WO2014200065A1 - Composition for treating and/or diagnosing colorectal cancer, and use therefor - Google Patents

Composition for treating and/or diagnosing colorectal cancer, and use therefor Download PDF

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WO2014200065A1
WO2014200065A1 PCT/JP2014/065615 JP2014065615W WO2014200065A1 WO 2014200065 A1 WO2014200065 A1 WO 2014200065A1 JP 2014065615 W JP2014065615 W JP 2014065615W WO 2014200065 A1 WO2014200065 A1 WO 2014200065A1
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mir
colorectal cancer
sequence
expression
expression level
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Japanese (ja)
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勝彦 能正
拓 鈴木
博幸 山本
恭久 篠村
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北海道公立大学法人札幌医科大学
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    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention relates to a technique for treating and / or diagnosing colorectal cancer, and more particularly to a technique for treating and / or diagnosing colorectal cancer having mutant KRAS or mutant BRAF. is there.
  • KRAS, BRAF and PIK3CA are all important effector proteins in the signal transduction pathway downstream of the receptor tyrosine kinase, and in particular, metastatic colon cancer (KRAS gene wild-type metastasis) in which the gene encoding the KRAS protein is a wild type.
  • an antibody against the EGF receptor is often used.
  • BRAF and PIK3CA are both effector proteins that function downstream of KRAS, but the signal transduction pathway from KRAS through BRAF and the signal transduction pathway from KRAS through PIK3CA are independent from each other. Therefore, even if the therapeutic target is focused on the signal transduction pathway downstream of the EGF receptor, a wide variety of therapeutic agents must be developed depending on the presence or absence of mutations in KRAS, BRAF, and PIK3CA. In the first place, since it is necessary to perform individual sequence analysis in order to detect mutations in these genes, it is not possible to easily develop therapeutic agents for each.
  • Micro RNA which is a functional RNA of non-coding RNA, is a short RNA of 16 to 25 bases, and more than 1,000 types of human miRNA are registered. miRNA suppressively controls the expression of various genes by inhibiting the translation of mRNAs of many target genes having homology into proteins. Analysis of miRNA in cancer has attracted attention not only for elucidating the mechanism of carcinogenesis but also for developing new cancer diagnostic methods and cancer treatment methods.
  • Non-Patent Document 1 the expression of 156 miRNAs in colorectal cancer cell lines was examined by real-time PCR, and 13 mature miRNAs that were differentially expressed between colorectal cancer and non-tumor tissues were identified. In particular, the difference between miR-31, miR-96, miR-133b, miR-135b, miR-145, and miR-183 was significant, and the expression level of miR-31 was It is disclosed that there is a clear difference between stage II and stage IV of colorectal cancer.
  • Non-patent document 2 describes that BRAF mutation is associated with anti-EGFR antibody resistance in colorectal cancer, and HSA-MIR31-3P is a metastatic colorectal cancer patient having a wild-type KRAS gene. It has been suggested that it is a tool for predicting anti-EGFR antibody responsiveness.
  • Patent Document 1 and Non-Patent Documents 3 and 4 disclose that miR-31 functions as a tumor suppressor in breast cancer and gastric cancer.
  • Non-Patent Document 1 The knowledge about miR-31 described in Non-Patent Document 1 is only a differential expression between colorectal cancer and non-tumor tissue, and it is between wild-type KRAS and mutant KRAS in colorectal cancer. It is not a result reflecting a difference or a result reflecting a difference between wild type BRAF and mutant BRAF in colorectal cancer.
  • Non-patent document 1 suggests that miR-9, which is one of miRNAs overexpressed in colorectal cancer cell lines having mutations in KRAS, is a miRNA for BRAF regulation. It is described that miRNA is overexpressed only in a colon cancer cell line having wild type BRAF. This suggests that miR-9 overexpression can be an indicator of mutant KRAS / wild-type BRAF, and among the miRNAs investigated in Non-Patent Document 1, there is a mutation in BRAF. Teaches that no miRNAs were found that correlate with.
