels and apply it to diagnosis and treatment, resulting in remarkable progress in cancer diagnosis and treatment. .. In particular, in terms of treatment, as a mechanism different from conventional anticancer agents, so-called molecular-targeted therapeutic agents targeting genes and proteins activated in cancer cells are advancing and developing. Such molecular-targeted therapeutic agents have been clinically applied by identifying molecules that are activated in cancer at the time of diagnosis.
　上記に関連して、例えば、国際公開第２０１９／１０７４８７号にはブロモドメインタンパク質（ＢＲＤ４）とがんとの関係に着目し、特定のマイクロＲＮＡを含み、腫瘍の処置に用いられる医薬組成物が提案されている。 In connection with the above, for example, WO 2019/107487 focuses on the relationship between bromodomain protein (BRD4) and cancer, and contains specific microRNAs and pharmaceutical compositions used in the treatment of tumors. Proposed.
　本発明は、ブロモドメインタンパク質（ＢＲＤ４）遺伝子の発現を抑制し、腫瘍の処置に用いることができる医薬組成物を提供することを目的とする。 An object of the present invention is to provide a pharmaceutical composition that suppresses the expression of a bromodomain protein (BRD4) gene and can be used for treating tumors.
　前記課題を解決するための具体的手段は以下の通りであり、本発明は以下の態様を包含する。第一態様は、ｍｉＲ－１２９３、ｍｉＲ－８７６－３ｐ、ｍｉＲ－４４３８、ｍｉＲ－６７５１、ｍｉＲ－６３４及びｍｉＲ－９２ａ－２－５ｐからなる群から選択される少なくとも１種のマイクロＲＮＡをコードする遺伝子の転写産物もしくはそのプロセッシング産物と同一の塩基配列、又は転写産物もしくはそのプロセッシング産物の塩基配列において１から５塩基が置換、欠損又は付加された塩基配列を有するポリヌクレオチドを含み、腫瘍の処置に用いられる医薬組成物である。 Specific means for solving the above problems are as follows, and the present invention includes the following aspects. The first aspect encodes at least one microRNA selected from the group consisting of miR-1293, miR-876-3p, miR-4438, miR-6751, miR-634 and miR-92a-2-5p. For the treatment of tumors, it contains a polynucleotide having the same base sequence as the transcript of a gene or its processing product, or a base sequence in which 1 to 5 bases are substituted, deleted or added in the base sequence of the transcript or its processing product. The pharmaceutical composition used.
　第二態様は、ｍｉＲ－１２９３、ｍｉＲ－８７６－３ｐ、ｍｉＲ－４４３８、ｍｉＲ－６７５１、ｍｉＲ－６３４及びｍｉＲ－９２ａ－２－５ｐからなる群から選択される少なくとも１種のマイクロＲＮＡをコードする遺伝子の転写産物もしくはそのプロセッシング産物と同一の塩基配列、又は転写産物もしくはそのプロセッシング産物の塩基配列において１から５塩基が置換、欠損又は付加された塩基配列を有するポリヌクレオチドを含み、ＢＲＤ４遺伝子関連疾患の処置に用いられる医薬組成物である。 The second aspect encodes at least one microRNA selected from the group consisting of miR-1293, miR-876-3p, miR-4438, miR-6751, miR-634 and miR-92a-2-5p. BRD4 gene-related disease containing a polynucleotide having the same base sequence as the transcript of a gene or its processing product, or a base sequence in which 1 to 5 bases are substituted, deleted or added in the base sequence of the transcript or its processing product. It is a pharmaceutical composition used for the treatment of.
　第三態様は、ｍｉＲ－１２９３、ｍｉＲ－８７６－３ｐ、ｍｉＲ－４４３８、ｍｉＲ－６７５１、ｍｉＲ－６３４及びｍｉＲ－９２ａ－２－５ｐからなる群から選択される少なくとも１種のマイクロＲＮＡをコードする遺伝子の転写産物もしくはそのプロセッシング産物と同一の塩基配列、又は転写産物もしくはそのプロセッシング産物の塩基配列において１から５塩基が置換、欠損又は付加された塩基配列を有するポリヌクレオチドを含む医薬組成物の有効量を対象に投与することを含む、腫瘍の処置方法である。 The third aspect encodes at least one microRNA selected from the group consisting of miR-1293, miR-876-3p, miR-4438, miR-6751, miR-634 and miR-92a-2-5p. Effectiveness of a pharmaceutical composition containing a polynucleotide having the same base sequence as the transcript of a gene or its processing product, or a base sequence in which 1 to 5 bases are substituted, deleted or added in the base sequence of the transcript or its processing product. A method of treating a tumor, including administering the dose to the subject.
　第四態様は、ｍｉＲ－１２９３、ｍｉＲ－８７６－３ｐ、ｍｉＲ－４４３８、ｍｉＲ－６７５１、ｍｉＲ－６３４及びｍｉＲ－９２ａ－２－５ｐからなる群から選択される少なくとも１種のマイクロＲＮＡをコードする遺伝子の転写産物もしくはそのプロセッシング産物と同一の塩基配列、又は転写産物もしくはそのプロセッシング産物の塩基配列において１から５塩基が置換、欠損又は付加された塩基配列を有するポリヌクレオチドを含む医薬組成物の有効量を対象に投与することを含む、ＢＲＤ４遺伝子関連疾患の処置方法である。 The fourth aspect encodes at least one microRNA selected from the group consisting of miR-1293, miR-876-3p, miR-4438, miR-6751, miR-634 and miR-92a-2-5p. Effectiveness of a pharmaceutical composition containing a polynucleotide having the same base sequence as the transcript of a gene or its processing product, or a base sequence in which 1 to 5 bases are substituted, deleted or added in the base sequence of the transcript or its processing product. A method of treating a BRD4 gene-related disorder, which comprises administering an amount to a subject.
　本発明によれば、ＢＲＤ４遺伝子の発現を抑制し、腫瘍の処置に用いることができる医薬組成物を提供することができる。 According to the present invention, it is possible to provide a pharmaceutical composition that suppresses the expression of the BRD4 gene and can be used for treating tumors.
大腸がん細胞株を用いた抗腫瘍性マイクロＲＮＡの１次スクリーニング結果を示す図である。It is a figure which shows the primary screening result of the antitumor microRNA using the colorectal cancer cell line. がん細胞株におけるＢＲＤ４の発現抑制を示すウエスタンブロットの結果を示す図である。It is a figure which shows the result of the Western blot which showed the expression suppression of BRD4 in a cancer cell line. ＢＲＤ４遺伝子の３’ＵＴＲ領域を組み込んだコンストラクトを用いたルシフェラーゼレポーターアッセイの結果を示す図である。It is a figure which shows the result of the luciferase reporter assay using the construct which incorporated the 3'UTR region of the BRD4 gene. 大腸がん細胞株に対してｓｉＲＮＡを用いて、ＢＲＤ４遺伝子をノックダウンした場合の細胞増殖に対する影響を示す図である。It is a figure which shows the effect on cell proliferation when the BRD4 gene is knocked down by using siRNA for a colorectal cancer cell line. がん細胞株に対して、ｍｉＲ－８７６－３ｐあるいはｍｉＲ－１２９３を導入した場合における細胞増殖曲線を示した図である。It is a figure which showed the cell growth curve at the time of introducing miR-876-3p or miR-1293 into a cancer cell line. 大腸がん細胞株に対して、ｍｉＲ－８７６－３ｐあるいはｍｉＲ－１２９３を導入した場合における細胞の状態を示した図である。It is a figure which showed the state of the cell when miR-876-3p or miR-1293 was introduced into the colorectal cancer cell line. がん細胞株に対して、ｍｉＲ－８７６－３ｐあるいはｍｉＲ－１２９３を導入した場合におけるアポトーシスの割合を示す図である。It is a figure which shows the rate of apoptosis at the time of introducing miR-876-3p or miR-1293 into a cancer cell line. がん細胞株に対して、ｍｉＲ－８７６－３ｐあるいはｍｉＲ－１２９３を導入した場合におけるアポトーシスマーカーの発現状態を示すウエスタンブロットである。6 is a Western blot showing the expression state of an apoptosis marker when miR-876-3p or miR-1293 is introduced into a cancer cell line. ｍｉＲ－８７６－３ｐをがん細胞株に導入した後に行った発現アレイ解析において発現が１．５倍以上低下した遺伝子数を示すベン図である。It is a Venn diagram showing the number of genes whose expression was reduced by 1.5 times or more in the expression array analysis performed after introducing miR-876-3p into a cancer cell line. ｍｉＲ－８７６－３ｐを導入した口腔がん細胞株における、ＲＳＫ１遺伝子、ｐ－ｐ７０Ｓ６Ｋ及びｐＳ６の発現状態を示すウエスタンブロットの結果を示す図である。It is a figure which shows the result of the Western blotting which shows the expression state of RSK1 gene, p-p70S6K and pS6 in the oral cancer cell line which introduced miR-876-3p. ＲＳＫ１遺伝子の３'ＵＴＲ領域を組み込んだコンストラクトを用いたｍｉＲ－８７６－３ｐのルシフェラーゼレポーターアッセイの結果を示す図である。It is a figure which shows the result of the luciferase reporter assay of miR-876-3p using the construct which incorporated the 3'UTR region of the RSK1 gene. 口腔がん細胞株に対してｓｉＲＮＡを用いて、ＲＳＫ１遺伝子及びＢＲＤ４遺伝子をノックダウンした場合の細胞増殖に対する影響を示す図である。It is a figure which shows the effect on cell proliferation when the RSK1 gene and the BRD4 gene are knocked down by using siRNA against an oral cancer cell line. ｍｉＲ－８７６－３ｐによる腫瘍増殖抑制の推定機序を示す概念図である。It is a conceptual diagram which shows the estimation mechanism of the tumor growth suppression by miR-876-3p. ｍｉＲ－１２９３をがん細胞株に導入した後に行った発現アレイ解析において発現が１．５倍以上低下した遺伝子数を示すベン図である。It is a Venn diagram showing the number of genes whose expression was reduced by 1.5 times or more in the expression array analysis performed after introducing miR-1293 into a cancer cell line. ｍｉＲ－１２９３を導入したがん細胞株における、ＡＰＥＸ１遺伝子、ＲＰＡ１遺伝子、ＰＯＬＤ４遺伝子の発現状態を示すウエスタンブロットの結果を示す図である。It is a figure which shows the result of the Western blotting which shows the expression state of APEX1 gene, RPA1 gene, and POLD4 gene in the cancer cell line which introduced miR-1293. ＡＰＥＸ１遺伝子、ＲＰＡ１遺伝子又はＰＯＬＤ４遺伝子の３'ＵＴＲ領域を組み込んだコンストラクトを用いたｍｉＲ－１２９３のルシフェラーゼレポーターアッセイの結果を示す図である。It is a figure which shows the result of the luciferase reporter assay of miR-1293 using the construct which incorporated the 3'UTR region of APEX1 gene, RPA1 gene or POLD4 gene. 大腸癌がん細胞株に対してｓｉＲＮＡを用いて、ＡＰＥＸ１遺伝子、ＰＲＡ１遺伝子、ＰＯＬＤ４遺伝子をノックダウンした場合の細胞増殖に対する影響を示す図である。FIG. 5 is a diagram showing the effect on cell proliferation when knockdown of APEX1 gene, PRA1 gene, and POLD4 gene is performed on colorectal cancer cell line using siRNA. ｍｉＲ－１２９３を導入したがん細胞株に対してＮＣＳを加えた後のＤＮＡ修復に対する影響を示す免疫蛍光染色の図である。It is a figure of immunofluorescence staining which shows the influence on the DNA repair after adding NCS to the cancer cell line which introduced miR-1293. ｍｉＲ－１２９３を導入したがん細胞株に対してＮＣＳを加えた後のＤＮＡ修復に対する影響を示す図である。It is a figure which shows the influence on the DNA repair after adding NCS to the cancer cell line which introduced miR-1293. ｍｉＲ－１２９３又はＢＲＤ４に対するｓｉＲＮＡを導入した大腸がん細胞株における相同組み換え修復を評価した結果を示す図である。It is a figure which shows the result of having evaluated the homologous recombination repair in the colorectal cancer cell line which introduced siRNA for miR-1293 or BRD4. ｍｉＲ－１２９３による腫瘍増殖抑制の推定機序を示す概念図である。It is a conceptual diagram which shows the estimation mechanism of the tumor growth suppression by miR-1293. マウスを用いたｉｎ　ｖｉｖｏ試験におけるｍｉＲ－８７６－３ｐあるいはｍｉＲ－１２９３の投与スケジュールを示した図である。It is a figure which showed the administration schedule of miR-876-3p or miR-1293 in an in vivo test using a mouse. 大腸がん細胞株の皮下注射から１９日後のマウスの皮下腫瘍の外観及び摘出された腫瘍を示した図である。It is a figure which showed the appearance of the subcutaneous tumor of the mouse 19 days after the subcutaneous injection of the colorectal cancer cell line, and the excised tumor. 上記ｉｎ　ｖｉｖｏ試験における、摘出された腫瘍の体積の測定結果を示す図である。It is a figure which shows the measurement result of the volume of the excised tumor in the said in vivo test. 上記ｉｎ　ｖｉｖｏ試験における、摘出された腫瘍の重量の測定結果を示す図である。It is a figure which shows the measurement result of the weight of the excised tumor in the said in vivo test. 上記ｉｎ　ｖｉｖｏ試験において摘出された腫瘍における、ｍｉＲ－８７６－３ｐ及びｍｉＲ－１２９３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 miR-876-3p and miR-1293 in the tumor excised in the above-mentioned in vivo test. 上記ｉｎ　ｖｉｖｏ試験において摘出された腫瘍におけるＢＲＤ４遺伝子の発現状態を免疫染色により示した図である。It is a figure which showed the expression state of the BRD4 gene in the tumor excised in the above-mentioned in vivo test by immunostaining. 上記ｉｎ　ｖｉｖｏ試験において摘出された腫瘍におけるＲＳＫ１遺伝子の発現状態を免疫染色により示した図である。It is a figure which showed the expression state of the RSK1 gene in the tumor excised in the above-mentioned in vivo test by immunostaining. 上記ｉｎ　ｖｉｖｏ試験において摘出された腫瘍におけるＡＰＥＸ１遺伝子、ＲＰＡ１遺伝子、ＰＯＬＤ４遺伝子の発現状態を免疫染色により示した図である。It is a figure which showed the expression state of APEX1 gene, RPA1 gene, and POLD4 gene in the tumor excised in the above-mentioned in vivo test by immunostaining. ｍｉＲ－１２９３をがん細胞株に導入した後の細胞生存率を示す図である。It is a figure which shows the cell viability after introducing miR-1293 into a cancer cell line. がん細胞株におけるＢＲＣＡ１／２病的変異の有無とｍｉＲ－１２９３感受性の関係を示す図である。It is a figure which shows the relationship between the presence or absence of BRCA1 / 2 pathological mutation and miR-1293 sensitivity in a cancer cell line.
　本明細書において組成物中の各成分の含有量は、組成物中に各成分に該当する物質が複数存在する場合、特に断らない限り、組成物中に存在する当該複数の物質の合計量を意味する。また、マイクロＲＮＡ（ｍｉＲＮＡ）には、リボヌクレオチドからなるポリヌクレオチド（オリゴヌクレオチド）に加えて、リボヌクレオチドと修飾ヌクレオチドとからなるポリヌクレオチド（オリゴヌクレオチド）及び修飾ヌクレオチドからなるポリヌクレオチド（オリゴヌクレオチド）が含まれる。更にマイクロＲＮＡは１本鎖であっても、２本鎖であってもよい。以下、本発明の実施形態を詳細に説明する。ただし、以下に示す実施形態は、本発明の技術思想を具体化するための、医薬組成物を例示するものであって、本発明は、以下に示す医薬組成物に限定されない。 In the present specification, the content of each component in the composition is the total amount of the plurality of substances present in the composition unless otherwise specified, when a plurality of substances corresponding to each component are present in the composition. means. Further, microRNA (miRNA) includes a polynucleotide (oligonucleotide) composed of a ribonucleotide, a polynucleotide (oligonucleotide) composed of a ribonucleotide and a modified nucleotide, and a polynucleotide (oligonucleotide) composed of a modified nucleotide. included. Further, the microRNA may be single-stranded or double-stranded. Hereinafter, embodiments of the present invention will be described in detail. However, the embodiments shown below exemplify a pharmaceutical composition for embodying the technical idea of the present invention, and the present invention is not limited to the pharmaceutical compositions shown below.
医薬組成物
　医薬組成物は、ｍｉＲ－１２９３、ｍｉＲ－８７６－３ｐ、ｍｉＲ－４４３８、ｍｉＲ－６７５１、ｍｉＲ－６３４及びｍｉＲ－９２ａ－２－５ｐからなる群から選択される少なくとも１種のマイクロＲＮＡ（以下、併せて特定マイクロＲＮＡともいう）をコードする遺伝子の転写産物もしくはそのプロセッシング産物と同一の塩基配列を有するポリヌクレオチド、又はそれらの転写産物もしくはそのプロセッシング産物の塩基配列において１から５塩基が置換、欠損又は付加された塩基配列を有するポリヌクレオチド（以下、併せて特定マイクロＲＮＡに由来するポリヌクレオチドともいう）を含み、腫瘍の処置に用いられる。医薬組成物はＢＲＤ４遺伝子の発現抑制剤であってよい。 Pharmaceutical Composition The pharmaceutical composition is at least one microRNA selected from the group consisting of miR-1293, miR-876-3p, miR-4438, miR-6751, miR-634 and miR-92a-2-5p. A polynucleotide having the same base sequence as a transcript of a gene encoding (hereinafter, also referred to as a specific microRNA) or a processing product thereof, or a polynucleotide having the same base sequence as the transcript thereof or a processing product thereof contains 1 to 5 bases. It contains a polynucleotide having a substituted, deleted or added base sequence (hereinafter, also referred to as a polynucleotide derived from a specific microRNA), and is used for treating a tumor. The pharmaceutical composition may be an agent that suppresses the expression of the BRD4 gene.
　一態様において、医薬組成物は、ｍｉＲ－１２９３、ｍｉＲ－８７６－３ｐ、ｍｉＲ－４４３８、ｍｉＲ－６７５１、ｍｉＲ－６３４及びｍｉＲ－９２ａ－２－５ｐからなる群から選択される少なくとも１種のマイクロＲＮＡをコードする遺伝子の転写産物もしくはそのプロセッシング産物と同一の塩基配列を有するポリヌクレオチド、又はそれらの転写産物もしくはそのプロセッシング産物の塩基配列において１から５塩基が置換、欠損又は付加された塩基配列を有し、少なくとも１種の修飾ヌクレオチドを含んでなるポリヌクレオチドを含み、腫瘍の処置に用いられる。処置される腫瘍は、ＢＲＤ４遺伝子を発現している腫瘍であってよい。 In one embodiment, the pharmaceutical composition is at least one micro selected from the group consisting of miR-1293, miR-876-3p, miR-4438, miR-6751, miR-634 and miR-92a-2-5p. A polynucleotide having the same base sequence as the transcript of the gene encoding RNA or its processing product, or a base sequence in which 1 to 5 bases are substituted, deleted or added in the base sequence of those transcripts or its processing product. Containing a polynucleotide having and containing at least one modified nucleotide, it is used in the treatment of tumors. The tumor to be treated may be a tumor expressing the BRD4 gene.
　一態様において、医薬組成物は、ｍｉＲ－１２９３、ｍｉＲ－８７６－３ｐ、ｍｉＲ－４４３８、ｍｉＲ－６７５１及びｍｉＲ－９２ａ－２－５ｐからなる群から選択される少なくとも１種のマイクロＲＮＡをコードする遺伝子の転写産物もしくはそのプロセッシング産物と同一の塩基配列を有するポリヌクレオチド、又はそれらの転写産物もしくはそのプロセッシング産物の塩基配列において１から５塩基が置換、欠損又は付加された塩基配列を有し、少なくとも１種の修飾ヌクレオチドを含んでなるポリヌクレオチドを含み、腫瘍の処置に用いられる。 In one embodiment, the pharmaceutical composition encodes at least one microRNA selected from the group consisting of miR-1293, miR-876-3p, miR-4438, miR-6751 and miR-92a-2-5p. A polynucleotide having the same base sequence as a gene transcript or its processing product, or a base sequence in which 1 to 5 bases are substituted, deleted or added in the base sequence of those transcripts or its processing product, and at least It contains a polynucleotide consisting of one modified nucleotide and is used in the treatment of tumors.
　特定マイクロＲＮＡに由来するポリヌクレオチドは、ＢＲＤ４遺伝子の発現を抑制することができる。これにより、腫瘍細胞における過剰な転写活性を抑制し、抗腫瘍性を示すことができる。ＢＲＤ４は、アセチル化修飾されたリジンに結合するブロモドメインを２つ有し、Ｃ末端側にはｐ－ＴＥＦｂ（positive transcription elongation factor b）と結合する領域を有しているタンパク質である。ヒストンのアセチル化修飾は転写活性と正の相関を認め、ｐ－ＴＥＦｂは転写を行うＲＮＡポリメラーゼIIを抑制するタンパク質を不活性化することで転写を活性化する。ＢＲＤ４はアセチル化修飾されたヒストンのリジンに結合することで、そこにｐ－ＴＥＦｂを動員し、転写を促進する働きがあるとされている。血液腫瘍がん、膵がん、乳がんを含む多くのがん種において、ＢＲＤ４遺伝子の発現の異常な活性化が腫瘍細胞の増殖に寄与すると考えられている。したがって、ＢＲＤ４遺伝子の発現を抑制することが、がん治療において有効である可能性がある。 A polynucleotide derived from a specific microRNA can suppress the expression of the BRD4 gene. As a result, excessive transcriptional activity in tumor cells can be suppressed and antitumor properties can be exhibited. BRD4 is a protein having two bromodomains that bind to acetylated modified lysine and a region that binds to p-TEFb (positive transcription evolution factor b) on the C-terminal side. Acetylation modification of histones is positively correlated with transcriptional activity, and p-TEFb activates transcription by inactivating a protein that suppresses transcriptional RNA polymerase II. BRD4 is said to have the function of mobilizing p-TEFb and promoting transcription by binding to the lysine of acetylated modified histones. In many cancer types, including hematological tumor cancer, pancreatic cancer, and breast cancer, abnormal activation of BRD4 gene expression is thought to contribute to tumor cell proliferation. Therefore, suppressing the expression of the BRD4 gene may be effective in the treatment of cancer.
　マイクロＲＮＡは標的転写産物（ｍＲＮＡ）のコーディング（ＣＤＳ）領域あるいは３'非翻訳領域（３'ＵＴＲ）への結合を通じて、その翻訳あるいは安定化を妨害することにより遺伝子発現を抑制する、内因性の小分子のノンコーディングＲＮＡである。１つのｍＲＮＡは複数のマイクロＲＮＡによって標的にされる一方、１つのマイクロＲＮＡは複数のｍＲＮＡを標的にすることができる。複数の発がん経路に寄与する複数の遺伝子を標的にすることができるマイクロＲＮＡの投与は、がん治療においてより効果的であると考えられる。 MicroRNAs are endogenous, suppressing gene expression by interfering with their translation or stabilization through binding of the target transcript (mRNA) to the coding (CDS) region or the 3'untranslated region (3'UTR). It is a small non-coding RNA. One mRNA can be targeted by multiple microRNAs, while one microRNA can target multiple mRNAs. Administration of microRNAs that can target multiple genes that contribute to multiple carcinogenic pathways may be more effective in cancer treatment.
　ｍｉＲ－１２９３、ｍｉＲ－８７６－３ｐ、ｍｉＲ－４４３８、ｍｉＲ－６７５１、ｍｉＲ－６３４及びｍｉＲ－９２ａ－２－５ｐは、ＢＲＤ４遺伝子の発現を抑制してＢＲＤ４遺伝子の発現が認められる腫瘍細胞に対する優れた増殖抑制効果を有する。また、少なくともｍｉＲ－８７６－３ｐは、ＲＳＫ１遺伝子の発現を抑制し、ＲＳＫ１遺伝子を発現が認められる腫瘍細胞に対して優れた増殖抑制効果を有する。また、少なくともｍｉＲ－１２９３は、ＡＰＥＸ１、ＲＰＡ１及びＰＯＬＤ４からなる群から選択される少なくとも１種の発現が認められる腫瘍細胞に対して優れた増殖抑制効果を有する。特定マイクロＲＮＡが、ＢＲＤ４遺伝子のみならず、他の腫瘍関連遺伝子の発現を抑制することで腫瘍細胞に対してより優れた増殖抑制効果を示すことができる。さらに、少なくともｍｉＲ－１２９３は、ＢＲＣＡ１／２遺伝子に病的変異を有する、又はＢＲＣＡｎｅｓｓ状態である腫瘍細胞に対して優れた増殖抑制効果を有する。 miR-1293, miR-876-3p, miR-4438, miR-6751, miR-634 and miR-92a-2-5p are excellent for tumor cells in which expression of BRD4 gene is observed by suppressing expression of BRD4 gene. Has a growth inhibitory effect. In addition, at least miR-876-3p suppresses the expression of the RSK1 gene and has an excellent growth-suppressing effect on tumor cells in which the expression of the RSK1 gene is observed. In addition, at least miR-1293 has an excellent growth inhibitory effect on tumor cells in which expression of at least one selected from the group consisting of APEX1, RPA1 and POLD4 is observed. By suppressing the expression of not only the BRD4 gene but also other tumor-related genes, the specific microRNA can exhibit a more excellent growth-suppressing effect on tumor cells. Furthermore, at least miR-1293 has an excellent growth inhibitory effect on tumor cells having a pathogenic mutation in the BRCA1 / 2 gene or in a BRCAness state.
　医薬組成物が含むポリヌクレオチドは、ｍｉＲ－１２９３、ｍｉＲ－８７６－３ｐ、ｍｉＲ－４４３８、ｍｉＲ－６７５１、ｍｉＲ－６３４及びｍｉＲ－９２ａ－２－５ｐからなる群から選択されるマイクロＲＮＡをコードする遺伝子の転写産物又はそのプロセッシング産物と同一の塩基配列を有していてよい。マイクロＲＮＡをコードする遺伝子の一次転写産物は初期転写産物（ｐｒｉ－ｍｉＲＮＡ）と呼ばれ、一般にステムループのヘアピン構造を有する。ｐｒｉ－ｍｉＲＮＡはＲＮａｓｅＩＩＩ様のＤｒｏｓｈａと呼ばれる酵素によってステムループ構造をもつ成熟したｍｉＲＮＡの前駆体（ｐｒｅ－ｍｉＲＮＡ）に変換される。ｐｒｅ－ｍｉＲＮＡは細胞核の外に輸送され、Ｄｉｃｅｒと呼ばれる酵素のスプライシングによって、２０から２５塩基長の２本鎖の成熟ｍｉＲＮＡとなる。したがって、マイクロＲＮＡをコードする遺伝子の転写産物又はそのプロセッシング産物は、ｐｒｉ－ｍｉＲＮＡ、ｐｒｅ－ｍｉＲＮＡ及び成熟ｍｉＲＮＡを包含する。なお、２本鎖の成熟ｍｉＲＮＡにおいては、一方の鎖のみが所望の効果を奏する場合、それぞれの鎖が所望の効果を奏する場合、及び２本鎖の状態で所望の効果を奏する場合の３態様がある。したがって、本明細書におけるｍｉＲＮＡに由来するポリヌクレオチドは、１本鎖及び２本鎖のいずれであってもよく、好ましくは２本鎖である。 The polynucleotide contained in the pharmaceutical composition encodes a microRNA selected from the group consisting of miR-1293, miR-876-3p, miR-4438, miR-6751, miR-634 and miR-92a-2-5p. It may have the same base sequence as the transcript of the gene or its processing product. The primary transcript of the gene encoding the microRNA is called the early transcript (pri-miRNA) and generally has a stem-loop hairpin structure. Pri-miRNAs are converted to mature miRNA precursors (pre-miRNAs) with a stem-loop structure by an RNase III-like enzyme called Drosha. The pre-miRNA is transported out of the cell nucleus and spliced with an enzyme called Dicer to become a 20 to 25 base long double-stranded mature miRNA. Thus, transcripts of genes encoding microRNAs or processing products thereof include tri-miRNAs, pre-miRNAs and mature miRNAs. In the double-stranded mature miRNA, there are three embodiments: when only one strand exerts a desired effect, when each strand exerts a desired effect, and when a double-stranded state exerts a desired effect. There is. Therefore, the miRNA-derived polynucleotides herein may be either single-stranded or double-stranded, preferably double-stranded.
　また、医薬組成物が含むポリヌクレオチドは、ｍｉＲ－１２９３、ｍｉＲ－８７６－３ｐ、ｍｉＲ－４４３８、ｍｉＲ－６７５１、ｍｉＲ－６３４及びｍｉＲ－９２ａ－２－５ｐからなる群から選択されるマイクロＲＮＡをコードする遺伝子の転写産物又はそのプロセッシング産物の機能を保持する変異体と同一の塩基配列を有していてもよい。変異体は、マイクロＲＮＡをコードする遺伝子の転写産物又はそのプロセッシング産物の塩基配列において１から５塩基が置換、欠損又は付加された塩基配列を有していてもよい。置換、欠損又は付加される塩基数は、好ましくは１から３であってよく、１又は２であってよい。また、変異体は元の塩基配列に対して、例えば８０％以上、好ましくは８５％以上、９０％以上又は９５％以上の配列相同性を有していてよい。配列相同性は、例えばＢＬＡＳＴを用いて算出される。 The polynucleotide contained in the pharmaceutical composition is a microRNA selected from the group consisting of miR-1293, miR-876-3p, miR-4438, miR-6751, miR-634 and miR-92a-2-5p. It may have the same base sequence as a mutant that retains the function of the transcript of the encoding gene or its processing product. The mutant may have a base sequence in which 1 to 5 bases are substituted, deleted or added in the base sequence of the transcript of the gene encoding microRNA or the processing product thereof. The number of bases substituted, deleted or added is preferably 1 to 3, and may be 1 or 2. Further, the mutant may have, for example, 80% or more, preferably 85% or more, 90% or more, or 95% or more sequence homology with respect to the original base sequence. Sequence homology is calculated using, for example, BLAST.
　ここで、特定マイクロＲＮＡが１本鎖である場合、ｍｉＲ－１２９３は、ｈｓａ－ｍｉＲ－１２９３であってよく、配列番号１の塩基配列を有していてよい。ｍｉＲ－８７６－３ｐは、ｈｓａ－ｍｉＲ－８７６－３ｐであってよく、配列番号２の塩基配列を有していてよい。ｍｉＲ－４４３８は、ｈｓａ－ｍｉＲ－４４３８であってよく、配列番号３の塩基配列を有していてよい。ｍｉＲ－６７５１は、ｈｓａ－ｍｉＲ－６７５１であってよく、ｈｓａ－ｍｉＲ－６７５１－５ｐであってよく、配列番号４の塩基配列を有していてよい。ｍｉＲ－６３４は、ｈｓａ－ｍｉＲ－６３４であってよく、配列番号５の塩基配列を有していてよい。ｍｉＲ－９２ａ－２－５ｐは、ｈｓａ－ｍｉＲ－９２ａ－２－５ｐであってよく、配列番号６の塩基配列を有していてよい。 Here, when the specific microRNA is single-stranded, miR-1293 may be hsa-miR-1293 and may have the base sequence of SEQ ID NO: 1. miR-876-3p may be hsa-miR-876-3p and may have the nucleotide sequence of SEQ ID NO: 2. miR-4438 may be hsa-miR-4438 and may have the nucleotide sequence of SEQ ID NO: 3. miR-6751 may be hsa-miR-6751, hsa-miR-6751-5p, and may have the nucleotide sequence of SEQ ID NO: 4. miR-634 may be hsa-miR-634 and may have the nucleotide sequence of SEQ ID NO: 5. miR-92a-2-5p may be hsa-miR-92a-2-5p and may have the base sequence of SEQ ID NO: 6.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　医薬組成物は、ポリヌクレオチドを１本鎖として含んでいてもよく、２本鎖として含んでいてもよい。ポリヌクレオチドを２本鎖として含む場合、少なくとも部分的に２本鎖を形成していればよく、２本鎖の末端の少なくとも一方に１本鎖部分を有していてもよく、２本鎖部分に少なくとも１対のミスマッチ塩基対又は塩基欠損を有していてもよい。２本鎖の末端に１本鎖部分を有する場合、１本鎖部分を少なくとも３’側に有することが好ましい。また、１本鎖部分の鎖長は、例えば、２残基以上２０残基以下、好ましくは２残基以上１２残基以下、又は２残基以上５残基以下である。一方、２本鎖部分に複数のミスマッチ塩基対を有する場合、ミスマッチ塩基対は連続して配置されてもよく、不連続に配置されてもよい、また、ミスマッチ塩基対の総数は例えば、１０塩基対以下、好ましくは６塩基対以下、又は４塩基対以下であり、また例えば、１塩基対以上、２塩基対以上、又は３塩基対以上であってよい。なお、相補的な塩基対には、熱力学的に安定な非ワトソンクリック型のゆらぎ塩基対（例えば、Ｇ－Ｕ）が含まれる。 The pharmaceutical composition may contain the polynucleotide as a single strand or may contain the polynucleotide as a double strand. When the polynucleotide is contained as a double strand, it may have at least a partially double strand, and at least one of the ends of the double strand may have a single strand portion. May have at least one pair of mismatched base pairs or base deficiencies. When the double-stranded portion has a single-stranded portion at the end, it is preferable to have the single-stranded portion at least on the 3'side. The chain length of the single-stranded portion is, for example, 2 residues or more and 20 residues or less, preferably 2 residues or more and 12 residues or less, or 2 residues or more and 5 residues or less. On the other hand, when there are a plurality of mismatched base pairs in the double-stranded portion, the mismatched base pairs may be arranged continuously or discontinuously, and the total number of mismatched base pairs is, for example, 10 bases. It may be pair or less, preferably 6 base pairs or less, or 4 base pairs or less, and may be, for example, 1 base pair or more, 2 base pairs or more, or 3 base pairs or more. Note that complementary base pairs include thermodynamically stable non-Watson click type wobble base pairs (eg, GU).
　医薬組成物が含む２本鎖のポリヌクレオチドは、下記（１）から（６）のいずれかの塩基配列又はその変異体の塩基配列を有していてよい。また、医薬組成物は、下記（１）から（６）のいずれかの塩基配列又はその変異体の塩基配列を有する２本鎖ポリヌクレオチドからなる群から選択される少なくとも１種を含んでいてよい。 The double-stranded polynucleotide contained in the pharmaceutical composition may have the base sequence of any of the following (1) to (6) or the base sequence of a mutant thereof. In addition, the pharmaceutical composition may contain at least one selected from the group consisting of double-stranded polynucleotides having the base sequence of any of the following (1) to (6) or the base sequence of a variant thereof. ..
（１）第１の２本鎖ポリヌクレオチドは、ｈｓａ－ｍｉＲ－１２９３をコードする遺伝子に由来する。第１の２本鎖ポリヌクレオチドは、塩基配列「ＵＧＧＧＵＧＧＵＣＵＧＧＡＧＡＵＵＵＧＵＧＣ」（配列番号１）の１本鎖ポリヌクレオチド及び塩基配列「ＧＣＡＣＡＡＡＵＣＵＣＣＧＧＡＣＣＡＣＵＵＡ」（配列番号７）の１本鎖ポリヌクレオチドからなる相補的な２本鎖ポリヌクレオチドである。配列番号１は、ｈｓａ－ｍｉＲ－１２９３（成熟ｍｉＲＮＡ）の塩基配列であり、以下に示す相補的構造を形成し得る。天然型の成熟ｍｉＲＮＡでは、配列番号１の塩基配列を有する５’側のＲＮＡがガイド鎖として、配列番号７の塩基配列を有する３’側のＲＮＡがパッセンジャー鎖として位置付けられている。 (1) The first double-stranded polynucleotide is derived from the gene encoding hsa-miR-1293. The first double-stranded polynucleotide consists of a single-stranded polynucleotide having the nucleotide sequence "UGGGUGGUCUGGAGAUUGUGGC" (SEQ ID NO: 1) and a single-stranded polynucleotide having the nucleotide sequence "GCACAAAUCUCCGGACCUUA" (SEQ ID NO: 7). It is a chain polynucleotide. SEQ ID NO: 1 is the base sequence of hsa-miR-1293 (mature miRNA) and can form the complementary structure shown below. In the natural mature miRNA, the RNA on the 5'side having the nucleotide sequence of SEQ ID NO: 1 is positioned as the guide strand, and the RNA on the 3'side having the nucleotide sequence of SEQ ID NO: 7 is positioned as the passenger strand.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-C000003
Figure JPOXMLDOC01-appb-C000003
（２）第２の２本鎖ポリヌクレオチドは、ｈｓａ－ｍｉＲ－８７６－３ｐをコードする遺伝子に由来する。第２の２本鎖ポリヌクレオチドは、塩基配列「ＵＧＧＵＧＧＵＵＵＡＣＡＡＡＧＵＡＡＵＵＣＡ」（配列番号２）の１本鎖ポリヌクレオチド及び塩基配列「ＵＧＧＡＵＵＵＣＵＵＵＧＵＧＡＡＵＣＡＣＣＡ」（配列番号８）の１本鎖ポリヌクレオチドからなる略相補的な２本鎖ポリヌクレオチドである。配列番号２は、ｈｓａ－ｍｉＲ－８７６－３ｐ（成熟ｍｉＲＮＡ）の塩基配列であり、以下に示す略相補的構造を形成し得る。天然型の成熟ｍｉＲＮＡでは、配列番号２の塩基配列を有する３’側のＲＮＡがガイド鎖として、配列番号８の塩基配列を有する５’側のＲＮＡがパッセンジャー鎖として位置付けられている。ここで「略相補的」とは、下記式のように、２本鎖ポリヌクレオチドの一部が１本鎖である部分、及び／又は、塩基対の一部が水素結合で結合していない部分（ミスマッチ）を含みつつ、全体としては２本鎖ポリヌクレオチドを構成している状態である。 (2) The second double-stranded polynucleotide is derived from the gene encoding hsa-miR-876-3p. The second double-stranded polynucleotide is a substantially complementary 2 consisting of a single-stranded polynucleotide having the nucleotide sequence "UGGUGUUUAGAAAGUAAUUCA" (SEQ ID NO: 2) and a single-stranded polynucleotide having the nucleotide sequence "UGGAUUUCUUGUAUCCA" (SEQ ID NO: 8). It is a main chain polynucleotide. SEQ ID NO: 2 is the base sequence of hsa-miR-876-3p (mature miRNA) and can form a substantially complementary structure shown below. In the natural mature miRNA, the RNA on the 3'side having the nucleotide sequence of SEQ ID NO: 2 is positioned as the guide strand, and the RNA on the 5'side having the nucleotide sequence of SEQ ID NO: 8 is positioned as the passenger strand. Here, "substantially complementary" means a portion in which a part of the double-stranded polynucleotide is a single strand and / or a portion in which a part of the base pair is not bonded by a hydrogen bond, as shown in the following formula. It is in a state of constituting a double-stranded polynucleotide as a whole while containing (mismatch).
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-C000005
Figure JPOXMLDOC01-appb-C000005
（３）第３の２本鎖ポリヌクレオチドは、ｈｓａ－ｍｉＲ－４４３８をコードする遺伝子に由来する。第３の２本鎖ポリヌクレオチドは、塩基配列「ＣＡＣＡＧＧＣＵＵＡＧＡＡＡＡＧＡＣＡＧＵ」（配列番号３）の１本鎖ポリヌクレオチド及び塩基配列「ＡＣＵＧＵＣＵＵＵＵＣＵＡＡＧＣＣＵＧＵＧ」（配列番号９）の１本鎖ポリヌクレオチドからなる相補的な２本鎖ポリヌクレオチドである。配列番号３は、ｈｓａ－ｍｉＲ－４４３８（成熟ｍｉＲＮＡ）の塩基配列であり、以下に示す相補的構造を形成し得る。天然型の成熟ｍｉＲＮＡでは、配列番号３の塩基配列を有する３’側のＲＮＡがガイド鎖として、配列番号９の塩基配列を有する５’側のＲＮＡがパッセンジャー鎖として位置付けられている。 (3) The third double-stranded polynucleotide is derived from the gene encoding hsa-miR-4438. The third double-stranded polynucleotide is a complementary two-stranded polynucleotide consisting of the single-stranded polynucleotide of the base sequence "CAAGGCUUAGAAAAGACAGU" (SEQ ID NO: 3) and the single-stranded polynucleotide of the base sequence "ACUGUCUUUCUAAGCCUGUG" (SEQ ID NO: 9). It is a chain polynucleotide. SEQ ID NO: 3 is the base sequence of hsa-miR-4438 (mature miRNA) and can form the complementary structure shown below. In the native mature miRNA, the RNA on the 3'side having the nucleotide sequence of SEQ ID NO: 3 is positioned as the guide strand, and the RNA on the 5'side having the nucleotide sequence of SEQ ID NO: 9 is positioned as the passenger strand.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-C000007
Figure JPOXMLDOC01-appb-C000007
（４）第４の２本鎖ポリヌクレオチドは、ｈｓａ－ｍｉＲ－６７５１－５ｐをコードする遺伝子に由来する。第４の２本鎖ポリヌクレオチドは、塩基配列「ＵＵＧＧＧＧＧＵＧＡＧＧＵＵＧＧＵＧＵＣＵＧＧ」（配列番号４）の１本鎖ポリヌクレオチド及び塩基配列「ACUGAGCCUCUCUCUCUCCAG」（配列番号１０）の１本鎖ポリヌクレオチドからなる略相補的な２本鎖ポリヌクレオチドである。配列番号４は、ｈｓａ－ｍｉＲ－６７５１－５ｐ（成熟ｍｉＲＮＡ）の塩基配列であり、以下に示す略相補的構造を形成し得る。天然型の成熟ｍｉＲＮＡでは、配列番号４の塩基配列を有する５’側のＲＮＡがガイド鎖として、配列番号１０の塩基配列を有する３’側のＲＮＡがパッセンジャー鎖として位置付けられている。 (4) The fourth double-stranded polynucleotide is derived from the gene encoding hsa-miR-6751-5p. The fourth double-stranded polynucleotide is a substantially complementary 2 consisting of a single-stranded polynucleotide having the nucleotide sequence "UGGGGGGUGAGGUGUGGUGUCUGUGG" (SEQ ID NO: 4) and a single-stranded polynucleotide having the nucleotide sequence "ACUGAGCCUCUCUCUCUCCAG" (SEQ ID NO: 10). It is a main chain polynucleotide. SEQ ID NO: 4 is the base sequence of hsa-miR-6751-5p (mature miRNA), which can form a substantially complementary structure shown below. In the natural mature miRNA, the RNA on the 5'side having the nucleotide sequence of SEQ ID NO: 4 is positioned as the guide strand, and the RNA on the 3'side having the nucleotide sequence of SEQ ID NO: 10 is positioned as the passenger strand.