  • microRNA31 (miR-31) is significantly correlated with KRAS mutation and BRAF mutation by microRNA analysis using 781 samples including colon cancer, miR-31. was found to be associated with proximal colon cancer and poor prognosis. Further, when miR-31 mimics were introduced into colon cancer cell lines, the cell invasion ability increased and an inhibitor against miR-31 (hereinafter referred to as miR-31) , MiR-31 inhibitor) was administered to colorectal cancer cells, and cell growth was suppressed, and the present invention was completed.
  • miR-31 an inhibitor against miR-31
  • the first composition of the present invention is characterized by containing a miR-31 inhibitor for treating colorectal cancer.
  • the first composition of the present invention preferably contains the first polynucleotide or an expression vector containing the first polynucleotide as the miR-31 inhibitor.
  • the first composition of the present invention is preferably used for the treatment of colorectal cancer cells in which at least one of the KRAS gene and the BRAF gene is mutated, and the colorectal cancer cells or metastasis resistant to treatment with an anti-EGFR antibody It may be used for the treatment of invasive colorectal cancer or may be used for combination therapy with an anti-EGFR antibody.
  • the first composition of the present invention may further contain an anti-EGFR antibody, and may be configured to be provided in a kit together with the anti-EGFR antibody. .
  • the method for diagnosing colorectal cancer of the present invention comprises the steps of measuring the expression level of miR-31 in a biological sample derived from a colorectal cancer patient, and comparing the measured expression level with a control value. It is characterized by that.
  • This method can also be a method of acquiring data for diagnosing colorectal cancer as a method for assisting in the diagnosis of colorectal cancer.
  • the miR-31 is preferably a polynucleotide consisting of any one of SEQ ID NOs: 1 to 16.
  • the second composition of the present invention is characterized by containing a second polynucleotide in order to diagnose colorectal cancer.
  • Non-Patent Document 2 considers that mir-31-3p expression in patients with wild-type KRAS is a predictor of anti-EGF response, mir-31-3p expression level, age, sex, and BRAF mutation status The established nomogram is described as a tool for selecting patients with wild-type KRAS for anti-EGFR therapy. Such findings do not teach or suggest that miR-31 inhibitors are effective against colon cancer cells in which at least one of the KRAS gene and BRAF gene has been mutated. In the first place, one of the double strands cut out from the precursor microRNA molecule having a hairpin structure is degraded, and the other functions as a mature microRNA molecule.
  • Non-Patent Document 2 strongly teaches that mir-31-3p is a functional mature microRNA molecule in patients with wild-type KRAS.
  • Non-Patent Document 2 not only describes or suggests the involvement of mir-31-5p, but also prevents the motivation to arrive at the involvement of mir-31-5p by the above teaching. That is, by using mir-31-5p, it is possible to know whether a colon cancer patient has colon cancer cells in which at least one of the KRAS gene and the BRAF gene has a mutation. It cannot be predicted by those skilled in the art from Non-Patent Document 2. Thus, the fact that the second composition of the present invention can diagnose colorectal cancer is a new finding discovered by the inventors' original viewpoint and inventive idea.
  • the diagnosis is preferably at least one of a companion diagnosis for anti-EGFR antibody treatment for colorectal cancer patients and a prognosis determination for colorectal cancer patients.
  • the control value used in the method of the present invention is the expression level of miR-31 in a biological sample derived from a healthy subject, or a biological sample collected from a colon cancer patient to be diagnosed for a certain period of time before Expression level of miR-31 in the normal colon tissue collected from the colon cancer patient at the same time as the colon cancer tissue collection from the colon cancer patient to be diagnosed, or It can be the expression level of miR-31 in normal colon tissue collected from healthy individuals.
  • the screening method of the present invention uses a colon cancer cell having a mutation in at least one of the KRAS gene and BRAF gene as a candidate substance in order to screen a drug for treating colon cancer that is resistant to treatment with an anti-EGFR antibody. And a step of selecting a candidate substance whose miR-31 expression level after incubation is lower than the miR-31 expression level before incubation.
  • the screened drug may be used for combination therapy with an anti-EGFR antibody, and metastatic colorectal cancer may be the subject of treatment.
  • the agent is preferably provided in the form of a composition or kit with the anti-EGFR antibody.
  • Micro RNA is important not only for basic research of cell physiology but also as a biomarker for disease diagnosis or prognosis of treatment.