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-C000009
Figure JPOXMLDOC01-appb-C000009
（５）第５の２本鎖ポリヌクレオチドは、ｈｓａ－ｍｉＲ－６３４をコードする遺伝子に由来する。第５の２本鎖ポリヌクレオチドは、塩基配列「ＡＡＣＣＡＧＣＡＣＣＣＣＡＡＣＵＵＵＧＧＡＣ」（配列番号５）の１本鎖ポリヌクレオチド及び塩基配列「ＡＵＣＧＡＧＧＧＵＵＧＧＧＧＣＵＵＧＧＵ」（配列番号１１）の１本鎖ポリヌクレオチドからなる略相補的な２本鎖ポリヌクレオチドである。配列番号５は、ｈｓａ－ｍｉＲ－６３４（成熟ｍｉＲＮＡ）の塩基配列であり、以下に示す略相補的構造を形成し得る。天然型の成熟ｍｉＲＮＡでは、配列番号５の塩基配列を有する３’側のＲＮＡがガイド鎖として、配列番号１１の塩基配列を有する５’側のＲＮＡがパッセンジャー鎖として位置付けられている。 (5) The fifth double-stranded polynucleotide is derived from the gene encoding hsa-miR-634. The fifth double-stranded polynucleotide is a substantially complementary 2 consisting of a single-stranded polynucleotide having the nucleotide sequence "AACCAGCACCCCAAACUUGGAC" (SEQ ID NO: 5) and a single-stranded polynucleotide having the nucleotide sequence "AUCGAGGGUUGGGGGCUUGGU" (SEQ ID NO: 11). It is a main chain polynucleotide. SEQ ID NO: 5 is the base sequence of hsa-miR-634 (mature miRNA) and can form a substantially complementary structure as shown below. In the native mature miRNA, the RNA on the 3'side having the nucleotide sequence of SEQ ID NO: 5 is positioned as the guide strand, and the RNA on the 5'side having the nucleotide sequence of SEQ ID NO: 11 is positioned as the passenger strand.
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-C000011
Figure JPOXMLDOC01-appb-C000011
（６）第６の２本鎖ポリヌクレオチドは、ｈｓａ－ｍｉＲ－９２ａ－２をコードする遺伝子に由来する。第６の２本鎖ポリヌクレオチドは、塩基配列「ＧＧＧＵＧＧＧＧＡＵＵＵＧＵＵＧＣＡＵＵＡＣ」（配列番号６）の１本鎖ポリヌクレオチド及び塩基配列「ＵＡＵＵＧＣＡＣＵＵＧＵＣＣＣＧＧＣＣＵＧＵ」（配列番号１２）の１本鎖ポリヌクレオチドからなる略相補的な２本鎖ポリヌクレオチドである。配列番号６は、ｈｓａ－ｍｉＲ－９２ａ－２（成熟ｍｉＲＮＡ）の塩基配列であり、以下に示す略相補的構造を形成し得る。天然型の成熟ｍｉＲＮＡでは、配列番号６の塩基配列を有する５’側のＲＮＡがガイド鎖として、配列番号１２の塩基配列を有する３’側のＲＮＡがパッセンジャー鎖として位置付けられている。 (6) The sixth double-stranded polynucleotide is derived from the gene encoding hsa-miR-92a-2. The sixth double-stranded polynucleotide is a substantially complementary 2 consisting of a single-stranded polynucleotide having the nucleotide sequence "GGGUGGGAUUUGUGCAUUAC" (SEQ ID NO: 6) and a single-stranded polynucleotide having the nucleotide sequence "UAUUGCACUUGUCCGCGCCUGU" (SEQ ID NO: 12). It is a main chain polynucleotide. SEQ ID NO: 6 is the base sequence of hsa-miR-92a-2 (mature miRNA) and can form a substantially complementary structure as shown below. In the native mature miRNA, the RNA on the 5'side having the nucleotide sequence of SEQ ID NO: 6 is positioned as the guide strand, and the RNA on the 3'side having the nucleotide sequence of SEQ ID NO: 12 is positioned as the passenger strand.
Figure JPOXMLDOC01-appb-T000012
Figure JPOXMLDOC01-appb-T000012
Figure JPOXMLDOC01-appb-C000013
Figure JPOXMLDOC01-appb-C000013
　ポリヌクレオチドを構成するヌクレオチドは、少なくとも１個のリボヌクレオチドを含んでいてもよく、リボヌクレオチドに代えて対応するデオキシリボヌクレオチド及び修飾されたヌクレオチドの少なくとも１種を含んでいてもよい。修飾されたヌクレオチドには、リン酸部分が修飾されたヌクレオチド、糖部分が修飾されたヌクレオチド、塩基部分が修飾されたヌクレオチド等が含まれる。修飾されたヌクレオチドは、リン酸部分、糖部分及び塩基部分のいずれかが修飾されたものであってもよく、２種以上の修飾が組み合わされたものであってもよい。修飾されたヌクレオチドについては、例えば、特開平１０－３０４８８９号公報、国際公開第２００５／０２１５７０号、特開平１０－１９５０９８号公報、特表２００２－５２１３１０号公報、国際公開第２００７／１４３３１５号、国際公開第２００８／０４３７５３号、国際公開第２００８／０２９６１９号、国際公開第２００８／０４９０８５号等を参照することができる。 The nucleotide constituting the polynucleotide may contain at least one ribonucleotide, and may contain at least one of the corresponding deoxyribonucleotide and modified nucleotide in place of the ribonucleotide. Modified nucleotides include nucleotides with a modified phosphate moiety, nucleotides with a modified sugar moiety, nucleotides with a modified base moiety, and the like. The modified nucleotide may be one in which any of the phosphoric acid moiety, the sugar moiety and the base moiety is modified, and may be a combination of two or more kinds of modifications. Regarding the modified nucleotides, for example, JP-A-10-304889, WO 2005/021570, JP-A-10-195098, JP-A-2002-521310, WO 2007/143315, International Publication No. Publication No. 2008/043753, International Publication No. 2008/029619, International Publication No. 2008/049085, etc. can be referred to.
　リン酸部分の修飾としては、酸素原子を硫黄原子に置換した「ホスホロチオエート修飾」（Ｓ化）等が知られている。糖部分の修飾としては、非架橋化型修飾と、架橋型修飾が知られている。非架橋型修飾としては、２'位のフッ素（Ｆ）化、Ｏ－メチル化、ＭＯＥ化等の２'位水酸基の修飾、モルフォリノ核酸化等が挙げられる。また、架橋化型修飾としては、ＬＮＡ（２’，４’－ＢＮＡ）化、ＥＮＡ化等が挙げられる。 As a modification of the phosphoric acid moiety, "phosphorothioate modification" (Sification) in which an oxygen atom is replaced with a sulfur atom is known. Non-crosslinked modification and crosslinked modification are known as modifications of the sugar moiety. Examples of the non-crosslinking type modification include modification of the 2'-hydroxyl group such as fluorination (F) at the 2'position, O-methylation, and MOE formation, and morpholinonucleation. Further, examples of the crosslinked modification include LNA (2', 4'-BNA) conversion, ENA conversion and the like.
　本実施形態に係るポリヌクレオチドは、公知ポリヌクレオチドの合成法等を用いて合成することが可能である。ポリヌクレオチドの合成法としては、ホスホロアミダイド法及びその改良法、Ｈ－ホスホネート法及びその改良法、酵素合成法（ｉｎ　ｖｉｔｒｏ転写法）等が挙げられる。また、市販品の当該核酸、委託製造された当該核酸を適応することも可能である。 The polynucleotide according to this embodiment can be synthesized by using a known method for synthesizing a polynucleotide or the like. Examples of the method for synthesizing a polynucleotide include a phosphoromidide method and an improved method thereof, an H-phosphonate method and an improved method thereof, and an enzyme synthesis method (in vitro transcription method). It is also possible to apply the nucleic acid of a commercially available product or the nucleic acid of which is manufactured by consignment.
　医薬組成物は、有効成分として、ポリヌクレオチドに加えて、他の抗腫瘍性化合物を更に含んでいてもよい。他の抗腫瘍性化合物としては、例えば、代謝拮抗剤、分子標的薬、アルキル化剤、植物アルカロイド剤、抗がん性抗生物質、プラチナ製剤、ホルモン剤、生物学的応答調節剤、免疫チェックポイント阻害剤などが挙げられる。 The pharmaceutical composition may further contain other antitumor compounds as an active ingredient in addition to the polynucleotide. Other antitumor compounds include, for example, antimetabolites, molecular targeted drugs, alkylating agents, plant alkaloids, anticancer antibiotics, platinum preparations, hormonal agents, biological response regulators, immune checkpoints. Inhibitors and the like can be mentioned.
　医薬組成物は、上記の有効成分としての核酸（ポリヌクレオチド）とともに適切な医薬製剤担体を含んで、製剤組成物の形態に調製されてもよい。当該製剤担体としては、使用形態に応じた担体を選択することが可能であり、充填剤、増量剤、結合剤、付湿剤、崩壊剤、界面活性剤等の賦形剤、希釈剤等を使用することができる。また、上記の有効成分としての核酸に対して適切な担体を添加することで使用時に液状となしうる乾燥剤とすることも可能である。 The pharmaceutical composition may be prepared in the form of a pharmaceutical composition by containing a suitable pharmaceutical pharmaceutical carrier together with the nucleic acid (polynucleotide) as the active ingredient described above. As the pharmaceutical carrier, a carrier can be selected according to the mode of use, and excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, surfactants, diluents and the like can be used. Can be used. It is also possible to prepare a desiccant that can be made liquid at the time of use by adding an appropriate carrier to the nucleic acid as the active ingredient.
　医薬組成物は、有効成分であるポリヌクレオチドを薬学的に許容される担体に内包、又は結合した状態で含んでいてもよい。例えば、高分子ミセル、シクロデキストリン含有ポリマーで構成されたナノ粒子、安定核酸脂質粒子、多機能エンベローブ型ナノ構造体等のドラッグデリバリーシステムを活用して、医薬組成物の効果をより向上させることが可能である。 The pharmaceutical composition may contain a polynucleotide as an active ingredient in a pharmaceutically acceptable carrier in a state of being encapsulated or bound. For example, drug delivery systems such as high molecular weight micelles, nanoparticles composed of cyclodextrin-containing polymers, stable nucleic acid lipid particles, and multifunctional envelope-type nanostructures can be utilized to further improve the effects of pharmaceutical compositions. It is possible.
　医薬組成物の形態は、上記の有効成分としての核酸を効果的に含有可能な形態であれば、特に限定されるものではなく、錠剤、粉末剤、顆粒剤、丸剤等の固剤、軟膏剤、ハップ剤であってもよい。また、液剤、懸濁剤、乳剤等の注射剤形態とするのも好適である。現状において医薬組成物は、局所への投与が効果的である。この投与形式に適した剤型は、通常は注射剤、軟膏剤、又はハップ剤の形態である。注射剤を用いる局所への投与は、腫瘍に対して、静脈内、皮下、皮内、筋肉内注射等の体外からの注射により、直接有効成分を注入し、さらに、内視鏡を用いて体内（消化管内、子宮内、膀胱内等）の腫瘍に対して、直接有効成分を注入する、等の態様で行われる。軟膏剤又はハップ剤を用いる局所への投与は、皮膚がんに対して直接有効成分を浸潤させる等の態様で行われる。 The form of the pharmaceutical composition is not particularly limited as long as it can effectively contain the nucleic acid as the active ingredient, and is a solid agent such as a tablet, a powder, a granule, or a pill, or an ointment. It may be an agent or a pill agent. It is also preferable to use an injection form such as a liquid, a suspension, or an emulsion. At present, topical administration of pharmaceutical compositions is effective. Dosage forms suitable for this dosage form are usually in the form of injections, ointments, or haps. For local administration using an injection, the active ingredient is directly injected into the tumor by injection from outside the body such as intravenous, subcutaneous, intradermal, or intramuscular injection, and further, the active ingredient is injected into the body using an endoscope. It is performed by directly injecting the active ingredient into a tumor (intradigestive tract, uterus, bladder, etc.). Topical administration using an ointment or a happing agent is carried out in such a manner that the active ingredient is directly infiltrated against skin cancer.
　上記の有効成分としての核酸の人体に対する投与量は、一日成人１人あたり、例えば、０．０１μｇ以上１０００ｍｇ以下程度である。この投与は１日１回、又は２回以上５回以下であってよく、さらに連日又は数日おきに行うことも可能である。 The dose of the nucleic acid as the active ingredient to the human body is, for example, 0.01 μg or more and 1000 mg or less per adult per day. This administration may be performed once a day, or twice or more and 5 times or less, and may be performed every day or every few days.
　医薬組成物は、腫瘍の処置に用いられる。ここで腫瘍の処置とは、腫瘍について施される何らかの処置であればよく、例えば、腫瘍の治療、改善、進行の抑制（悪化の防止）、予防、腫瘍に起因する症状の緩和等が挙げられる。医薬組成物は、ＢＲＤ４遺伝子の発現を抑制するポリヌクレオチドを含むことから、処置の対象となる腫瘍は、ＢＲＤ４遺伝子を発現している腫瘍細胞を含むことが好ましい。処置の対象となる腫瘍としては、食道がん、肺がん、口腔がん、胃がん、大腸がん、子宮がん、皮膚がん、脳腫瘍、神経芽腫、膠芽腫、乳がん、膵がん、卵巣がん、前立腺がん、膀胱がん、食道がん、肝がん、腎がん等の固形がん、及び血液腫瘍等が例示される。処置の対象となる腫瘍は、好ましくは、大腸がん、膵がん、口腔がん、肺がん、食道がん、胃がん、子宮がん、皮膚がん、血液腫瘍、脳腫瘍、神経芽腫、膠芽腫、乳がん、卵巣がん、前立腺がん、膀胱がん、食道がん、肝がん及び腎がんからなる群から選ばれる少なくとも１種であってよい。また、有効成分であるポリヌクレオチドが確実に腫瘍に到達可能であることから、局所投与、すなわち、医薬組成物を直接腫瘍に接触させることが可能な腫瘍が、処置対象として好適である。 The pharmaceutical composition is used in the treatment of tumors. Here, the treatment of the tumor may be any treatment performed on the tumor, and examples thereof include treatment of the tumor, improvement, suppression of progression (prevention of deterioration), prevention, alleviation of symptoms caused by the tumor, and the like. .. Since the pharmaceutical composition contains a polynucleotide that suppresses the expression of the BRD4 gene, the tumor to be treated preferably contains a tumor cell expressing the BRD4 gene. Tumors to be treated include esophageal cancer, lung cancer, oral cancer, gastric cancer, colon cancer, uterine cancer, skin cancer, brain tumor, neuroblastoma, glioblastoma, breast cancer, pancreatic cancer, and ovary. Examples thereof include solid cancers such as cancer, prostate cancer, bladder cancer, esophageal cancer, liver cancer and renal cancer, and hematological malignancies. The tumors to be treated are preferably colon cancer, pancreatic cancer, oral cancer, lung cancer, esophageal cancer, gastric cancer, uterine cancer, skin cancer, hematological tumor, brain tumor, neuroblastoma, collagen bud. It may be at least one selected from the group consisting of tumor, breast cancer, ovarian cancer, prostate cancer, bladder cancer, esophageal cancer, liver cancer and renal cancer. In addition, since the polynucleotide as an active ingredient can reach the tumor with certainty, topical administration, that is, a tumor in which the pharmaceutical composition can be brought into direct contact with the tumor is suitable as a treatment target.
　医薬組成物は、ＲＳＫ１遺伝子を発現している腫瘍の処置に用いられてもよい。医薬組成物が有効成分として、例えばｍｉＲ－８７６－３ｐをコードする遺伝子に由来するポリヌクレオチドを含む場合に特に好適である。 The pharmaceutical composition may be used in the treatment of tumors expressing the RSK1 gene. It is particularly suitable when the pharmaceutical composition contains, for example, a polynucleotide derived from a gene encoding miR-876-3p as an active ingredient.
　ＲＳＫ１は、ＲＳＫ(リボソームＳ６キナーゼ)ファミリーメンバーであり、増殖因子制御型セリンスレオニンキナーゼである。ＲＳＫ１は***促進因子活性化キナーゼ（ＭＡＰＫ）カスケード活性化に関与し、細胞増殖や分化を刺激する。また、ＭＡＰＫカスケードによって活性化されたＲＳＫ１は、Ｓ６キナーゼを活性化することが知られており、その結果、ｍＴＯＲ経路が活性化され、細胞増殖や生存を促進する。なお、このｍＴＯＲ経路の阻害剤はすでに癌の治療においても臨床応用されている。 RSK1 is a member of the RSK (ribosomal S6 kinase) family and is a growth factor-controlled serine threonine kinase. RSK1 is involved in mitogen-activated kinase (MAPK) cascade activation and stimulates cell proliferation and differentiation. In addition, RSK1 activated by the MAPK cascade is known to activate S6 kinase, and as a result, the mTOR pathway is activated to promote cell proliferation and survival. Inhibitors of this mTOR pathway have already been clinically applied in the treatment of cancer.
　医薬組成物は、ＡＰＥＸ１、ＲＰＡ１及びＰＯＬＤ４からなる群から選択される少なくとも１種の遺伝子を発現している腫瘍の処置に用いられてもよい。医薬組成物が有効成分として、例えばｍｉＲ－１２９３をコードする遺伝子に由来するポリヌクレオチドを含む場合に特に好適である。 The pharmaceutical composition may be used in the treatment of tumors expressing at least one gene selected from the group consisting of APEX1, RPA1 and POLD4. It is particularly suitable when the pharmaceutical composition contains, for example, a polynucleotide derived from a gene encoding miR-1293 as an active ingredient.
　ＡＰＥＸ１、ＲＰＡ１及びＰＯＬＤ４は、いずれもＤＮＡの修復に関わる遺伝子である。癌においては遺伝子の変異、構造異常が蓄積されていることから複製ストレスがかかっていると考えられており、ＤＮＡ修復経路を阻害する治療薬としてＰＡＲＰ阻害剤が臨床応用されている。 APEX1, RPA1 and POLD4 are all genes involved in DNA repair. It is considered that replication stress is applied to cancer due to the accumulation of gene mutations and structural abnormalities, and PARP inhibitors are clinically applied as therapeutic agents that inhibit the DNA repair pathway.
　さらに「がんとの共存」を目指す場合も医薬組成物の適応対象となりうる。すなわち、がんにおけるＢＲＤ４遺伝子の発現をはじめ、ＲＳＫ１、ＡＰＥＸ１、ＲＰＡ１、ＰＯＬＤ４等の遺伝子の発現を抑制することにより、がんの悪性化を防ぎ、天寿を全うすることを目指す場合にも医薬組成物は適していると考えられる。 Furthermore, when aiming for "coexistence with cancer", it can be an indication target for pharmaceutical compositions. That is, the pharmaceutical composition is also used when aiming to prevent malignant transformation of cancer and complete natural life by suppressing the expression of genes such as RSK1, APEX1, RPA1, and POLD4, as well as the expression of BRD4 gene in cancer. Things are considered suitable.
　なお、上記の遺伝子発現の定量は、常法に従って行うことが可能である。例えば、ＢＲＤ４遺伝子、ＲＳＫ１遺伝子、ＡＰＥＸ１遺伝子、ＲＰＡ１遺伝子、ＰＯＬＤ４遺伝子等の発現の定量方法としては、ノーザンブロッティング法、ウェスタンブロティング法、ｉｎ　ｓｉｔｕハイブリダイゼーション法（ＩＳＨ法）、定量的ＲＴ－ＰＣＲ法（リアルタイムＰＣＲ法を含む）、ＲＮａｓｅプロテクションアッセイ、ｉｎ　ｖｉｔｒｏ転写法、発現アレイ解析法、免疫組織化学染色法等、又はこれらの方法の変法が挙げられる。 The above gene expression can be quantified according to a conventional method. For example, as a method for quantifying the expression of BRD4 gene, RSK1 gene, APEX1 gene, RPA1 gene, POLD4 gene and the like, Northern blotting method, Western blotting method, in situ hybridization method (ISH method), quantitative RT-PCR method and the like. Examples thereof include (including real-time PCR method), RNase protection assay, in situ transcription method, expression array analysis method, immunohistochemical staining method, and modifications of these methods.
　処置の対象となる腫瘍におけるＢＲＤ４、ＲＳＫ１、ＡＰＥＸ１、ＲＰＡ１、ＰＯＬＤ４等の遺伝子の発現は、過剰発現であってもよい。所定の遺伝子の過剰発現の有無は、以下のようにして特定することができる。それぞれの遺伝子の正常な発現量を基にして適切なカットオフ値を包括的に又は腫瘍個別的に設定する。上述した遺伝子発現の定量法を、処置対象の腫瘍に対して適用して求められる当該腫瘍における所定の遺伝子の発現定量値に対して、前記カットオフ値を当て嵌めることで、所定の遺伝子の過剰発現の有無を特定することができる。このような所定の遺伝子の過剰発現が認められるがんを、医薬組成物の適切な適用対象として特定することができる。 The expression of genes such as BRD4, RSK1, APEX1, RPA1, and POLD4 in the tumor to be treated may be overexpressed. The presence or absence of overexpression of a predetermined gene can be specified as follows. Appropriate cutoff values are set comprehensively or individually for each tumor based on the normal expression level of each gene. By applying the above-mentioned gene expression quantification method to a tumor to be treated and applying the cutoff value to the expression quantification value of a predetermined gene in the tumor, an excess of the predetermined gene is applied. The presence or absence of expression can be specified. Cancers in which such overexpression of a predetermined gene is observed can be specified as an appropriate application target of the pharmaceutical composition.
　医薬組成物は、ＢＲＣＡ１／２遺伝子に病的変異を有する腫瘍の処置に用いられてもよい。医薬組成物が有効成分として、例えばｍｉＲ－１２９３をコードする遺伝子に由来するポリヌクレオチドを含む場合に特に好適である。 The pharmaceutical composition may be used in the treatment of tumors having a pathogenic mutation in the BRCA1 / 2 gene. It is particularly suitable when the pharmaceutical composition contains, for example, a polynucleotide derived from a gene encoding miR-1293 as an active ingredient.
　ＢＲＣＡ１／２遺伝子は、ＤＮＡ損傷時の相同組換え修復において重要な役割を果たす遺伝子であり、ＢＲＣＡ１とＢＲＣＡ２が知られている。ＢＲＣＡ１／２遺伝子の病的変異としては、フレームシフト変異、ナンセンス変異、メチル化等を挙げることができる。また、処置の対象となる腫瘍は、いわゆるＢＲＣＡｎｅｓｓであってもよい。ＢＲＣＡｎｅｓｓとは、ＢＲＣＡ１／２遺伝子自体には病的変異が認められなくても、他の因子の変調によって相同組換え機能不全に陥り、ＢＲＣＡ１／２遺伝子の病的変異と同様にＤＮＡ損傷修復機構の異常が惹起されている状態をいう。ＢＲＣＡ１／２遺伝子の病的変異が陽性の腫瘍にはＰＡＰＲ（ポリ（ＡＤＰリボース）ポリメラーゼ）阻害剤が効果を有することが知られている。したがって、ｍｉＲ－１２９３をコードする遺伝子に由来するポリヌクレオチドを含む医薬組成物は、ＰＡＰＲ阻害剤に抵抗性になった腫瘍に対しても効果が期待される。 The BRCA1 / 2 gene is a gene that plays an important role in homologous recombination repair at the time of DNA damage, and BRCA1 and BRCA2 are known. Examples of pathogenic mutations in the BRCA1 / 2 gene include frameshift mutations, nonsense mutations, and methylation. Further, the tumor to be treated may be a so-called BRCAness. BRCAness is a DNA damage repair mechanism similar to the pathological mutation of the BRCA1 / 2 gene, which causes homologous recombination dysfunction due to modulation of other factors even if no pathological mutation is found in the BRCA1 / 2 gene itself. The state in which the abnormality of is caused. PAPR (poly (ADP ribose) polymerase) inhibitors are known to be effective against tumors positive for pathological mutations in the BRCA1 / 2 gene. Therefore, a pharmaceutical composition containing a polynucleotide derived from a gene encoding miR-1293 is also expected to be effective against tumors that have become resistant to PAPR inhibitors.
　医薬組成物の処置の対象は、腫瘍に限られず、上述した遺伝子的特徴を示す疾患であってよい。遺伝子的特徴に基づいた処置対象となる疾患としては、例えば、ＢＲＤ４遺伝子に関連する疾患として、急性骨髄性白血病（acute myelogenous leukemia：ＡＭＬ）、混合系統白血病（Mixed Lineage Leukemia：ＭＬＬ）、バーキットリンパ腫（Burkitt lymphoma）、小児期に最も一般的な脳腫瘍である髄芽腫（medulloblastoma）、ＭＹＣＮ増幅神経芽細胞腫（MYCN-amplified neuroblastoma）、去勢抵抗性前立腺がん（Castration Resistant Prostate Cancer：ＣＲＰＣ）、ユーイング肉腫（Ewing's sarcoma）、結腸直腸がん（colorectal cancer）、リンパ腫（lymphoma）、白血病（Leukemia）、多発性骨髄腫（multiple myeloma）、固形腫瘍（solid tumor）（例えば、膵臓及び前立腺のがん）、乳がん（breast cancer）、非小細胞肺がん（non-small cell lung carcinoma：ＮＳＣＬＣ）、骨髄異形成症候群（myelodysplastic syndromes：ＭＤＳ）、骨髄増殖性新生物（Myeloproliferative neoplasms）、分類できない葉状腫瘍（Phyllodes tumor）、びまん性大細胞型Ｂ細胞リンパ腫（diffuse large B-cell lymphoma：ＤＬＢＣＬ）、トリプルネガティブ乳がん（triple negative breast cancer：ＴＮＢＣ）、膵管腺がん（pancreatic ductal adenocarcinoma）、多形性神経膠芽腫（glioblastoma multiforme）、２型糖尿病(Type 2 diabetes mellitus)、虚血性心疾患（Ischemic heart diseases）（例えば、Int. J. Mol. Sci. 2016, 17,_1849；Cold Spring Harb Perspect Med. 2017; 7:a026674 参照）等が挙げられる。 The target of treatment of the pharmaceutical composition is not limited to the tumor, but may be a disease exhibiting the above-mentioned genetic characteristics. Diseases to be treated based on genetic characteristics include, for example, acute myelogenous leukemia (AML), mixed lineage leukemia (MLL), and barkit lymphoma as diseases related to the BRD4 gene. (Burkitt lymphoma), the most common brain tumors in childhood, medulloblastoma, MYCN-amplified neuroblastoma, castration-resistant prostate cancer (CRPC), Ewing's sarcoma, colorectal cancer, lymphoma, leukemia, multiple myeloma, solid tumor (eg, cancer of the pancreas and prostate) ), Breast cancer, non-small cell lung carcinoma (NSCLC), myelodysplastic syndromes (MDS), myeloproliferative neoplasms, foliar tumors that cannot be classified (Phyllodes) tumor), diffuse large B-cell lymphoma (DLBCL), triple negative breast cancer (TNBC), pancreatic ductal adenocarcinoma, polymorphic glioma Cancer (glioblastoma multiforme), type 2 diabetes (Type 2 diabetes mellitus), ischemic heart diseases (for example, Int. J. Mol. Sci. 2016, 17, _1849; Cold Spring Harb Perspect Med. 2017; 7: a026674) and so on.
腫瘍の処置方法
　腫瘍の処置方法は、有効量の前記医薬組成物を、対象に投与することを含む腫瘍を処置する方法である。処置の対象となる腫瘍は既述の通りであり、例えば、ＢＲＤ４、ＲＳＫ１、ＡＰＥＸ１、ＲＰＡ１及びＰＯＬＤ４からなる群から選択される少なくとも１種の遺伝子を発現している腫瘍である。また、ＢＲＣＡ１／２遺伝子に病的変異を有する、又はＢＲＣＡｎｅｓｓ状態である腫瘍であってもよい。なお、医薬組成物の詳細及び投与方法は既述の通りである。処置の対象は、例えば、哺乳動物であり、哺乳動物はヒトを含む。また、処置の対象は、非ヒト動物であってもよく、ヒトであってもよい。 Tumor Treatment Method A tumor treatment method is a method of treating a tumor, which comprises administering an effective amount of the pharmaceutical composition to a subject. The tumor to be treated is as described above, and is, for example, a tumor expressing at least one gene selected from the group consisting of BRD4, RSK1, APEX1, RPA1 and POLD4. Further, the tumor may have a pathological mutation in the BRCA1 / 2 gene or is in a BRCAness state. The details of the pharmaceutical composition and the administration method are as described above. The subject of treatment is, for example, a mammal, which includes a human. In addition, the target of treatment may be a non-human animal or a human.
　本発明は、別の態様として、腫瘍の処置に用いられる医薬組成物の製造における前記ポリヌクレオチドの使用、腫瘍の処置における前記ポリヌクレオチドの使用、腫瘍の処置に使用される前記ポリヌクレオチドをも包含する。 The present invention also includes, as another aspect, the use of the polynucleotide in the production of a pharmaceutical composition used in the treatment of a tumor, the use of the polynucleotide in the treatment of a tumor, and the polynucleotide used in the treatment of a tumor. To do.
　以下、本発明を実施例により具体的に説明するが、本発明はこれらの実施例に限定されるものではない。 Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited to these Examples.
１．材料と方法
（１）マイクロＲＮＡ及びｓｉＲＮＡ
　ｈｓａ－ｍｉＲ－１２９３（成熟ｍｉＲＮＡ）をガイド鎖（配列番号１）とする２本鎖マイクロＲＮＡの合成品を、アンビオン社（Ａｍｂｉｏｎ、Ｉｎｃ．：米国）より入手した（商品名：ＭＣ１３６９８）。当該マイクロＲＮＡは、下記の天然型と同一の塩基配列の２本鎖ＲＮＡであり、修飾ヌクレオチドを含んでいる。以下、本実施例では、この２本鎖ＲＮＡを、特に断らない限り「ｍｉＲ－１２９３」と記載する。 1. 1. Materials and methods (1) MicroRNA and siRNA
A synthetic product of double-stranded microRNA using hsa-miR-1293 (mature miRNA) as a guide strand (SEQ ID NO: 1) was obtained from Ambion (Ambion, Inc .: USA) (trade name: MC13698). The microRNA is a double-stranded RNA having the same base sequence as the following natural type, and contains a modified nucleotide. Hereinafter, in this example, this double-stranded RNA will be referred to as "miR-1293" unless otherwise specified.
Figure JPOXMLDOC01-appb-T000014
Figure JPOXMLDOC01-appb-T000014
　ｈｓａ－ｍｉＲ－８７６－３ｐ（成熟ｍｉＲＮＡ）をガイド鎖（配列番号２）とする２本鎖マイクロＲＮＡの合成品を、アンビオン社より入手した（商品名：ＭＣ１２８８６）。当該マイクロＲＮＡは、下記の天然型と同一の塩基配列の２本鎖ＲＮＡであり、修飾ヌクレオチドを含んでいる。以下、本実施例では、この２本鎖ＲＮＡを、特に断らない限り「ｍｉＲ－８７６－３ｐ」と記載する。 A synthetic product of double-stranded microRNA using hsa-miR-876-3p (mature miRNA) as a guide strand (SEQ ID NO: 2) was obtained from Ambion (trade name: MC12886). The microRNA is a double-stranded RNA having the same base sequence as the following natural type, and contains a modified nucleotide. Hereinafter, in this example, this double-stranded RNA will be referred to as "miR-876-3p" unless otherwise specified.
Figure JPOXMLDOC01-appb-T000015
Figure JPOXMLDOC01-appb-T000015
　ｈｓａ－ｍｉＲ－４４３８（成熟ｍｉＲＮＡ）をガイド鎖（配列番号３）とする２本鎖マイクロＲＮＡの合成品を、アンビオン社より入手した（商品名：ＭＣ２２６３４）。当該マイクロＲＮＡは、下記の天然型と同一の塩基配列の２本鎖ＲＮＡであり、修飾ヌクレオチドを含んでいる。以下、本実施例では、この２本鎖ＲＮＡを、特に断らない限り「ｍｉＲ－４４３８」と記載する。 A synthetic product of double-stranded microRNA using hsa-miR-4438 (mature miRNA) as a guide strand (SEQ ID NO: 3) was obtained from Ambion (trade name: MC22634). The microRNA is a double-stranded RNA having the same base sequence as the following natural type, and contains a modified nucleotide. Hereinafter, in this example, this double-stranded RNA will be referred to as "miR-4438" unless otherwise specified.
Figure JPOXMLDOC01-appb-T000016
Figure JPOXMLDOC01-appb-T000016
　ｈｓａ－ｍｉＲ－６７５１－５ｐ（成熟ｍｉＲＮＡ）をガイド鎖（配列番号４）とする２本鎖マイクロＲＮＡの合成品を、アンビオン社より入手した（商品名：ＭＣ２７１９４）。当該マイクロＲＮＡは、下記の天然型と同一の塩基配列の２本鎖ＲＮＡであり、修飾ヌクレオチドを含んでいる。以下、本実施例では、この２本鎖ＲＮＡを、特に断らない限り「ｍｉＲ－６７５１」と記載する。 A synthetic product of double-stranded microRNA using hsa-miR-6751-5p (mature miRNA) as a guide strand (SEQ ID NO: 4) was obtained from Ambion (trade name: MC27194). The microRNA is a double-stranded RNA having the same base sequence as the following natural type, and contains a modified nucleotide. Hereinafter, in this example, this double-stranded RNA will be referred to as "miR-6751" unless otherwise specified.
Figure JPOXMLDOC01-appb-T000017
Figure JPOXMLDOC01-appb-T000017
　ｈｓａ－ｍｉＲ－６３４（成熟ｍｉＲＮＡ）をガイド鎖（配列番号５）とする２本鎖マイクロＲＮＡの合成品を、アンビオン社より入手した（商品名：ＭＣ１１５３８）。当該マイクロＲＮＡは、下記の天然型と同一の塩基配列の２本鎖ＲＮＡであり、修飾ヌクレオチドを含んでいる。以下、本実施例では、この２本鎖ＲＮＡを、特に断らない限り「ｍｉＲ－６３４」と記載する。 A synthetic product of double-stranded microRNA using hsa-miR-634 (mature miRNA) as a guide strand (SEQ ID NO: 5) was obtained from Ambion (trade name: MC11538). The microRNA is a double-stranded RNA having the same base sequence as the following natural type, and contains a modified nucleotide. Hereinafter, in this example, this double-stranded RNA will be referred to as "miR-634" unless otherwise specified.
Figure JPOXMLDOC01-appb-T000018
Figure JPOXMLDOC01-appb-T000018
　ｈｓａ－ｍｉＲ－９２ａ－２（成熟ｍｉＲＮＡ）をガイド鎖（配列番号６）とする２本鎖マイクロＲＮＡの合成品を、アンビオン社より入手した（商品名：ＭＣ１２５２４）。当該マイクロＲＮＡは、下記の天然型と同一の塩基配列の２本鎖ＲＮＡであり、修飾ヌクレオチドを含んでいる。以下、本実施例では、この２本鎖ＲＮＡを、特に断らない限り「ｍｉＲ－９２ａ－２」と記載する。 A synthetic product of double-stranded microRNA using hsa-miR-92a-2 (mature miRNA) as a guide strand (SEQ ID NO: 6) was obtained from Ambion (trade name: MC12524). The microRNA is a double-stranded RNA having the same base sequence as the following natural type, and contains a modified nucleotide. Hereinafter, in this example, this double-stranded RNA will be referred to as "miR-92a-2" unless otherwise specified.
Figure JPOXMLDOC01-appb-T000019
Figure JPOXMLDOC01-appb-T000019
　対照のマイクロＲＮＡには、コントロールｍｉＲＮＡ(ネガティブコントロール＃１；ｍｉＲ－ＮＣ)（アンビオン社）を用いた。 Control miRNA (negative control # 1; miR-NC) (Ambion) was used as the control microRNA.
　ＲＳＫ１（ｓｉＧＥＮＯＭＥ　ＳＭＡＲＴｐｏｏｌ：Ｍ－００３０２５－０４）、ＡＰＥＸ１（ｓｉＧＥＮＯＭＥ　ＳＭＡＲＴｐｏｏｌ：Ｍ－０１０２３７－０１）、ＢＲＤ４（ｓｉＧＥＮＯＭＥ　ＳＭＡＲＴｐｏｏｌ：Ｍ－００４９３７－０２－０００５）、ＲＰＡ１（ｓｉＧＥＮＯＭＥ　ＳＭＡＲＴｐｏｏｌ：Ｍ－０１５７４９－０１）、ＰＯＬＤ４（ｓｉＧＥＮＯＭＥ　ＳＭＡＲＴｐｏｏｌ：Ｍ－０１４０１３－０１）に対するｓｉＲＮＡは、各々ダーマコン社から入手した。 RSK1 (siGENOME SMARTpool: M-003025-04), APEX1 (siGENOME SMARTpool: M-01237-01), BRD4 (siGENOME SMARTpool: M-004937-02-0005), RPA1 (siGENOME49ol) The siRNAs for POLD4 (siGENOME SMARTpool: M-014013-1) were obtained from Dermacon.
（２）がん細胞株
　膵がん細胞株（ＭＩＡＰａＣａ２細胞）、口腔がん細胞株（ＨＯＣ３１３細胞、ＨＳＣ２細胞）、肺がん細胞株（Ａ５４９細胞）、大腸がん細胞株（ＨＣＴ１１６＋／＋細胞、ＳＷ４８細胞、ＨＴ２９細胞）、胃がん細胞株（ＡＧＳ細胞）はアメリカンタイプカルチャーコレクション（米国）から購入した。大腸がん細胞株ＨＣＴ１１６＋／＋細胞及びＨＣＴ１１６－／－細胞はＶｏｇｅｌｓｔｅｉｎ研究室より供与を受けた。食道がん細胞株（ＫＹＳＥ１５０細胞）は嶋田裕博士（富山大学）より供与を受けた。 (2) Cancer cell line Pancreatic cancer cell line (MIAPaCa2 cell), oral cancer cell line (HOC313 cell, HSC2 cell), lung cancer cell line (A549 cell), colon cancer cell line (HCT116 + // cell, SW48) Cells, HT29 cells), gastric cancer cell line (AGS cells) were purchased from the American Type Culture Collection (USA). Colorectal cancer cell lines HCT116 + / + cells and HCT116 − / − cells were donated by Vogelstein laboratory. The esophageal cancer cell line (KYSE150 cells) was donated by Dr. Hiroshi Shimada (University of Toyama).
　ＨＣＴ１１６＋／＋、ＨＣＴ１１６－／－、ＭＩＡＰａＣａ２、ＫＹＳＥ１５０、ＨＴ２９、ＳＷ４８、ＨＯＣ３１３、ＨＳＣ２細胞はダルベッコ改変イーグル培地に、Ａ５４９、ＡＧＳ細胞はＲＰＭＩ１６４０培地に、それぞれ１０％ウシ胎児血清を添加して、５％ＣＯ２・３７℃で培養した。 HCT116 + / +, HCT116 − / −, MIAPaCa2, KYSE150, HT29, SW48, HOC313, HSC2 cells in Dulbecco's modified Eagle's medium, A549, AGS cells in RPMI1640 medium, 5% each CO were cultured in 2 · 37 ° C..