  • the present invention it is possible not only to know the stage of colorectal cancer, but also to make a companion diagnosis for anti-EGFR antibody treatment for colorectal cancer patients.
  • colon cancer cells into which miR-31 has been introduced function as miR-31 high-expressing cells with enhanced cell invasion ability, the present invention can be used for screening therapeutic agents for miR-31 high-expressing cells. .
  • the presence or absence of a protein mutation in the signal transduction pathway downstream of the EGF receptor can be easily predicted.
  • Colon cancer cell lines (DLD-1 (KRAS mutation; c.38G> A) and SW480 (KRAS mutation; c.35G> T)) were transfected with miR-31 mimics and miR-31 It was confirmed that expression was up-regulated (A in the figure).
  • Colon cancer cell lines (HT-29 (BRAF mutation; c.1799T> A), HCT-116 (KRAS mutation; c.38G> A), RKO (BRAF mutation; c.1799T> A), DLD-1 and SW480 ) was transfected with a miR-31 inhibitor, and it was confirmed that the expression of miR-31 was down-regulated in these cells (B in the figure).
  • (A) shows that in the MTT proliferation assay, miR-31 inhibitors significantly reduced cell proliferation in HCT-116 cells.
  • (B) shows that in the Matrigel invasion assay, invasion in DLD-1 cells increased 8.5-fold after miR-31 was transfected into this cell line.
  • (C) shows that expression of BRAF protein was reduced by miR-31 inhibitor in Western blot analysis.
  • (B) shows the expression level of miR-31-3p in colorectal cancer cell lines.
  • miRNA is short single-stranded RNA like siRNA. Unlike siRNAs that bind to the translation region of the target gene mRNA, miRNAs bind to the 3 ′ untranslated region (UTR) of the target gene mRNA, thereby causing suppression of expression or translation or degradation of the target mRNA. In addition, siRNA completely binds to the target gene mRNA and suppresses gene expression, but miRNA binds only partially to the target gene mRNA, so that it is an incomplete complementary strand containing a mismatch to the target gene mRNA. Can also cause translational inhibition.
  • UTR untranslated region
  • microRNA 31 may be a precursor microRNA molecule (pre-miRNA) or a mature microRNA molecule.
  • pre-miRNA precursor microRNA molecule
  • the pri-miRNA is transcribed from the gene, then a pre-miRNA having a hairpin structure is generated from the pri-miRNA, and finally a mature miRNA is generated from the pre-miRNA as a single-stranded RNA by Dicer-mediated processing.
  • mature miRNA molecules exist primarily as single strands
  • pre-miRNA molecules are at least partially self-complementary structures that can form double-stranded structures.
  • the term “complementary” intends a condition in which the base of one nucleotide sequence and the base of the other nucleotide sequence can bind to each other to form a base pair.
  • the base sequence of the precursor molecule (pre-miRNA31) of miR-31 is “GGAGAGGAGGCAAGAUUGCUGGCAAUAGCUGUUGAACUGGGGAACCUGCUAUUGCCAACAUAUUGCCAUCUUCC (SEQ ID NO: 1)”.
  • mature miRNA 31 is intended to be miR-31-5p, the base sequence thereof is “GAGGCAAGAUUGCUGGCAAUAGCUGUU (SEQ ID NO: 2)” or a fragment thereof and “GGCAAGAUUGCUGCGCAU” (SEQ ID NO: 3) "is intended, and those containing" AGGCAAGAUUGCUGGCAAUAGCU (SEQ ID NO: 7) "are preferred.
  • Examples of the mature miRNA 31 include “GAGGCAAGAUUGCUGGCAAUAGCU (SEQ ID NO: 4)”, “AGGCAAGAUUGCUGGCAAUAGCUUG (SEQ ID NO: 5)”, “AGGCAAGUGCUGGCAUGAGUGUG (SEQ ID NO: 6)”, “AGGCAAGUGUG sequence”. ) ",” AGGGCAAGAUUGCUGGCAAUAG (SEQ ID NO: 9) "," AGGGCAAGAUUGCUGGCAAUAGCUGUU (SEQ ID NO: 10) ",” AGGCAAGAUGCUGGCAAU (SEQ ID NO: 11) ",” AGGCAAGAUGCUGGCAAU (Guide No.