（３）抗体
　ウエスタンブロット用の抗体として、抗ＢＲＤ４抗体（ｃｅｌｌ　ｓｉｇｎａｌｉｎｇ社）、抗ｃｌｅａｖｅｄ　ＰＡＲＰ抗体（ｃｅｌｌ　ｓｉｇｎａｌｉｎｇ社）、抗ＲＳＫ１抗体（Ｒ＆Ｄ　Ｓｙｓｔｅｍｓ社）、抗ＡＰＥＸ１抗体（ｐｒｏｔｅｉｎｔｅｃｈ社）、抗リン酸化ＲＳＫ１抗体（Ｒ＆Ｄ Ｓｙｓｔｅｍｓ社）、抗リン酸化Ｐ７０Ｓ６Ｋ抗体（ｃｅｌｌ　ｓｉｇｎａｌｉｎｇ社）、抗リン酸化Ｓ６抗体（ｃｅｌｌ　ｓｉｇｎａｌｉｎｇ社）、抗ＭＹＣ抗体（ｃｅｌｌ　ｓｉｇｎａｌｉｎｇ社）、抗ＰＯＬＤ４抗体（ｐｒｏｔｅｉｎｔｅｃｈ社）、抗γＨ２ＡＸ抗体（ｃｅｌｌ　ｓｉｇｎａｌｉｎｇ社）、抗βアクチン抗体（シグマ社）を用いた。免疫組織染色用の抗ＢＲＤ４抗体（Ａｔｌａｓ　ａｎｔｉｂｏｄｉｅｓ社）、抗ＲＳＫ1抗体（Ａｔｌａｓ　ａｎｔｉｂｏｄｉｅｓ社）、抗ＡＰＥＸ１抗体（ｐｒｏｔｅｉｎｔｅｃｈ社）、抗ＲＰＡ１抗体（ａｂｃａｍ社）、及び抗ＰＯＬＤ４抗体（ＭｙＢｉｏＳｏｕｒｃｅ社）を用いた。 (3) Antibodies As antibodies for Western blot, anti-BRD4 antibody (cell signing), anti-cleave PARP antibody (cell signing), anti-RSK1 antibody (R & D Systems), anti-APEX1 antibody (proteintech), anti-phosphorylation RSK1 antibody (R & D Systems), anti-phosphorylated P70S6K antibody (cell signing), anti-phosphorylated S6 antibody (cell signing), anti-MYC antibody (cell signing), anti-POLD4 antibody (proteintech), anti-γH2AX antibody (Cell signing) and anti-β-actin antibody (Sigma) were used. Anti-BRD4 antibody (Atlas antibodies), anti-RSK1 antibody (Atlas antibodies), anti-APEX1 antibody (proteintech), anti-RPA1 antibody (abcam), and anti-POLD4 antibody (MyBioSour) for immunohistochemical staining. ..
（４）統計解析
　サブグループ間の差はＡＮＯＶＡ、Ｓｔｕｄｅｎｔ　ｔ－ｔｅｓｔ及びＭａｎｎ－Ｗｈｉｔｎｅｙ’ｓ　Ｕ　ｔｅｓｔ（下記のｉｎ　ｖｉｖｏ試験におけるマウスの腫瘍の重量の解析において用いた）で解析した。計算されたＰ値が０．０５未満の場合、統計学的な有意差ありと判断した。 (4) Statistical analysis Differences between subgroups were analyzed by ANOVA, Student's t-test and Mann-Whitney's U test (used in the analysis of mouse tumor weight in the in vivo test below). When the calculated P value was less than 0.05, it was judged that there was a statistically significant difference.
＜実施例Ａ１＞　細胞増殖を抑制するｍｉＲＮＡの１次スクリーニング
　ＨＣＴ１１６＋／＋細胞及びＨＣＴ１１６－／－細胞（６０００個／ｗｅｌｌ）に、ｍｉＲＮＡライブラリー［ｍｉＲＶａｎａ　ｍｉＲＮＡ　ｍｉｍｉｃ　Ｌｉｂｒａｒｙ　Ｖ２１（アンビオン社）］に含まれる２５６５種類のｍｉＲＮＡ又はコントロールｍｉＲＮＡ（ｍｉＲ－ＮＣ）の１０ｎｍｏｌ／Ｌを、Ｌｉｐｏｆｅｃｔａｍｉｎｅ　ＲＮＡｉＭＡＸ（インビトロゲン社）を用い、添付の手順書に従って導入した。導入から３日後に細胞生存数をクリスタルバイオレット（ＣＶ）染色アッセイで評価した。なお、ＣＶ染色アッセイは以下のように実施した。細胞をＰＢＳで洗浄し、０．２％ＣＶ含有１０％ホルムアルデヒドＰＢＳで５分間固定した。過剰なＣＶ溶液は除去し、完全に空気乾燥した後、染色細胞に２％ＳＤＳ溶液を加えて、プレートを１時間振盪することで溶解した。吸光度をマイクロプレートリーダー（ＡＲＶＯｍｘ；パーキンエルマー社）を用いてウェル毎に５６０ｎｍで計測した。細胞にコントロールｍｉＲＮＡを導入したコントロールウェルにおける吸光度を１００％に設定した場合の各ウェルにおける吸光度から、生存細胞数の比を決定した。 <Example A1> Primary screening of miRNA that suppresses cell proliferation HCT116 +/ + cells and HCT116 − / − cells (6000 cells / well) are included in the miRNA library [miRVana miRNA mimic Library V21 (Ambion)]. 10 nmol / L of 2565 types of miRNA or control miRNA (miR-NC) was introduced using Lipofectamine RNAiMAX (Invitrogen) according to the attached procedure manual. Cell viability was evaluated by crystal violet (CV) staining assay 3 days after introduction. The CV staining assay was performed as follows. Cells were washed with PBS and fixed with 10% formaldehyde PBS containing 0.2% CV for 5 minutes. Excess CV solution was removed and completely air dried, then 2% SDS solution was added to the stained cells and the plate was lysed by shaking for 1 hour. Absorbance was measured at 560 nm per well using a microplate reader (ARVOmx; PerkinElmer). The ratio of the number of surviving cells was determined from the absorbance in each well when the absorbance in the control well into which the control miRNA was introduced into the cells was set to 100%.
　図１に、上記のＨＣＴ１１６＋／＋、ＨＣＴ１１６－／－細胞における１次スクリーニングの結果を示す。２５６５種のｍｉＲＮＡをそれぞれ導入したときの、コントロールｍｉＲＮＡと比べた時の細胞生存数の比を示している。横軸がＨＣＴ１１６＋／＋細胞の結果、縦軸がＨＣＴ１１６－／－細胞の結果を示す。コントロールｍｉＲＮＡをトランスフェクションした場合の生存細胞数を１としたときの相対的な生存細胞数を示す。なお、細胞増殖抑制効果の基準としてコントロールとの比を０．３未満とした。 FIG. 1 shows the results of the primary screening in the above HCT116 +/+ and HCT116 − / − cells. The ratio of the number of surviving cells when compared with the control miRNA when 2565 kinds of miRNAs are introduced is shown. The horizontal axis shows the result of HCT116 +/ + cells, and the vertical axis shows the result of HCT116 − / − cells. The relative number of viable cells when the number of viable cells when transfected with the control miRNA is 1. The ratio to the control was set to less than 0.3 as a standard for the cell proliferation inhibitory effect.
　１次スクリーニングにおいてＨＣＴ１１６＋／＋、ＨＣＴ１１６－／－細胞とも増殖抑制効果を認めたのは１３８種類のｍｉＲＮＡであった。なお、ＨＣＴ１１６＋／＋において細胞増殖抑制効果を認めたのは６３０種類であり、ＨＣＴ１１６－／－において増殖抑制効果を認めたのは１４１種類であり、両方に共通するｍｉＲＮＡが１３８種類であった。 In the primary screening, 138 types of miRNAs showed a growth inhibitory effect on both HCT116 + / + and HCT116 − / − cells. The cell proliferation inhibitory effect was observed in 630 types in HCT116 + / +, 141 types were observed in HCT116 − / −, and 138 types of miRNAs were common to both.
　２次スクリーニングとして、１次スクリーニングにおいてＨＣＴ１１６＋／＋、ＨＣＴ１１６－／－細胞とも増殖抑制をみとめた１３８種類のｍｉＲＮＡ又はコントロールｍｉＲＮＡの１０ｎｍｏｌ／Ｌを、ＨＣＴ１１６＋／＋、ＨＣＴ１１６－／－、ＭＩＡＰａＣａ２、ＨＯＣ３１３、ＨＳＣ２、ＳＷ４８、ＨＴ２９、ＡＧＳ、Ａ５４９、及びＫＹＳＥ１５０のそれぞれの細胞にＬｉｐｏｆｅｃｔａｍｉｎｅ　ＲＮＡｉＭＡＸを用いて、添付の手順書に従って導入した。導入から３日後に細胞生存数をクリスタルバイオレット（ＣＶ）染色アッセイで上記と同様にして評価した。 As the secondary screening, 10 nmol / L of 138 types of miRNAs or control miRNAs, which were found to suppress the growth of both HCT116 + / + and HCT116 − / − cells in the primary screen, were used in HCT116 +/+, HCT116 − / −, MIAPaCa2, HOC313, etc. Each cell of HSC2, SW48, HT29, AGS, A549, and KYSE150 was introduced using Lipofectamine RNAiMAX according to the attached procedure manual. Three days after introduction, cell viability was evaluated by crystal violet (CV) staining assay in the same manner as above.
　表１４は、２次スクリーニングの結果を表している。１次スクリーニングで抽出した１３８種のｍｉＲＮＡをそれぞれ導入したときの、コントロールｍｉＲＮＡと比べた時の細胞生存数の比を示している。コントロールｍｉＲＮＡをトランスフェクションした場合の生存細胞数を１としたときの相対的な生存細胞数を示す。なお、増殖抑制効果の基準としてコントロールとの比で０．６未満とした。ここでは１０種類の癌細胞すべてにおいて増殖抑制効果を認めた７種類のｍｉＲＮＡを示している。 Table 14 shows the results of the secondary screening. The ratio of the number of surviving cells when compared with the control miRNA when each of the 138 kinds of miRNAs extracted in the primary screening is introduced is shown. The relative number of viable cells when the number of viable cells when transfected with the control miRNA is 1. As a standard for the growth inhibitory effect, the ratio with the control was set to less than 0.6. Here, 7 types of miRNAs showing a growth inhibitory effect in all 10 types of cancer cells are shown.
Figure JPOXMLDOC01-appb-T000020
Figure JPOXMLDOC01-appb-T000020
＜実施例Ａ２＞
（ウエスタンブロット）
　ｍｉＲ－ＮＣ、ｍｉＲ－８７６－３ｐ、ｍｉＲ－１２９３、ｍｉＲ－４４３８、ｍｉＲ－６７５１－５ｐ、ｍｉＲ－９２ａ－５ｐ、ｍｉＲ－６３４、又はｍｉＲ－３１４０－３ｐを導入した、ＨＣＴ１１６＋／＋細胞、ＨＣＴ１１６－／－細胞、及びＨＯＣ３１３細胞から得られた試料に対して、ウエスタンブロットを行い、ＢＲＤ４の発現量を調べた。ウエスタンブロットは以下のようにして行った。全細胞の溶解物に対してＳＤＳ－ＰＡＧＥを行い、タンパクをＰＶＤＦ膜（ＧＥヘルスケア社）に転写した。０．０５％Ｔｗｅｅｎ２０と５％スキムミルクを含有するＴＢＳを用いて室温で１時間ブロッキングを行った後、当該膜を抗体と共に４℃で一晩反応させた。１次抗体の希釈倍率は、抗ＢＲＤ４抗体（１／１０００）、抗β－アクチン抗体（１／５０００）であった。当該膜を洗浄後、ＨＲＰ結合抗マウス又は抗ウサギＩｇＧ抗体（ともに１／５０００）に室温１時間暴露した。結合した抗体を、ＳｕｐｅｒＳｉｇｎａｌ　Ｗｅｓｔ　Ｆｅｍｔｏ　Ｓｕｂｓｔｒａｔｅ（サーモフィッシャーサイエンティフィック社）を用いて視覚化した。結果を図２－１に示す。 <Example A2>
(Western blot)
HCT116 +/ + cells, HCT116 into which miR-NC, miR-876-3p, miR-1293, miR-4438, miR-6751-5p, miR-92a-5p, miR-634, or miR-3140-3p were introduced. Western blots were performed on the samples obtained from − / − cells and HOC313 cells to examine the expression level of BRD4. Western blotting was performed as follows. SDS-PAGE was performed on the lysates of all cells, and the protein was transferred to a PVDF membrane (GE Healthcare). After blocking for 1 hour at room temperature with TBS containing 0.05 % Tween 20 and 5% skim milk, the membrane was reacted with the antibody overnight at 4 ° C. The dilution ratio of the primary antibody was anti-BRD4 antibody (1/1000) and anti-β-actin antibody (1/5000). After washing the membrane, it was exposed to HRP-binding anti-mouse or anti-rabbit IgG antibody (both 1/5000) at room temperature for 1 hour. The bound antibody was visualized using the SuperSignal West Femto Substrate (Thermo Fisher Scientific). The results are shown in Fig. 2-1.
　図２－１に示すように、これら３種類のがん細胞株において、ｍｉＲ－８７６－３ｐ、ｍｉＲ－１２９３、ｍｉＲ－４４３８、ｍｉＲ－６７５１－５ｐ、ｍｉＲ－９２ａ－５ｐ、ｍｉＲ－６３４、又はｍｉＲ－３１４０－３ｐを導入することによるＢＲＤ４遺伝子の発現の抑制が認められた。 As shown in FIG. 2-1 in these three types of cancer cell lines, miR-876-3p, miR-1293, miR-4438, miR-6751-5p, miR-92a-5p, miR-634, or Suppression of BRD4 gene expression was observed by introducing miR-3140-3p.
＜実施例Ａ３＞
（ルシフェラーゼレポーターアッセイ）
　ＢＲＤ４遺伝子の３’ＵＴＲ領域をルシフェラーゼベクターに組み込んだコンストラクトを用いるルシフェラーゼレポーターアッセイによって、ｍｉＲＮＡとＢＲＤ４遺伝子の相互作用を評価した。 <Example A3>
(Luciferase Reporter Assay)
The interaction between miRNA and the BRD4 gene was evaluated by a luciferase reporter assay using a construct in which the 3'UTR region of the BRD4 gene was incorporated into a luciferase vector.
　図２－２（Ａ）は、ｍｉＲ－１２９３のルシフェラーゼレポーターアッセイによる検証の結果を示している。ｍｉＲ－１２９３のガイド鎖の塩基配列と相同する領域は、ＢＲＤ４遺伝子に５つ存在し（Ｒ１からＲ５）、当該アッセイに用いるルシフェラーゼレポータープラスミドは、ｐｍｉＲＧｌｏ　Ｄｕａｌ－Ｌｕｃｉｆｅｒａｓｅ　ｍｉＲＮＡ　Ｔａｒｇｅｔ　Ｅｘｐｒｅｓｓｉｏｎ　Ｖｅｃｔｏｒ（プロメガ社）のルシフェラーゼ遺伝子の下流に、当該相同領域を含むＢＲＤ４遺伝子の３’ＵＴＲ領域に対応するオリゴヌクレオチド（Ｗｔ）、あるいは、この領域の塩基配列に変異を入れたオリゴヌクレオチド（Ｍｔ）を挿入することで作製した。全ての部位特異的変異は、ＫＯＤ　ｍｕｔａｇｅｎｅｓｉｓ　ｋｉｔ（ＴＯＹＯＢＯ社）を用いて作製した。ルシフェラーゼレポータープラスミドを、ＨＯＣ３１３細胞に、Ｌｉｐｏｆｅｃｔａｍｉｎｅ２０００（インビトロゲン社）を用いて添付の手順文書に沿って導入し、次の日にｍｉＲ－１２９３又はコントロールｍｉＲＮＡを導入した。２日後に、ホタルルシフェラーゼ活性とウミシイタケルシフェラーゼ活性を、Ｄｕａｌ－Ｌｕｃｉｆｅｒａｓｅ　Ｒｅｐｏｒｔｅｒ　Ａｓｓａｙ　Ｓｙｓｔｅｍ（プロメガ社）を用いて測定した。相対的ルシフェラーゼ活性は、対応する内部標準コントロールのウミシイタケルシフェラーゼ活性で補正することで、ホタルルシフェラーゼ活性を標準化して算出した。 FIG. 2-2 (A) shows the results of verification of miR-1293 by the luciferase reporter assay. There are five regions in the BRD4 gene that are homologous to the nucleotide sequence of the guide chain of miR-1293 (R1 to R5), and the luciferase reporter plasmid used for the assay is pmiRGlo Dual-Luciferase miRNA Target Expression Vector (Promega). Created by inserting an oligonucleotide (Wt) corresponding to the 3'UTR region of the BRD4 gene containing the homologous region or an oligonucleotide (Mt) having a mutation in the base sequence of this region downstream of the luciferase gene. did. All site-specific mutations were made using the KOD mutagenesis kit (TOYOBO). The luciferase reporter plasmid was introduced into HOC313 cells using Lipofectamine 2000 (In vitrogen) according to the attached procedural document, and the next day miR-1293 or control miRNA was introduced. Two days later, firefly luciferase activity and sea shiitake mushroom luciferase activity were measured using Dual-Luciferase Reporter Assay System (Promega). Relative luciferase activity was calculated by standardizing firefly luciferase activity by correcting for the corresponding internal standard control, Shiitake mushroom luciferase activity.
　ｍｉＲ－１２９３の導入により、ＢＲＤ４の３’ＵＴＲ領域のＲ１とＲ４の２か所において、野生型の３’ＵＴＲ領域に対応するルシフェラーゼ活性の低下を認め、ｍｉＲ－１２９３のガイド鎖の塩基配列とマッチする領域に変異を入れたベクターではルシフェラーゼ活性は回復した。これらの結果から、ｍｉＲ－１２９３は、ＢＲＤ４遺伝子のＲ１とＲ４の２か所の３’ＵＴＲ領域への作用により、直接ＢＲＤ４遺伝子の発現を有意に抑制していることが示された。 With the introduction of miR-1293, a decrease in luciferase activity corresponding to the wild-type 3'UTR region was observed at two locations, R1 and R4, in the 3'UTR region of BRD4, and the nucleotide sequence of the guide chain of miR-1293 was observed. Luciferase activity was restored in the vector in which the matching region was mutated. From these results, it was shown that miR-1293 significantly suppressed the expression of the BRD4 gene directly by the action of the BRD4 gene on the two 3'UTR regions of R1 and R4.
　図２－２（Ｂ）は、ｍｉＲ－８７６－３ｐのルシフェラーゼレポーターアッセイによる検証の結果を示している。ｍｉＲ－８７６－３ｐのガイド鎖の塩基配列と相同する領域は、ＢＲＤ４遺伝子に１つ存在する。その領域の配列に変異を入れたこと以外は上記と同様にしてアッセイを行った。 FIG. 2-2 (B) shows the results of verification by the luciferase reporter assay of miR-876-3p. There is one region in the BRD4 gene that is homologous to the nucleotide sequence of the guide strand of miR-876-3p. The assay was performed in the same manner as above except that the sequence of the region was mutated.
　ｍｉＲ－８７６－３ｐの導入により、野生型の３’ＵＴＲ領域に対応するルシフェラーゼ活性の低下を認め、ｍｉＲ－８７６－３ｐのガイド鎖の塩基配列とマッチする領域に変異を入れたベクターではルシフェラーゼ活性は回復した。これらの結果から、ｍｉＲ－８７６－３ｐは、ＢＲＤ４遺伝子の３’ＵＴＲ領域への作用により、直接ＢＲＤ４遺伝子の発現を有意に抑制していることが示された。 The introduction of miR-876-3p resulted in a decrease in luciferase activity corresponding to the wild-type 3'UTR region, and the vector in which a mutation was added to the region matching the base sequence of the guide chain of miR-876-3p showed luciferase activity. Has recovered. From these results, it was shown that miR-876-3p significantly suppressed the expression of the BRD4 gene directly by the action of the BRD4 gene on the 3'UTR region.
　図２－２（Ｃ）は、ｍｉＲ－６７５１－５ｐのルシフェラーゼレポーターアッセイによる検証の結果を示している。ｍｉＲ－６７５１－５ｐのガイド鎖の塩基配列と相同する領域は、ＢＲＤ４遺伝子に１つ存在する。その領域の配列に変異を入れたこと以外は上記と同様にしてアッセイを行った。 FIG. 2-2 (C) shows the results of verification by the luciferase reporter assay of miR-6751-5p. There is one region in the BRD4 gene that is homologous to the nucleotide sequence of the guide strand of miR-6751-5p. The assay was performed in the same manner as above except that the sequence of the region was mutated.
　ｍｉＲ－６７５１－５ｐの導入により、野生型の３’ＵＴＲ領域に対応するルシフェラーゼ活性の低下を認め、ｍｉＲ－６７５１－５ｐのガイド鎖の塩基配列とマッチする領域に変異を入れたベクターではルシフェラーゼ活性は回復した。これらの結果から、ｍｉＲ－６７５１－５ｐは、ＢＲＤ４遺伝子の３’ＵＴＲ領域への作用により、直接ＢＲＤ４遺伝子の発現を有意に抑制していることが示された。 The introduction of miR-6751-5p showed a decrease in luciferase activity corresponding to the wild-type 3'UTR region, and the vector in which a mutation was added to the region matching the base sequence of the guide chain of miR-6751-5p showed luciferase activity. Has recovered. From these results, it was shown that miR-6751-5p significantly suppressed the expression of the BRD4 gene directly by the action of the BRD4 gene on the 3'UTR region.
＜比較例１＞
　大腸がん細胞株（ＨＣＴ１１６－／－細胞）にＢＲＤ４遺伝子に対するｓｉＲＮＡを作用させて、ＢＲＤ４遺伝子をノックダウンしたときの細胞増殖に対する効果を図２－３（Ａ）に示す。比較としてコントロールｓｉＲＮＡ（ｓｉ－ＮＣ）を導入した場合の細胞増殖に対する効果を併せて示す。また、実施例Ａ２と同様にしてウエスタンブロットを行った結果を図２－３（Ｂ）に示す。 <Comparative example 1>
Figure 2-3 (A) shows the effect on cell proliferation when siRNA against the BRD4 gene is allowed to act on a colon cancer cell line (HCT116 − / − cells) and the BRD4 gene is knocked down. For comparison, the effect on cell proliferation when control siRNA (si-NC) is introduced is also shown. The results of Western blotting performed in the same manner as in Example A2 are shown in FIG. 2-3 (B).
　ウエスタンブロットの結果から、ｓｉ－ＢＲＤ４を導入することでＢＲＤ４遺伝子の発現が抑制されていることが示された。また、ＨＣＴ１１６－／－細胞においては、ＢＲＤ４遺伝子のノックダウンに伴い、細胞増殖が抑制されることが示された。これにより、ＢＲＤ４遺伝子の発現が実際にこれらの大腸がん細胞株の増殖に寄与していることが確認された。また、ｓｉ－ＢＲＤ４を作用させた場合の細胞増殖抑制効果は、本実施形態に係るｍｉＲＮＡを作用させた場合よりも小さいことが分かる。 From the results of Western blotting, it was shown that the expression of the BRD4 gene was suppressed by introducing si-BRD4. It was also shown that in HCT116 − / − cells, cell proliferation was suppressed with knockdown of the BRD4 gene. This confirmed that the expression of the BRD4 gene actually contributed to the growth of these colorectal cancer cell lines. Further, it can be seen that the cell proliferation inhibitory effect when si-BRD4 is allowed to act is smaller than that when miRNA according to the present embodiment is allowed to act.
＜実施例Ａ４＞
（細胞増殖抑制能の評価）
　標的がん細胞株として、大腸がん細胞株（ＨＣＴ１１６＋／＋細胞、ＨＣＴ１１６－／－細胞）、口腔がん細胞株（ＨＯＣ３１３細胞、ＨＳＣ２細胞）、肺がん細胞株（Ａ５４９細胞（非小細胞性肺がん細胞株））及び食道がん細胞株（ＫＹＳＥ１５０細胞）を用いて、ｍｉＲＮＡの導入による細胞増殖抑制効果を評価した。ｍｉＲ－８７６－３ｐ、ｍｉＲ－１２９３又は対照であるコントロールｍｉＲＮＡ(ｍｉＲ－ＮＣ)の１０ｎｍｏｌ／Ｌを、Ｌｉｐｏｆｅｃｔａｍｉｎｅ　ＲＮＡｉＭＡＸを用い、添付文書の手順書に従って、それぞれの標的がん細胞株に導入した。導入から２日後及び４日後に生存細胞数を評価した。細胞増殖曲線を図３－１に示す。また、導入７２時間後の大腸がん細胞株（ＨＣＴ１１６－／－細胞）の顕微鏡写真を図３－２に示す。 <Example A4>
(Evaluation of cell proliferation inhibitory ability)
Target cancer cell lines include colon cancer cell lines (HCT116 + / + cells, HCT116 − / − cells), oral cancer cell lines (HOC313 cells, HSC2 cells), and lung cancer cell lines (A549 cells (non-small cell lung cancer)). Cell line)) and esophageal cancer cell line (KYSE150 cells) were used to evaluate the cell growth inhibitory effect of the introduction of miRNA. 10 nmol / L of miR-876-3p, miR-1293 or control miRNA (miR-NC) as a control was introduced into each target cancer cell line using Lipofectamine RNAiMAX according to the procedure manual in the package insert. The number of viable cells was evaluated 2 days and 4 days after the introduction. The cell proliferation curve is shown in FIG. 3-1. In addition, a micrograph of a colorectal cancer cell line (HCT116 − / − cells) 72 hours after introduction is shown in FIG. 3-2.
　図３－１に示すように、いずれの細胞株においても、ｍｉＲ－８７６－３ｐ又はｍｉＲ－１２９３の導入により、有意に細胞増殖が抑制され、顕著な増殖抑制効果が認められた。また、図３－２に示すように、大腸がん細胞株において、ｍｉＲ－８７６－３ｐ又はｍｉＲ－１２９３を導入することで死細胞が多数確認され、生存細胞が対照と比べて少ない。 As shown in FIG. 3-1 the cell proliferation was significantly suppressed by the introduction of miR-876-3p or miR-1293 in any of the cell lines, and a remarkable proliferation inhibitory effect was observed. Further, as shown in FIG. 3-2, in the colorectal cancer cell line, a large number of dead cells were confirmed by introducing miR-876-3p or miR-1293, and the number of viable cells was smaller than that of the control.
＜実施例Ａ５＞
（アポトーシス評価１）
　大腸がん細胞株（ＨＣＴ１１６＋／＋、ＨＣＴ１１６－／－細胞）、口腔がん細胞株（ＨＯＣ３１３細胞）に対して、ｍｉＲ－ＮＣ、ｍｉＲ－８７６－３ｐ又はｍｉＲ－１２９３の１０ｎｍｏｌ／Ｌを、Ｌｉｐｏｆｅｃｔａｍｉｎｅ　ＲＮＡｉＭＡＸを用い、添付文書の手順書に従って、それぞれのがん細胞株に導入した。導入から７２時間後のアポトーシス細胞の割合をアネキシンＶ／ＰＩ染色で評価した。アネキシンＶ／ＰＩ陽性細胞の割合はＡｃｃｕｒｉ　Ｃ６　Ｆｌｏｗ　Ｃｙｔｏｍｅｔｅｒ（ＢＤ　Ｂｉｏｓｃｉｅｎｃｅｓ社）を用いて評価した。結果を図３－３に示す。 <Example A5>
(Apoptosis evaluation 1)
10 nmol / L of miR-NC, miR-876-3p or miR-1293 was added to the colorectal cancer cell line (HCT116 + / +, HCT116 − / − cells) and oral cancer cell line (HOC313 cells). RNAiMAX was used and introduced into each cancer cell line according to the procedure manual in the package insert. The proportion of apoptotic cells 72 hours after introduction was evaluated by Annexin V / PI staining. The proportion of annexin V / PI positive cells was evaluated using Accuri C6 Flow Cytometry (BD Biosciences). The results are shown in Fig. 3-3.
　図３－３に示すように、ｍｉＲ－８７６－３ｐ又はｍｉＲ－１２９３の導入により、それぞれの細胞において、コントロール（ｍｉＲ－ＮＣ）と比較してアポトーシス細胞の割合が増加している。 As shown in FIG. 3-3, the introduction of miR-876-3p or miR-1293 increased the proportion of apoptotic cells in each cell as compared with the control (miR-NC).
（アポトーシス評価２）
　大腸がん細胞株（ＨＣＴ１１６＋／＋、ＨＣＴ１１６－／－細胞）又は口腔がん細胞株（ＨＯＣ３１３細胞）に対して、ｍｉＲ－ＮＣ、ｍｉＲ－８７６－３ｐ又はｍｉＲ－１２９３の１０ｎｍｏｌ／Ｌを、Ｌｉｐｏｆｅｃｔａｍｉｎｅ　ＲＮＡｉＭＡＸを用い、添付文書の手順書に従って、それぞれのがん細胞株に導入した。導入から７２時間後の細胞についてウエスタンブロットにより、アポトーシスのマーカーであるｃｌｅａｖｅｄ　ＰＡＲＰの発現量を評価した。なお、ウエスタンブロットは、上記と同様の手順で実施した。結果を図３－４に示す。 (Apoptosis evaluation 2)
10 nmol / L of miR-NC, miR-876-3p or miR-1293 to colorectal cancer cell line (HCT116 +/+, HCT116 − / − cells) or oral cancer cell line (HOC313 cells), Lipofectamine RNAiMAX was used and introduced into each cancer cell line according to the procedure manual in the package insert. The expression level of cleared PARP, which is a marker of apoptosis, was evaluated by Western blotting on cells 72 hours after introduction. Western blotting was performed in the same procedure as described above. The results are shown in Figure 3-4.
　図３－４に示すように、ｍｉＲ－８７６－３ｐ又はｍｉＲ－１２９３の導入により、それぞれの細胞において、コントロール（ｍｉＲ－ＮＣ）と比較して、ｃｌｅａｖｅｄ　ＰＡＲＰの発現量が増加していることが示された。 As shown in FIG. 3-4, the introduction of miR-876-3p or miR-1293 increased the expression level of cleared PARP in each cell as compared with the control (miR-NC). Shown.
　以上の結果から、ｍｉＲ－８７６－３ｐ及びｍｉＲ－１２９３は、難治性のがんを含めて様々ながん種において、アポトーシスを誘導し、著明に増殖抑制効果を示すことが示された。 From the above results, it was shown that miR-876-3p and miR-1293 induce apoptosis in various cancer types including intractable cancers and show a remarkable growth inhibitory effect.
＜実施例Ａ５＞
　大腸がん細胞株（ＨＣＴ１１６－／－細胞）、口腔がん細胞株（ＨＳＣ２、ＨＯＣ３１３細胞）、肺がん細胞株（Ａ５４９細胞）、食道がん細胞株（ＫＹＳＥ１５０細胞）に対して、実施例Ａ１に記した要領でｍｉＲ－ＮＣ又はｍｉＲ－８７６－３ｐを導入した。導入から４８時間後に、以下のようにして遺伝子発現アレイ解析を行った。遺伝子発現アレイ解析は、Ａｇｉｌｅｎｔ　４ｘ４４Ｋ遺伝子発現アレイ（アジレントテクノロジー社）を用いて、その操作マニュアルに従って行った。遺伝子発現アレイ解析のデータはＧｅｎｅＳｐｒｉｎｇソフト（アジレントテクノロジー社）を用いて解析した。 <Example A5>
Example A1 for colon cancer cell line (HCT116 − / − cells), oral cancer cell line (HSC2, HOC313 cells), lung cancer cell line (A549 cells), esophageal cancer cell line (KYSE150 cells) MiR-NC or miR-876-3p was introduced as described. Forty-eight hours after introduction, gene expression array analysis was performed as follows. Gene expression array analysis was performed using an Agilent 4x44K gene expression array (Agilent Technologies, Inc.) according to its operating manual. The data of the gene expression array analysis was analyzed using GeneSpring software (Agilent Technologies).
　図４－１には、これらのｍｉＲ導入細胞に対して行った発現アレイの結果、遺伝子発現が１．５倍を超えて低下した遺伝子の数をベン図で示している。ｍｉＲ－ＮＣ導入群と比較して１．５倍を超えて発現が抑制されている遺伝子のうち、当該５種類のがん細胞株のうち、４種類以上で共通していた遺伝子は１２２３個であった。この共通遺伝子１２２３個について、ＤＡＶＩＤ　ｓｏｆｔｗａｒｅ（https://david.ncifcrf.gov）を用いてパスウェイ解析を行ったところ、下表に示すｍＴＯＲ経路に関わる複数の遺伝子が有意に抑制され、その結果としてｍＴＯＲ経路が抑制されていることが示された。 FIG. 4-1 shows the number of genes whose gene expression decreased by more than 1.5 times as a result of the expression array performed on these miR-introduced cells. Among the genes whose expression was suppressed by more than 1.5 times compared to the miR-NC introduction group, 1223 genes were common to 4 or more of the 5 types of cancer cell lines. there were. A pathway analysis of 1223 of these common genes using DAVID software (https://david.ncifcrf.gov) revealed that multiple genes involved in the mTOR pathway shown in the table below were significantly suppressed, resulting in significant suppression. It was shown that the mTOR pathway was suppressed.
Figure JPOXMLDOC01-appb-T000021
Figure JPOXMLDOC01-appb-T000021
＜実施例Ａ６＞
（ウエスタンブロット）
　大腸がん細胞株（ＨＣＴ１１６－／－細胞）又は口腔がん細胞株（ＨＯＣ３１３細胞、ＫＯＳＣ３細胞）に対して、実施例Ａ１に記した要領でｍｉＲ－ＮＣ又はｍｉＲ－８７６－３ｐを導入した。導入から４８時間後に、ＲＳＫ１、リン酸化ＲＳＫ１（ｐ－ＲＳＫ１）、ｍＴＯＲシグナル経路の活性化の指標であるリン酸化ｐ７０Ｓ６Ｋ（ｐ－ｐ７０Ｓ６Ｋ）及びｐＳ６（ｐ－Ｓ６）の発現量を上記と同様にしてβ－ａｃｔｉｎタンパクの発現をポジティブコントロールとしたウエスタンブロットを行うことにより評価した。結果を図４－２に示す。 <Example A6>
(Western blot)
MiR-NC or miR-876-3p was introduced into a colon cancer cell line (HCT116 − / − cells) or an oral cancer cell line (HOC313 cells, KOSC3 cells) in the manner described in Example A1. Forty-eight hours after introduction, the expression levels of RSK1, phosphorylated RSK1 (p-RSK1), phosphorylated p70S6K (p-p70S6K) and pS6 (p-S6), which are indicators of activation of the mTOR signal pathway, were adjusted in the same manner as above. It was evaluated by performing Western blotting with the expression of β-actin protein as a positive control. The results are shown in FIG. 4-2.
　図４－２に示すように、ＨＣＴ１１６－／－細胞とＨＯＣ３１３細胞、ＫＯＳＣ３細胞において、ｍｉＲ－８７６－３ｐの導入によるＲＳＫ１の発現抑制が顕著に認められ、さらに、ｍＴＯＲシグナル経路のｐ－ｐ７０Ｓ６Ｋ、ｐ－Ｓ６タンパクの発現抑制が認められ、ｍｉＲ－８７６－３ｐの導入により、ＲＳＫ１ならびにｍＴＯＲシグナル経路の抑制が確認できた。　 As shown in FIG. 4-2, the expression of RSK1 was remarkably suppressed by the introduction of miR-876-3p in HCT116 − / − cells, HOC313 cells, and KOSC3 cells, and further, p-p70S6K of the mTOR signal pathway, Suppression of the expression of p-S6 protein was observed, and suppression of RSK1 and mTOR signal pathway was confirmed by the introduction of miR-876-3p.
＜実施例Ａ７＞
（ルシフェラーゼレポーターアッセイ）
　ＲＳＫ１遺伝子の３’ＵＴＲ領域をルシフェラーゼベクターに組み込んだコンストラクトを用いるルシフェラーゼレポーターアッセイによって、ｍｉＲ－８７６－３ｐとＲＳＫ１遺伝子の相互作用を評価した。ｍｉＲ－８７６－３ｐのガイド鎖の塩基配列と相同する領域は、ＲＳＫ１遺伝子に１つ存在する。その領域に変異を入れたこと以外は上記と同様にしてルシフェラーゼレポーターアッセイを実施した。結果を図４－３に示す。 <Example A7>
(Luciferase Reporter Assay)
The interaction between miR-876-3p and the RSK1 gene was evaluated by a luciferase reporter assay using a construct in which the 3'UTR region of the RSK1 gene was incorporated into a luciferase vector. There is one region in the RSK1 gene that is homologous to the nucleotide sequence of the guide strand of miR-876-3p. A luciferase reporter assay was performed in the same manner as above except that the region was mutated. The results are shown in Figure 4-3.
　図４－３に示すように、ｍｉＲ－８７６－３ｐの導入により、野生型の３’ＵＴＲ領域に対応するルシフェラーゼ活性の低下を認め、ｍｉＲ－８７６－３ｐのガイド鎖の塩基配列とマッチする領域に変異を入れたベクターではルシフェラーゼ活性は回復した。これらの結果から、ｍｉＲ－８７６－３ｐは、ＲＳＫ１遺伝子の３’ＵＴＲ領域への作用により、直接ＲＳＫ１遺伝子の発現を抑制していることが示された。 As shown in FIG. 4-3, the introduction of miR-876-3p showed a decrease in luciferase activity corresponding to the wild-type 3'UTR region, and a region matching the base sequence of the guide chain of miR-876-3p. The luciferase activity was restored in the vector mutated in. From these results, it was shown that miR-876-3p directly suppressed the expression of the RSK1 gene by the action of the RSK1 gene on the 3'UTR region.
＜参考例１＞
　口腔がん細胞株（ＫＯＳＣ３）に、ＢＲＤ４遺伝子に対するｓｉＲＮＡ及びＲＳＫ１に対するｓｉ－ＲＮＡを作用させて、ＢＲＤ４遺伝子及びＲＳＫ１遺伝子の少なくとも一方をノックダウンしたときの遺伝子発現と細胞増殖に対する効果を、コントロールｓｉＲＮＡ（ｓｉ－ＮＣ）を用いた場合と比較して評価した。遺伝子発現は上記と同様にしてウエスタンブロットによって評価した。結果を図４－４（Ａ）示す。また、細胞増殖に対する効果を図４－４（Ｂ）に示す。 <Reference example 1>
Control siRNA to control the effect on gene expression and cell proliferation when at least one of the BRD4 gene and RSK1 gene is knocked down by allowing siRNA against the BRD4 gene and si-RNA against RSK1 to act on the oral cancer cell line (KOSC3). It was evaluated in comparison with the case where (si-NC) was used. Gene expression was evaluated by Western blot in the same manner as above. The results are shown in FIG. 4-4 (A). The effect on cell proliferation is shown in FIG. 4-4 (B).
　図４－４（Ａ）に示すようにＢＲＤ４遺伝子及びＲＳＫ１遺伝子の少なくとも一方をノックダウンすることでノックダウンした遺伝子の発現が抑制される。また、ＢＲＤ４遺伝子及びＲＳＫ１遺伝子の両方を同時にノックダウンすることで、アポトーシスの指標であるｃｌｅａｖｅｄ　ＰＡＲＰの発現量が相乗的に増加した。図４－４（Ｂ）に示すように細胞増殖が、ＢＲＤ４遺伝子及びＲＳＫ１遺伝子の少なくとも一方をノックダウンすることで抑制され、両方をノックダウンしたときに相乗的に抑制された。 As shown in FIG. 4-4 (A), knockdown of at least one of the BRD4 gene and the RSK1 gene suppresses the expression of the knocked down gene. In addition, by knocking down both the BRD4 gene and the RSK1 gene at the same time, the expression level of cleared PARP, which is an index of apoptosis, was synergistically increased. As shown in FIG. 4-4 (B), cell proliferation was suppressed by knocking down at least one of the BRD4 gene and the RSK1 gene, and synergistically suppressed when both were knocked down.
　図４－５は、上記の実施例の結果から導かれるｍｉＲ－８７６－３ｐが腫瘍細胞の増殖を抑制する推定機序を模式的に示す概念図である。 FIG. 4-5 is a conceptual diagram schematically showing the presumed mechanism by which miR-876-3p suppresses the growth of tumor cells, which is derived from the results of the above examples.
＜実施例Ａ８＞
　大腸がん細胞株（ＨＣＴ１１６－／－細胞）、口腔がん細胞株（ＨＳＣ２、ＨＯＣ３１３細胞）、肺がん細胞株（Ａ５４９細胞）、食道がん細胞株（ＫＹＳＥ１５０細胞）に、実施例Ａ１に記した要領でｍｉＲ－ＮＣ又はｍｉＲ－１２９３を導入した。導入から４８時間後に、上記と同様にして遺伝子発現アレイ解析を行った <Example A8>
Examples A1 were described in a colon cancer cell line (HCT116 − / − cells), an oral cancer cell line (HSC2, HOC313 cells), a lung cancer cell line (A549 cells), and an esophageal cancer cell line (KYSE150 cells). MiR-NC or miR-1293 was introduced in the same manner. Forty-eight hours after introduction, gene expression array analysis was performed in the same manner as above.
　図５－１には、これらのｍｉＲ導入細胞に対して行った発現アレイの結果、遺伝子発現が１．５倍を超えて低下した遺伝子の数をベン図で示している。ｍｉＲ－ＮＣ導入群と比較して１．５倍を超えて発現が抑制されている遺伝子のうち、当該５種類のがん細胞株のうち、４種類以上で共通していた遺伝子は１２４８個であった。この共通遺伝子１２４８個について、ＤＡＶＩＤ　ｓｏｆｔｗａｒｅを用いてパスウェイ解析を行ったところ、ＤＮＡ修復に関わる複数の経路、すなわち、Ｈｏｍｏｌｏｇｏｕｓ　ｒｅｃｏｍｂｉｎａｔｉｏｎ（相同組み換え修復）、ｂａｓｅ　ｅｘｃｉｓｉｏｎ　ｒｅｐａｉｒ（塩基除去修復）、Ｍｉｓｍａｔｃｈ　ｒｅｐａｉｒ、ｎｕｃｌｅｏｔｉｄｅ　ｅｘｃｉｓｉｏｎ　ｒｅｐａｉｒが抑制されていることが示された。下表に各経路とその経路に関わる遺伝子を示す。 FIG. 5-1 shows the number of genes whose gene expression decreased by more than 1.5 times as a result of the expression array performed on these miR-introduced cells. Among the genes whose expression was suppressed by more than 1.5 times compared to the miR-NC introduction group, 1248 genes were common to 4 or more of the 5 types of cancer cell lines. there were. When pathway analysis was performed on 1248 of these common genes using DAVID software, multiple pathways involved in DNA repair, that is, Homologous recombination (homologous recombination repair), base excision repair (base excision repair), and Mismatch repair, nail It was shown that excision repair was suppressed. The table below shows each pathway and the genes involved in that pathway.