  • G AAGAUGCUGGCAUAGCU SEQ ID NO: 14
  • GGCAAGAUGCUGGCAUAGCUGUU SEQ ID NO: 15
  • GGCAAGAUGCUGGCAUAGCUGU SEQ ID NO: 16
  • miR-31 inhibitor refers to miR-31 function by reducing the intracellular expression or activity of miR-31 in the cell.
  • a substance that inhibits is intended, more specifically, a substance that acts directly on miR-31 to reduce the expression level of miR-31 or its activity, or acts on a regulator upstream of miR-31, Indirectly miR-31 expression level or its activity by reducing miR-31 expression at the transcriptional level, increasing degradation of expressed miR-31, or suppressing the activity of miR-31 It is intended to reduce substances.
  • the target of the miR-31 inhibitor is preferably pre-miR-31 and mature miR-31, more preferably mature miR-31.
  • various suppliers for example, Ambion, Cosmo Bio, BizScience, etc.
  • receiving orders for the synthesis of miR-31 inhibitors are well known, and those skilled in the art can easily obtain miR-31 inhibitors. can do.
  • the miR-31 inhibitor that can be used in the present invention is not particularly limited as long as it is a substance having an activity that inhibits the function of miR-31, but it is a biomolecule (eg, nucleic acid or protein), compound, or extract from animals or plants.
  • a biomolecule eg, nucleic acid or protein
  • an antisense nucleic acid molecule having a sequence complementary to the base sequence of miR-31 an antisense nucleic acid molecule of miR-31
  • a siRNA specific to miR-31 an RNA aptamer and a ribozyme It can be.
  • the miR-31 inhibitor is a miR-31 antisense nucleic acid molecule.
  • Antisense nucleic acid molecules that can be used in the present invention are non-enzymatic nucleic acid compounds that bind to miR-31 through the interaction of RNA-RNA, RNA-DNA, or RNAPNA (protein nucleic acid) and modulate these activities.
  • the antisense nucleic acid molecule of miR-31 is complementary to a single stranded fragment of pre-miR-31 or is complementary to mature miR-31.
  • the miR-31 antisense nucleic acid molecule is preferably in the form of an anti-oligonucleotide, which can inhibit the expression of miR-31 when introduced into a cell, and is capable of inhibiting endogenous nucleases (eg, Exonuclease and endonuclease), stable in vivo, and optionally modified oligonucleotides.
  • Modified oligonucleotides include oligomeric mimics (eg, peptide nucleic acids (PNA) and immobilized nucleic acids (LNA)) that have been modified to increase the affinity of the oligonucleotide for the target and confer resistance to target sequence mismatches.
  • PNA peptide nucleic acids
  • LNA immobilized nucleic acids
  • introduction is intended to introduce a foreign gene into a cell using various techniques known in the art, and thus the cell into which the foreign gene has been introduced is transformed.
  • the antisense nucleic acid molecule of miR-31 one produced using a standard molecular biological method (for example, a chemical synthesis method or a recombinant method) or a commercially available one can be used.
  • the antisense nucleic acid molecule of miR-31 is preferably 15 to 40 bases in length, and may be a polynucleotide of 16 to 40 bases in length containing the complementary sequence of any one of SEQ ID NOs: 1 to 16.
  • MiR-31 antisense nucleic acid molecules may be provided in an expression vector for expression in cells.
  • Such expression vectors can be introduced into cells using various techniques well known in the art.
  • the present invention provides a composition (first composition) for treating colorectal cancer, which contains a miR-31 inhibitor.
  • a correlation was shown between high expression of miR-31 and colorectal cancer with KRAS and BRAF variants. This strongly suggests that miR-31 expression suppression is effective in the treatment of colorectal cancer cells in which at least one of the KRAS gene and BRAF gene is mutated.
  • the first composition is preferably used for the treatment of colorectal cancer cells in which at least one of the KRAS gene and the BRAF gene is mutated, and for the treatment of colorectal cancer cells that are resistant to treatment with an anti-EGFR antibody. Or may be used in combination therapy with anti-EGFR antibodies.