Figure JPOXMLDOC01-appb-T000022
Figure JPOXMLDOC01-appb-T000022
＜実施例Ａ９＞
（ウエスタンブロット）
　ｍｉＲ－１２９３を導入した大腸がん細胞株（ＨＣＴ１１６－／－細胞）又は口腔がん細胞株（ＨＯＣ３１３細胞）に対して、実施例Ａ１に記した要領でｍｉＲ－ＮＣ又はｍｉＲ－１２９３を導入した。導入から４８時間後に、ＡＰＥＸ１、ＲＰＡ１及びＰＯＬＤ４タンパクの発現量を上記と同様にしてβ－ａｃｔｉｎタンパクの発現をポジティブコントロールとしたウエスタンブロットを行うことにより評価した。結果を図５－２に示す。 <Example A9>
(Western blot)
MiR-NC or miR-1293 was introduced into a colon cancer cell line (HCT116 − / − cells) or an oral cancer cell line (HOC313 cells) into which miR-1293 was introduced, in the manner described in Example A1. .. Forty-eight hours after the introduction, the expression levels of APEX1, RPA1 and POLD4 proteins were evaluated by performing Western blotting in the same manner as above with the expression of β-actin protein as a positive control. The results are shown in Figure 5-2.
　図５－２において示すように、ＨＣＴ１１６－／－細胞とＨＯＣ３１３細胞において、ｍｉＲ－１２９３の導入によるＡＰＥＸ１、ＲＰＡ１及びＰＯＬＤ４の発現抑制が顕著に認められた。 As shown in FIG. 5-2, the expression of APEX1, RPA1 and POLD4 was remarkably suppressed by the introduction of miR-1293 in HCT116 − / − cells and HOC313 cells.
＜実施例Ａ１０＞
（ルシフェラーゼレポーターアッセイ）
　ＡＰＥＸ１遺伝子、ＲＰＡ１遺伝子又はＰＯＬＤ４遺伝子の３’ＵＴＲ領域をルシフェラーゼベクターに組み込んだコンストラクトを用いるルシフェラーゼレポーターアッセイによって、ｍｉＲ－１２９３と、ＡＰＥＸ１遺伝子、ＲＰＡ１遺伝子又はＰＯＬＤ４遺伝子の相互作用を評価した。ｍｉＲ－１２９３のガイド鎖の塩基配列と相同する領域は、ＡＰＥＸ１、ＲＰＡ１及びＰＯＬＤ４遺伝子のそれぞれに１つずつ存在する。その領域にそれぞれ変異を入れたこと以外は上記と同様にしてルシフェラーゼレポーターアッセイを実施した。結果を図５－３に示す。 <Example A10>
(Luciferase Reporter Assay)
The interaction of miR-1293 with the APEX1, RPA1 or POLD4 gene was evaluated by a luciferase reporter assay using a construct that incorporated the 3'UTR region of the APEX1, RPA1 or POLD4 gene into a luciferase vector. There is one region homologous to the nucleotide sequence of the guide strand of miR-1293 for each of the APEX1, RPA1 and POLD4 genes. A luciferase reporter assay was performed in the same manner as above, except that mutations were placed in each region. The results are shown in Figure 5-3.
　図５－３に示すように、ｍｉＲ－１２９３導入により、ＡＰＥＸ１、ＲＰＡ１及びＰＯＬＤ４遺伝子のいずれにおいても野生型の３’ＵＴＲ領域に対応するルシフェラーゼ活性の有意な低下を認め、ｍｉＲ－１２９３のガイド鎖の塩基配列とマッチする領域に変異を入れたベクターではルシフェラーゼ活性は有意に回復した。これらの結果から、ｍｉＲ－１２９３は、ＡＰＥＸ１、ＲＰＡ１及びＰＯＬＤ４遺伝子の３’ＵＴＲ領域への作用により、直接ＡＰＥＸ１、ＲＰＡ１及びＰＯＬＤ４遺伝子の発現を抑制していることが示された。 As shown in FIG. 5-3, the introduction of miR-1293 significantly reduced the luciferase activity corresponding to the wild-type 3'UTR region in all of the APEX1, RPA1 and POLD4 genes, and the guide chain of miR-1293 was observed. The luciferase activity was significantly restored in the vector in which the mutation was added to the region matching the nucleotide sequence of. From these results, it was shown that miR-1293 directly suppresses the expression of APEX1, RPA1 and POLD4 genes by the action of APEX1, RPA1 and POLD4 genes on the 3'UTR region.
＜参考例２＞
　大腸がん細胞株（ＨＣＴ１１６－／－細胞）にｓｉＲＮＡを作用させて、ＢＲＤ４遺伝子、ＡＰＥＸ１遺伝子又はＢＲＤ４遺伝子とＡＰＥＸ１遺伝子の両方を同時にノックダウンしたとき（Ａ）、ＢＲＤ４遺伝子、ＲＰＡ１遺伝子又はＢＲＤ４遺伝子とＲＰＡ１遺伝子の両方を同時にノックダウンしたとき（Ｂ）、ＢＲＤ４遺伝子、ＰＯＬＤ４遺伝子又はＢＲＤ４遺伝子とＰＯＬＤ４遺伝子の両方を同時にノックダウンしたときの（Ｃ）、遺伝子発現と細胞増殖に対する効果を、コントロールｓｉＲＮＡ（ｓｉ－ＮＣ）を用いた場合と比較して評価した。遺伝子発現は上記と同様にしてウエスタンブロットによって評価した。ウエスタンブロットの結果を図５－４（Ａ）から（Ｃ）を示す。また細胞増殖に対する効果を図５－４（Ｄ）に示す。 <Reference example 2>
When siRNA is allowed to act on a colon cancer cell line (HCT116 − / − cells) and both the BRD4 gene, APEX1 gene or BRD4 gene and APEX1 gene are knocked down at the same time (A), the BRD4 gene, RPA1 gene or BRD4 gene Control siRNA of the effect on gene expression and cell proliferation when both the RPA1 gene and the RPA1 gene are knocked down at the same time (B) and when both the BRD4 gene, POLD4 gene or both the BRD4 gene and the POLD4 gene are knocked down at the same time (C) It was evaluated in comparison with the case where (si-NC) was used. Gene expression was evaluated by Western blot in the same manner as above. The results of Western blotting are shown in FIGS. 5-4 (A) to (C). The effect on cell proliferation is shown in FIG. 5-4 (D).
　図５－４（Ａ）から（Ｃ）のウエスタンブロットの結果に示されるように、ｓｉＲＮＡに対応する遺伝子の発現が抑制されている。また、ＢＲＤ４遺伝子とＡＰＥＸ１遺伝子、ＢＲＤ４遺伝子とＲＰＡ１遺伝子、又はＢＲＤ４遺伝子とＰＯＬＤ４遺伝子の両方を同時にノックダウンしたときに、アポトーシスの指標であるｃｌｅａｖｅｄ　ＰＡＲＰの相乗的な増加が認められた。図５－４（Ｄ）の細胞増殖の結果に示されるように、それぞれの遺伝子をノックダウンしたときに細胞増殖が抑制され、両方をノックダウンしたときに相乗的に細胞増殖が抑制された。 As shown in the results of Western blotting in FIGS. 5-4 (A) to (C), the expression of the gene corresponding to siRNA is suppressed. In addition, when both the BRD4 gene and APEX1 gene, the BRD4 gene and the RPA1 gene, or the BRD4 gene and the POLD4 gene were knocked down at the same time, a synergistic increase in cleared PARP, which is an index of apoptosis, was observed. As shown in the cell proliferation results in FIG. 5-4 (D), cell proliferation was suppressed when each gene was knocked down, and cell proliferation was synergistically suppressed when both genes were knocked down.
＜実施例Ａ１１＞
　大腸がん細胞株（ＨＣＴ１１６－／－細胞）に対して、実施例Ａ１に記した要領でｍｉＲ－ＮＣ又はｍｉＲ－１２９３を導入した。その後ネオカルチノスタチン（ＮＣＳ）を２００ｎｇ／ｍＬ添加して、ＤＮＡ切断を行った。２４時間後に以下のようにして免疫蛍光染色でＤＮＡ障害のマーカーであるγＨ２ＡＸの染色を行った。細胞を３．７％ホルムアルデヒド含有ＰＢＳで固定し、ＴｒｉｔｏｎＸ－１００含有ＰＢＳで処理した。３％ウシ血清含有ＰＢＳでブロッキングを行い、その後、抗γＨ２ＡＸ抗体（１／４００希釈）を１時間室温で反応させた。結合した抗体をＡｌｅｘａ　Ｆｌｕｏｒ５５５　ａｎｔｉ－ｒａｂｂｉｔ　ＩｇＧ抗体（１／５００希釈、Ｌｉｆｅ　Ｔｅｃｈｎｏｌｏｇｉｅｓ社）を用いて可視化した。その後、当該切片をＶＥＣＴＡＳＨＩＬＤ　ｃｏｎｔａｉｎｉｎｇ　ＤＡＰＩ（Ｖｅｃｔｏｒ　Ｌａｂｏｒａｔｏｒｉｅｓ社）で対比染色し、Ｎｉｋｏｎ社の蛍光顕微鏡で観察撮影した。結果を図５－５に示す。また、蛍光顕微鏡観察におけるランダムに抽出した３視野におけるγＨ２ＡＸ陽性細胞の割合を測定した。結果を図５－６に示す。 <Example A11>
MiR-NC or miR-1293 was introduced into a colorectal cancer cell line (HCT116 − / − cells) in the manner described in Example A1. Then, neocarzinostatin (NCS) was added at 200 ng / mL to perform DNA cleavage. Twenty-four hours later, γH2AX, which is a marker for DNA damage, was stained by immunofluorescence staining as follows. Cells were fixed with PBS containing 3.7% formaldehyde and treated with PBS containing Triton X-100. Blocking was performed with PBS containing 3% bovine serum, and then anti-γH2AX antibody (1/400 dilution) was reacted at room temperature for 1 hour. The bound antibody was visualized using an Alexa Fluor555 anti-rabbit IgG antibody (1/500 dilution, Life Technologies). Then, the section was counter-stained with VECTASHILD connecting DAPI (Vector Laboratories), and observed and photographed with a fluorescence microscope of Nikon. The results are shown in Figure 5-5. In addition, the proportion of γH2AX-positive cells in three randomly extracted visual fields in fluorescence microscope observation was measured. The results are shown in Figure 5-6.
　図５－５及び図５－６に示されるように、ｍｉＲ－１２９３導入細胞において、γＨ２ＡＸが多く認められた。すなわち、ｍｉＲ－１２９３導入により、ネオカルチノスタチンによるＤＮＡ障害からのＤＮＡ修復が抑制されたことを示す結果である。また、図５－５及び図５－６に示すようにｍｉＲ－１２９３のターゲットであるＡＰＥＸ１、ＲＰＡ１、ＰＯＬＤ４およびＢＲＤ４を抑制するとｍｉＲ－１２９３同様、ＤＮＡ修復が抑制される結果であった。これらの結果からｍｉＲ－１２９３によるＤＮＡ修復抑制効果がＡＰＥＸ１、ＲＰＡ１、ＰＯＬＤ４およびＢＲＤ４の抑制を介したものであることが示された。 As shown in FIGS. 5-5 and 5-6, a large amount of γH2AX was observed in miR-1293-introduced cells. That is, it is a result showing that the introduction of miR-1293 suppressed DNA repair from DNA damage caused by neocarzinostatin. Further, as shown in FIGS. 5-5 and 5-6, suppression of APEX1, RPA1, POLD4 and BRD4, which are targets of miR-1293, resulted in suppression of DNA repair as in miR-1293. From these results, it was shown that the DNA repair inhibitory effect of miR-1293 was mediated by the inhibition of APEX1, RPA1, POLD4 and BRD4.
＜実施例Ａ１２＞
　大腸がん細胞株（ＨＣＴ１１６－／－細胞）に対して、ｓｉＲＮＡを用いてＢＲＤ４遺伝子のノックダウンした場合と、ｍｉＲ－１２９３を導入した場合とにおける相同組み換え修復を評価した。相同組み換え修復の評価は、ＤＲ－ＧＦＰベクターを用いて、公知の方法（Genes Dev. 2001 Dec 15. 15(24):3237-42.）に従っておこなった。ここでＤＲ－ＧＦＰベクターは蛍光色素ＧＦＰの配列にエンドヌクレアーゼＳｃｅｌで切断される配列を組み込んだベクターであり、Ｓｃｅｌで切断されたのち、相同組み換え修復により切断されたベクターのＤＮＡが修復されるとＧＦＰを発現するベクターである。ＨＣＴ１１６－／－細胞に対してＤＲ－ＧＦＰベクターを導入し、ネオマイシンによってＤＲ－ＧＦＰベクターが安定的に発現している細胞を樹立した。ＤＲ－ＧＦＰベクターにはネオマイシン耐性遺伝子が組み込まれており、ＤＲ－ＧＦＰベクターを発現する細胞はネオマイシンに対して耐性を持つことで生存できるが、逆にＤＲ－ＧＦＰベクターを発現していない細胞はネオマイシン耐性がないため、生存できない。このＤＲ－ＧＦＰ発現ＨＣＴ１１６－／－細胞に対してｍｉＲ－１２９３あるいはコントロールとしてｍｉＲ－ＮＣを導入し、その後、エンドヌクレアーゼＳｃｅｌで処理して２４時間後にＤＲ－ＧＦＰを発色している細胞数をフローサイトメトリーで解析し、ｍｉＲ－ＮＣ投与群の細胞数を１とした時の相対的な細胞数を計算した。また、同様にＤＲ－ＧＦＰ発現ＨＣＴ１１６－／－細胞に対してｓｉ－ＢＲＤ４あるいはコントロールとしてｓｉ－ＮＣを導入し、その後、エンドヌクレアーゼＳｃｅｌで処理して２４時間後にＤＲ－ＧＦＰを発色している細胞数をフローサイトメトリーで解析し、ｍｉＲ－ＮＣ投与群の細胞数を１とした時の相対的な細胞数を計算した。結果を図５－７に示す。 <Example A12>
Homologous recombination repair was evaluated for colorectal cancer cell lines (HCT116 − / − cells) when the BRD4 gene was knocked down using siRNA and when miR-1293 was introduced. Evaluation of homologous recombination repair was performed using a DR-GFP vector according to a known method (Genes Dev. 2001 Dec 15.15 (24): 3237-42.). Here, the DR-GFP vector is a vector in which a sequence cleaved by endonuclease Scel is incorporated into the sequence of the fluorescent dye GFP, and when the DNA of the vector cleaved by homologous recombination repair is repaired after being cleaved by Scel It is a vector expressing GFP. The DR-GFP vector was introduced into HCT116 − / − cells, and cells in which the DR-GFP vector was stably expressed by neomycin were established. The neomycin resistance gene is integrated into the DR-GFP vector, and cells expressing the DR-GFP vector can survive by having resistance to neomycin, but conversely, cells expressing the DR-GFP vector do not. It cannot survive because it is not resistant to neomycin. MiR-1293 or miR-NC as a control was introduced into the DR-GFP expressing HCT116 − / − cells, and then treated with endonuclease Scel, and 24 hours later, the number of cells developing DR-GFP was flowed. The analysis was performed by cytometry, and the relative number of cells was calculated when the number of cells in the miR-NC administration group was 1. Similarly, cells in which si-BRD4 or si-NC as a control is introduced into DR-GFP expressing HCT116 − / − cells and then treated with endonuclease Scel to develop DR-GFP color 24 hours later. The number was analyzed by flow cytometry, and the relative number of cells was calculated when the number of cells in the miR-NC administration group was 1. The results are shown in Figure 5-7.
　図５－７に示されるように、ＢＲＤ４遺伝子のノックダウン、又はｍｉＲ－１２９３の導入により、ＧＦＰ発現細胞の数はコントロールよりも低くなった。すなわち、ｍｉＲ－１２９３の導入により、相同組み換え修復が抑制されることが示された。 As shown in FIG. 5-7, the number of GFP-expressing cells became lower than that of the control due to knockdown of the BRD4 gene or introduction of miR-1293. That is, it was shown that the introduction of miR-1293 suppresses homologous recombination repair.
　図５－８は、上記の実施例の結果から導かれるｍｉＲ－１２９３が腫瘍細胞の増殖を抑制する推定機序を模式的に示す概念図である。 FIG. 5-8 is a conceptual diagram schematically showing the presumed mechanism by which miR-1293, which is derived from the results of the above examples, suppresses the growth of tumor cells.
＜実施例Ｂ１＞
ｍｉＲ－８７６－３ｐ、ｍｉＲ－１２９３の投与によるｉｎ　ｖｉｖｏでの腫瘍細胞の増殖抑制効果
　７週齢のＢａｌｂ／ｃヌードマウスをオリエンタルバイオサービス社から購入し、無菌状態で飼育した。１０×１０６個の大腸がん細胞株（ＨＣＴ１１６－／－細胞）を含むＰＢＳ１００μｌをマウスの背側横腹に皮下注射した。この皮下注射以降のｉｎ　ｖｉｖｏ試験のスケジュールを図６－１に示す。１ｎｍｏｌのｍｉＲ－８７６－３ｐ、ｍｉＲ－１２９３又はコントロールｍｉＲＮＡと、２００μｌのＡｔｅｒｏＧｅｎｅ（ＫＯＫＥＮ社）の混合物を、腫瘍と皮膚の間の隙間に、計５回投与した（ＨＣＴ１１６－／－細胞の注射から３、７、１０、１４、１７日後）。細胞投与後１９日後、マウスを安楽死させ、腫瘍を摘出した。なお、すべてのマウスに対して行った実験手順は東京医科歯科大学の動物実験委員会の承認を受けた。 <Example B1>
In vivo tumor cell growth inhibitory effect by administration of miR-876-3p and miR-1293 7-week-old Balb / c nude mice were purchased from Oriental Bioservice and bred in a sterile condition. 100 μl of PBS containing 10 × 10 6 colorectal cancer cell lines (HCT116 − / − cells) was subcutaneously injected into the dorsal flank of mice. The schedule of the in vivo test after this subcutaneous injection is shown in FIG. 6-1. A mixture of 1 nmol miR-876-3p, miR-1293 or control miRNA and 200 μl AteroGene (KOKEN) was administered into the gap between the tumor and the skin a total of 5 times (from HCT116 − / − cell injection). After 3, 7, 10, 14, 17 days). Nineteen days after cell administration, mice were euthanized and tumors were removed. The experimental procedure performed on all mice was approved by the Animal Experiment Committee of Tokyo Medical and Dental University.
　図６－２に、ＨＣＴ１１６－／－細胞の注射から１９日後の代表的なマウスの皮下腫瘍の外観及び摘出された腫瘍の写真を示す。 FIG. 6-2 shows the appearance of a typical mouse subcutaneous tumor 19 days after injection of HCT116 − / − cells and a photograph of the excised tumor.
　図６－３に、腫瘍体積の推移を示す。腫瘍の体積は、（長径）×（短径）２×０．５で計算した。図６－３に示されるように、腫瘍の体積は、ｍｉＲ－８７６－３ｐ、ｍｉＲ－１２９３の投与により、コントロールｍｉＲＮＡ（ｍｉＲ－ＮＣ）の投与と比較して、有意に減少した。 Figure 6-3 shows the transition of tumor volume. Tumor volume was calculated as (major) x (minor) 2 x 0.5. As shown in FIG. 6-3, tumor volume was significantly reduced by administration of miR-876-3p, miR-1293 compared to administration of control miRNA (miR-NC).
　マウス毎に摘出した腫瘍の重量を計量した。結果を図６－４に示す。図６－４に示されるように、ｍｉＲ－８７６－３ｐ又はｍｉＲ－１２９３の投与群の腫瘍の重量は、コントロールｍｉＲＮＡの投与群に比べて、有意に軽い結果であった。 The weight of the removed tumor was weighed for each mouse. The results are shown in Figure 6-4. As shown in FIG. 6-4, the tumor weight in the miR-876-3p or miR-1293-administered group was significantly lighter than that in the control miRNA-administered group.