  • the first composition of the present invention may further contain an anti-EGFR antibody, and may be configured to be provided in a kit together with the anti-EGFR antibody. .
  • composition is intended to be a form in which various components are contained in one substance.
  • kit is intended to be a form in which at least one of various components is contained in another substance.
  • a composition is intended to be "a substance in which two or more components are present homogeneously as a whole and grasped as one substance", for example, a single substance containing substance A as a main component, a substance as a main component It may be a single substance containing A and substance B.
  • Such a composition may contain other components (for example, a pharmaceutically acceptable carrier) in addition to the substances A and B.
  • the composition is characterized by containing an active ingredient as substance A, and may be used alone or in combination with other substances or compositions.
  • another substance or other composition to be used in combination may be provided in the single composition of interest, and the other substance or other to be used in combination with the single composition of interest. May be provided separately as a single kit.
  • kit is intended to include packaging with containers (eg, bottles, plates, tubes, dishes, etc.) containing specific materials. Preferably, instructions for using each material are provided.
  • containers eg, bottles, plates, tubes, dishes, etc.
  • instructions for using each material are provided.
  • “comprising” is intended to mean being contained within any of the individual containers that make up the kit.
  • the kit may be a package in which a plurality of different compositions are packed together, where the form of the composition may be a form as described above, and in the case of a solution form, it is contained in a container. Also good.
  • the kit may comprise substance A and substance B mixed in the same container or in separate containers.
  • “Instructions” indicate procedures for applying each component in the kit to treatment and / or diagnosis.
  • the “instructions” may be written or printed on paper or other media, or attached to an electronic medium such as magnetic tape, computer-readable disk or tape, CD-ROM, etc. Also good.
  • the kit may also comprise a container containing a diluent, solvent, washing solution or other reagent.
  • the kit may also be equipped with the necessary equipment for application to therapy and / or diagnosis.
  • the miR-31 inhibitor is preferably an miR-31 antisense nucleic acid molecule (first polynucleotide) or an expression vector containing a first polynucleotide.
  • the first polynucleotide is a polynucleotide that hybridizes with miR-31 in an intracellular environment, has an activity of suppressing the growth of colon cancer cells, and its base sequence is that of the base sequence of miR-31.
  • it consists of a complementary sequence or a sequence in which one or several bases are substituted or deleted in the complementary sequence
  • the base sequence of miR-31 is a sequence consisting of the base sequence of SEQ ID NO: 2.
  • it is a partial sequence of SEQ ID NO: 2 and a sequence containing the base sequence of SEQ ID NO: 3.
  • the total number of substitution or deletion bases described above is preferably 3 or less, more preferably 1 or 2.
  • compositions to be introduced into a patient to treat colorectal cancer include a liposome preparation.
  • the compositions of the present invention can be administered topically directly to the cancerous tissue of the patient. In order to facilitate the presence of the miR-31 inhibitor around the cancer tissue, it is also possible to place a sustained-release preparation near the cancer tissue. Alternatively, it can be gradually and gradually administered to a cancer tissue using a pump or the like. Furthermore, even in a method of directly introducing an expression vector into cancer tissue of a cancer patient, a colon cancer cell that is a target cell is removed from the patient, the expression vector is introduced into the cell outside the patient's body, and the cell is It may be a method of returning to the body.
  • a method for treating colorectal cancer or colorectal cancer cells comprising the step of administering a miR-31 inhibitor or the first composition to a patient is also within the scope of the present invention. It can be said.
  • the administration step may be realized by directly localizing the miR-31 inhibitor or the first composition to the cancer tissue of the patient, or by arranging a sustained-release preparation in the vicinity of the cancer tissue.
  • the above-described pump or the like or an expression vector may be used.
  • a serrated lesion of the large intestine is a lesion in which the gland duct has a serrated luminal structure. Histopathologically, it is classified into three categories: hyperplastic polyp (HP), sessile serrated adenoma / polyp (SSA / P) and traditional serrated adenoma (TSA).
  • HP hyperplastic polyp
  • SSA / P sessile serrated adenoma / polyp
  • TSA traditional serrated adenoma
  • HP is often found in the left colon to rectum and is a white-toned basic polyp lesion of 5 mm or less in size.