＜実施例Ｂ２＞
ｍｉＲ－８７６－３ｐ及びｍｉＲ－１２９３の発現解析
　定量ＲＴ－ＰＣＲを用いて、摘出された腫瘍組織におけるｍｉＲ－８７６－３ｐ及びｍｉＲ－１２９３の発現解析を実施した。腫瘍組織より全ＲＮＡを、ＴＲＩｓｕｒｅ試薬（バイオライン社）を用いて標準的方法で分離した。当該全ＲＮＡから調製された１本鎖ＲＮＡをｍｉＲ－８７６－３ｐ又はｍｉＲ－１２９３に特異的なプライマーを用いて増幅した。ｍｉＲ－８７６－３ｐ及びｍｉＲ－１２９３に対するリアルタイムＲＴ－ＰＣＲは、ＡＢＩ　Ｐｒｉｓｍ　７５００　Ｆａｓｔ　Ｒｅａｌ－ｔｉｍｅ　ＰＣＲ　ｓｙｓｔｅｍ（アプライドバイオシステム社）、Ｔａｑｍａｎ　Ｕｎｉｖｅｒｓａｌ　ＰＣＲ　Ｍａｓｔｅｒ　Ｍｉｘ　（アプライドバイオシステム社）、Ｔａｑｍａｎ　Ｕｎｉｖｅｒｓａｌ　ＰＣＲ　Ｍａｓｔｅｒ　Ｍｉｘ（アプライドバイオシステム社）、Ｔａｑｍａｎ　ｍｉｃｒｏＲＮＡ　Ａｓｓａｙｓ（アプライドバイオシステム社）を用いて、これらの操作マニュアルに従って行った。ｍｉＲ－８７６－３ｐ及びｍｉＲ－１２９３の発現レベルは、全ＲＮＡの初期量の標準化コントロールとしてＲＮＵ６Ｂの発現量により補正することで標準化した。結果を図６－５に示す。 <Example B2>
Expression analysis of miR-876-3p and miR-1293 Expression analysis of miR-876-3p and miR-1293 was performed in the excised tumor tissue using quantitative RT-PCR. Total RNA was separated from the tumor tissue by a standard method using a TRIsure reagent (Bioline). The single-stranded RNA prepared from the total RNA was amplified using a primer specific to miR-876-3p or miR-1293. Real-time RT-PCR for miR-876-3p and miR-1293 includes ABI Prism 7500 Fast Real-time PCR system (Applied Biosystems), Taqman Universal PCR Master Mix (Applied Biosystems), TaqMan MultiMix (Applied Biosystems). Using Applied Biosystems) and Taqman microRNA Assays (Applied Biosystems), these procedures were performed according to the operating manuals. The expression levels of miR-876-3p and miR-1293 were standardized by adjusting for the expression level of RNU6B as a standardization control for the initial amount of total RNA. The results are shown in FIG. 6-5.
　図６－５に示されるように、定量ＲＴ－ＰＣＲによる検討の結果、腫瘍細胞におけるｍｉＲ－８７６－３ｐ又はｍｉＲ－１２９３の発現量は、それぞれのｍｉＲＮＡ投与群において顕著に増加していることが確認できた。 As shown in FIG. 6-5, as a result of examination by quantitative RT-PCR, the expression level of miR-876-3p or miR-1293 in tumor cells was significantly increased in each miRNA-administered group. It could be confirmed.
＜実施例Ｂ３＞　
　免疫染色によって、摘出された腫瘍組織におけるＢＲＤ４遺伝子、ＲＳＫ１遺伝子、ＡＰＥＸ１遺伝子、ＲＰＡ１遺伝子及びＰＯＬＤ４遺伝子の発現量を評価した。腫瘍組織を１０％ホルムアルデヒド含有ＰＢＳで固定し、パラフィン包埋を行い、４μｍ厚の切片に薄切した。腫瘍切片を以下に示すように、アビジン－ビオチン－ペルオキシダーゼ法を用いて、ＢＲＤ４、ＲＳＫ１、ＡＰＥＸ１、ＲＰＡ１、ＰＯＬＤ４に対して免疫組織化学染色を行った。パラフィンで包埋された腫瘍切片について、キシレンで脱パラフィン後、エタノールで再水和をおこなった。１０ｍＭのクエン酸緩衝液（ｐＨ６．０）中で煮沸することで抗原の賦活化を行い、内因性ペルオキシダーゼを不活性化するために０．３％過酸化水素含有メタノールで処理した。その後、当該切片について、抗ＢＲＤ４抗体（１／５００希釈）、抗ＲＳＫ１抗体（１／１００希釈）、抗ＡＰＥＸ１抗体（１／２００希釈）、ＲＰＡ１抗体（１／１００希釈）又は抗ＰＯＬＤ４抗体（１／１００希釈）を用いて、４℃で一晩反応させた。結合した抗体をジアミノベンジジン（ＶＥＣＴＡＳＴＡＩＮ－ＥｌｕｔｅＡＢＣｋｉｔ：ベクターラボラトリー社）を用いて可視化した。その後、当該切片をヘマトキシリンで対比染色した。 <Example B3>
The expression levels of the BRD4 gene, RSK1 gene, APEX1 gene, RPA1 gene and POLD4 gene in the excised tumor tissue were evaluated by immunostaining. Tumor tissue was fixed with PBS containing 10% formaldehyde, embedded in paraffin, and sliced into 4 μm thick sections. Tumor sections were immunohistochemically stained for BRD4, RSK1, APEX1, RPA1 and POLD4 using the avidin-biotin-peroxidase method as shown below. Tumor sections embedded in paraffin were deparaffinized with xylene and then rehydrated with ethanol. The antigen was activated by boiling in 10 mM citrate buffer (pH 6.0) and treated with methanol containing 0.3% hydrogen peroxide to inactivate the endogenous peroxidase. Then, for the section, anti-BRD4 antibody (1/500 dilution), anti-RSK1 antibody (1/100 dilution), anti-APEX1 antibody (1/200 dilution), RPA1 antibody (1/100 dilution) or anti-POLD4 antibody (1). / 100 dilution) was used to react overnight at 4 ° C. The bound antibody was visualized using diaminobenzidine (VECTASTAIN-EluteABCkit: Vector Laboratory). The sections were then counterstained with hematoxylin.
　抗ＢＲＤ４抗体を用いた免疫染色の結果を図６－６に示す。抗ＲＳＫ１抗体を用いた免疫染色の結果を図６－７に示す。抗ＡＰＥＸ１抗体、ＲＰＡ１抗体又は抗ＰＯＬＤ４抗体を用いた免疫染色の結果を図６－８に示す。 The results of immunostaining using the anti-BRD4 antibody are shown in FIG. 6-6. The results of immunostaining using the anti-RSK1 antibody are shown in FIG. 6-7. The results of immunostaining using an anti-APEX1 antibody, an RPA1 antibody or an anti-POLD4 antibody are shown in FIG. 6-8.
　図６－６及び図６－７に示す結果から、ｍｉＲ－８７６－３ｐの投与により、インビボの腫瘍組織において、ＢＲＤ４遺伝子及びＲＳＫ１遺伝子の発現が抑制され、腫瘍増殖が抑制されたことが示された。すなわち、ｍｉＲ－８７６－３ｐを含む医薬組成物は、ＢＲＤ４及びＲＳＫ１からなる群から選択される少なくとも１種の発現に起因する種々の腫瘍に対して効果を有することが期待される。 From the results shown in FIGS. 6-6 and 6-7, it was shown that the administration of miR-876-3p suppressed the expression of the BRD4 gene and the RSK1 gene and suppressed the tumor growth in the in vivo tumor tissue. It was. That is, the pharmaceutical composition containing miR-876-3p is expected to have an effect on various tumors resulting from the expression of at least one selected from the group consisting of BRD4 and RSK1.
　図６－６及び図６－８に示す結果から、ｍｉＲ－１２９３の投与により、インビボの腫瘍組織において、ＢＲＤ４遺伝子、ＡＰＥＸ１遺伝子、ＲＰＡ１遺伝子、及びＰＯＬＤ４遺伝子の発現が抑制され、腫瘍増殖が抑制されたことが示された。すなわち、ｍｉＲ－１２９３を含む医薬組成物は、ＢＲＤ４、ＡＰＥＸ１、ＲＰＡ１、及びＰＯＬＤ４からなる群から選択される少なくとも１種の発現に起因する種々の腫瘍に対して効果を有することが期待される。 From the results shown in FIGS. 6-6 and 6-8, administration of miR-1293 suppressed the expression of the BRD4 gene, APEX1 gene, RPA1 gene, and POLD4 gene in the in vivo tumor tissue, and suppressed tumor growth. It was shown that That is, the pharmaceutical composition containing miR-1293 is expected to have an effect on various tumors resulting from the expression of at least one selected from the group consisting of BRD4, APEX1, RPA1, and POLD4.
　従来の研究においても、１つのｍｉＲＮＡは各々の遺伝子のコーディング領域あるいは３'ＵＴＲ領域に直接結合することにより、複数の標的の発現を阻害することができることが示されている。このことは、がんの活性化に関与する複数の経路を１つのｍｉＲＮＡが標的とすることができることを示している。例えば、ｈｓａ－ｍｉＲ－３４ａは、ＣＣＮＤ１遺伝子、ＣＤＫ６遺伝子、ＭＹＣ遺伝子、ｃ－ＭＥＴ遺伝子、ＮＯＴＣＨ遺伝子といった複数の標的を介して、腫瘍増殖を抑制することが知られている。上記の実施例に示されるように、ｍｉＲ－８７６－３ｐは、ＢＲＤ４遺伝子及びＲＳＫ１遺伝子を標的とすることができる。また、ｍｉＲ－１２９３はＢＲＤ４遺伝子、ＡＰＥＸ１遺伝子、ＲＰＡ１遺伝子及びＰＯＬＤ４遺伝子を標的とすることができる。これらのｍｉＲＮＡの導入により、ヌードマウスの皮下に形成された大腸がん細胞株であるＨＣＴ１１６－／－細胞由来の腫瘍の増大を抑制する効果が認められた。 Previous studies have also shown that one miRNA can inhibit the expression of multiple targets by directly binding to the coding region or 3'UTR region of each gene. This indicates that a single miRNA can target multiple pathways involved in cancer activation. For example, hsa-miR-34a is known to suppress tumor growth via a plurality of targets such as CCND1 gene, CDK6 gene, MYC gene, c-MET gene, and NOTCH gene. As shown in the above examples, miR-876-3p can target the BRD4 and RSK1 genes. In addition, miR-1293 can target the BRD4 gene, APEX1 gene, RPA1 gene and POLD4 gene. The introduction of these miRNAs was found to have the effect of suppressing the growth of tumors derived from HCT116 − / − cells, which are colon cancer cell lines formed subcutaneously in nude mice.
　したがって本実施形態は、ｍｉＲ－８７６－３ｐに由来するポリヌクレオチドを含み、ＢＲＤ４遺伝子及びＲＳＫ１遺伝子が活性化した腫瘍に対する有効性が認められる医薬組成物を提供するものである。また、ｍｉＲ－１２９３に由来するポリヌクレオチドを含み、ＢＲＤ４遺伝子、ＡＰＥＸ１遺伝子、ＲＰＡ１遺伝子及びＰＯＬＤ４遺伝子が活性化した腫瘍に対する有効性が認められる医薬組成物を提供するものである。 Therefore, the present embodiment provides a pharmaceutical composition containing a polynucleotide derived from miR-876-3p and which is found to be effective against a tumor in which the BRD4 gene and the RSK1 gene are activated. Further, the present invention provides a pharmaceutical composition containing a polynucleotide derived from miR-1293 and having an effect on a tumor in which the BRD4 gene, APEX1 gene, RPA1 gene and POLD4 gene are activated.
＜実施例Ｃ＞
　細胞株として、乳がん１４株、大腸がん１０株、子宮体がん５株、食道がん４３株、胃がん４株、肝臓がん２株、肺がん３株、神経芽腫１株、口腔がん２２株、卵巣がん２２株、膵臓がん１株、前立腺がん４株、肉腫８株、甲状腺がん１２株、及び尿管がん１株を準備した。準備した細胞株においてＢＲＣＡ１またはＢＲＣＡ２（以下、ＢＲＣＡ１／２と略記することがある）に病的変異を持つ細胞株を下表に例示する。なお、ＢＲＣＡ１／２の病的変異としては、フレームシフト変異、ナンセンス変異、及びメチル化を意図した。 <Example C>
Cell lines include breast cancer 14 strains, colon cancer 10 strains, uterine body cancer 5 strains, esophageal cancer 43 strains, gastric cancer 4 strains, liver cancer 2 strains, lung cancer 3 strains, neuroblastoma 1 strain, oral cancer Twenty-two strains, ovarian cancer 22 strains, pancreatic cancer 1 strain, prostate cancer 4 strains, sarcoma 8 strains, thyroid cancer 12 strains, and urinary tract cancer 1 strain were prepared. The table below exemplifies cell lines having a pathological mutation in BRCA1 or BRCA2 (hereinafter, may be abbreviated as BRCA1 / 2) in the prepared cell lines. The pathological mutations of BRCA1 / 2 were intended to be frameshift mutations, nonsense mutations, and methylation.
Figure JPOXMLDOC01-appb-T000023
Figure JPOXMLDOC01-appb-T000023
　９６ｗｅｌｌ　ｐｌａｔｅの１ｗｅｌｌに８，０００個の細胞を播種し、それぞれの細胞株に適した培地で３７℃・５％ＣＯ２環境もしくはＣＯ２が含まれない環境で培養した。２４時間後にｍｉＲ－Ｎｅｇａｔｉｖｅ　Ｃｏｎｔｒｏｌ（ｍｉＲ－ＮＣ；２０μＭ）、またはｍｉＲ－１２９３（２．５，５，１０，もしくは２０μＭ）をＬｉｐｏｆｅｃｔａｍｉｎｅ　ＲＮＡｉＭＡＸ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　ｓｃｉｅｎｔｉｆｉｃ）と混合し、トランスフェクションした。 8,000 cells were seeded in 1 well of 96 well plate and cultured in a medium suitable for each cell line in a 37 ° C., 5% CO 2 environment or an environment free of CO 2. After 24 hours, miR-Negative Control (miR-NC; 20 μM) or miR-1293 (2.5, 5, 10, or 20 μM) was mixed with Lipofectamine RNAiMAX (Thermo Fisher scientific) and transfected.
　トランスフェクションしてから７２時間後にクリスタルバイオレット溶液（１０％ホルマリン、０．１％クリスタルバイオレット）で染色した。その後、水で洗浄し、完全に乾燥した後、２％ＳＤＳ溶液で溶解した。溶解液は、５６０ｎｍの波長を用いて吸光度計で計測し、得られた値を生存細胞の量とした。ｍｉＲ－ＮＣを基準として、ｍｉＲ－１２９３各濃度での細胞生存率を計算し、グラフ化した。結果の一部を図７－１に示す。 72 hours after transfection, the cells were stained with a crystal violet solution (10% formalin, 0.1% crystal violet). Then, it was washed with water, completely dried, and then dissolved in a 2% SDS solution. The lysate was measured with an absorptiometer using a wavelength of 560 nm, and the obtained value was taken as the amount of viable cells. Based on miR-NC, the cell viability at each concentration of miR-1293 was calculated and graphed. A part of the result is shown in FIG. 7-1.
　濃度２０ｎＭ未満で細胞生存率が０．５以下まで低下した細胞株を、ｍｉＲ－１２９３高感受性株として、ＢＲＣＡ１／２病的変異群（＋）と変異無し群（－）におけるｍｉＲ－１２９３感受性を評価した。結果を図７－２に示す。 The cell line whose cell viability decreased to 0.5 or less at a concentration of less than 20 nM was designated as a miR-1293 high-sensitivity strain, and the miR-1293 sensitivity in the BRCA1 / 2 pathological mutation group (+) and the non-mutation group (-) was determined. evaluated. The results are shown in Figure 7-2.
　また、ＢＲＣＡ１／２病的変異群（＋）と変異無し群（－）とでのｍｉＲ－１２９３感受性の有意差を２ｘ２　Ｃｈｉ　ｓｑｕａｒｅ　ｔｅｓｔを用いて解析した。その結果、ＢＲＣＡ１／２病的変異群でのｍｉＲ－１２９３の感受性は、有意差（Ｐ＝０．０４１６）を持って高いことが示された。 In addition, the significant difference in miR-1293 susceptibility between the BRCA1 / 2 pathological mutation group (+) and the non-mutation group (-) was analyzed using a 2x2 Chi square test. As a result, it was shown that the susceptibility of miR-1293 in the BRCA1 / 2 pathogenic mutant group was high with a significant difference (P = 0.0416).
　したがって本実施形態は、ｍｉＲ－１２９３に由来するポリヌクレオチドを含み、ＢＲＣＡ１／２に病的変異を有するか、またはＢＲＣＡｎｅｓｓの状態にある腫瘍に対する有効性が認められる医薬組成物を提供するものである。さらに、ＰＡＰＲ阻害剤に抵抗性を有する腫瘍に対する有効性が期待される医薬組成物を提供するものである。 Accordingly, the present embodiment provides a pharmaceutical composition comprising a polynucleotide derived from miR-1293 and found to be effective against tumors having a pathogenic variant in BRCA1 / 2 or in a state of BRCAness. .. Furthermore, it provides a pharmaceutical composition that is expected to be effective against tumors that are resistant to PAPR inhibitors.
　日本国特許出願２０１９－２３３２５０号（出願日：２０１９年１２月２４日）の開示はその全体が参照により本明細書に取り込まれる。本明細書に記載された全ての文献、特許出願、及び技術規格は、個々の文献、特許出願、及び技術規格が参照により取り込まれることが具体的かつ個々に記された場合と同程度に、本明細書に参照により取り込まれる。 The entire disclosure of Japanese Patent Application No. 2019-233250 (Filing Date: December 24, 2019) is incorporated herein by reference in its entirety. All documents, patent applications, and technical standards described herein are to the same extent as if the individual documents, patent applications, and technical standards were specifically and individually stated to be incorporated by reference. Incorporated herein by reference.

Claims (6)

  1. 　ｍｉＲ－１２９３、ｍｉＲ－８７６－３ｐ、ｍｉＲ－４４３８、ｍｉＲ－６７５１、ｍｉＲ－６３４及びｍｉＲ－９２ａ－２－５ｐからなる群から選択される少なくとも１種のマイクロＲＮＡをコードする遺伝子の転写産物もしくはそのプロセッシング産物と同一の塩基配列、又はそれらの塩基配列において１から５塩基が置換、欠損又は付加された塩基配列を有するポリヌクレオチドを含む、腫瘍の処置に用いられる医薬組成物。 A transcript of a gene encoding at least one microRNA selected from the group consisting of miR-1293, miR-876-3p, miR-4438, miR-6751, miR-634 and miR-92a-2-5p. A pharmaceutical composition used for treating a tumor, which comprises a nucleotide having the same base sequence as the processing product, or a base sequence in which 1 to 5 bases are substituted, deleted or added in the base sequence.
  2. 　前記マイクロＲＮＡが、ｍｉＲ－１２９３、ｍｉＲ－８７６－３ｐ、ｍｉＲ－４４３８、ｍｉＲ－６７５１及びｍｉＲ－９２ａ－２－５ｐからなる群から選択される少なくとも１種である請求項１に記載の医薬組成物。 The pharmaceutical composition according to claim 1, wherein the microRNA is at least one selected from the group consisting of miR-1293, miR-876-3p, miR-4438, miR-6751 and miR-92a-2-5p. Stuff.
  3. 　処置の対象となる腫瘍は、ＢＲＤ４遺伝子を発現している腫瘍細胞を含む、請求項１又は２に記載の医薬組成物。 The pharmaceutical composition according to claim 1 or 2, wherein the tumor to be treated contains tumor cells expressing the BRD4 gene.
  4. 　処置の対象となる腫瘍は、大腸がん、膵がん、口腔がん、肺がん、食道がん、胃がん、子宮がん、皮膚がん、血液腫瘍、脳腫瘍、神経芽腫、膠芽腫、乳がん、卵巣がん、前立腺がん、膀胱がん、食道がん、肝がん及び腎がんからなる群から選ばれる少なくとも１種である請求項１から３のいずれか１項に記載の医薬組成物。 The tumors to be treated are colon cancer, pancreatic cancer, oral cancer, lung cancer, esophageal cancer, gastric cancer, uterine cancer, skin cancer, blood tumor, brain tumor, neuroblastoma, glioblastoma, breast cancer. The pharmaceutical composition according to any one of claims 1 to 3, which is at least one selected from the group consisting of ovarian cancer, prostate cancer, bladder cancer, esophageal cancer, liver cancer and renal cancer. Stuff.
  5. 　前記ポリヌクレオチドは、少なくとも１種の修飾ヌクレオチドを含む請求項１から４のいずれか１項に記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 4, wherein the polynucleotide contains at least one modified nucleotide.
  6. 　前記ポリヌクレオチドを２本鎖として含む請求項１から５のいずれか１項に記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 5, which comprises the polynucleotide as a double strand.
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