  • HP is a lesion that is considered to maintain tissue organization and cell dynamics that are basically similar to the normal crypt of the large intestine.
  • SSA / P is frequently found in the right colon.
  • HP many exhibit sizes greater than 5 mm, and some exceed 10 mm.
  • it is recognized as a flat white-toning basic polyp lesion with slightly unclear borders, but may also have a more polypoid form. Although it consists of serrated crypts, it exhibits various structural abnormalities, and abnormal cell proliferation and maturation.
  • TSA is a serrated adenoma that exhibits a tumorous type similar to that of a normal type of adenoma and is referred to as SSA / P. It is commonly found in the left large intestine. Many exhibit lesions. HP, SSA / P and TSA may coexist within the same lesion, in which case they are referred to as a mixed polyp.
  • a polyp having a serrated gland duct structure of the large intestine was diagnosed as HP histopathologically and was considered to be a non-neoplastic lesion with no risk of canceration.
  • SSA / P lesion is a gene microsatellite instability (MSI) positive colon cancer that occurs in the right colon. It has been noted as a precursor lesion.
  • the miR-31 inhibitor is suitably used for the diagnosis of colorectal cancer.
  • the present invention provides a method for diagnosing colorectal cancer (or a method for assisting colorectal cancer diagnosis).
  • the method of the present invention comprises the steps of measuring the expression level of miR-31 in a biological sample derived from a colon cancer patient, and comparing the measured expression level with a control value. .
  • a biological sample derived from a colon cancer patient is intended to be a colon cancer tissue or a cell thereof collected from a colon cancer patient, and includes, for example, a colon cancer tissue or a cell removed during surgery.
  • the biological sample used as a control is intended to be normal colon tissue or cells thereof collected from a healthy person, or normal colon tissue or cells thereof derived from a colon cancer patient to be diagnosed.
  • a sample collected from the proximal colon may be used as the biological sample.
  • prognosis of colorectal cancer patients can be determined.
  • the expression level of miR-31 in a biological sample derived from a colorectal cancer patient, the expression level of miR-31 in a biological sample derived from a healthy subject, or a certain period of time from the colorectal cancer patient that is the subject of the diagnosis The expression level of miR-31 in the biological sample collected before, or in the normal colon tissue collected from the colon cancer patient at the same time as the collection of the colon cancer tissue from the colon cancer patient to be diagnosed
  • the expression level of miR-31 or the expression level of miR-31 in normal colon tissue collected from healthy subjects is compared.
  • the expression level of miR-31 in a biological sample derived from a colon cancer patient is significantly increased, it can be determined that the patient to be diagnosed has a poor prognosis.
  • the expression level of miR-31 in a biological sample derived from a colon cancer patient with a poor prognosis is 50% or more, preferably 80%, than the expression level of miR-31 in a biological sample derived from a healthy subject. More than that.
  • the method of the present invention it is possible to easily and reliably determine whether the prognosis after treatment (for example, after surgery) of a colorectal cancer patient is good or not. Furthermore, by applying the method of the present invention to a biological sample derived from a colorectal cancer patient before treatment (for example, before surgery), it is important to determine the treatment policy including surgery and the treatment policy after surgery. Information is provided.
  • miR-31 is significantly correlated with KRAS mutation and BRAF mutation.
  • functional mutations in KRAS and BRAF can be detected at a time, thereby making it possible to perform a companion diagnosis that predicts the effect of anti-EGFR antibody treatment.
  • the expression level of the polynucleotide may be measured using a well-known technique in the art, for example, DNA array method, Northern blot method, RT-PCR method, real-time RT-PCR method, RT-LAMP method and in situ. It is measured by a method selected from the group consisting of hybridization methods.
  • a hybridization probe (second polynucleotide) for miR-31 can be used. That is, this invention provides the composition (2nd composition) for diagnosing colon cancer containing the 2nd polynucleotide as a tool suitable for using for the said method.
  • the second polynucleotide consists of a complementary sequence of miR-31, where miR-31 is a polynucleotide consisting of a sequence consisting of the base sequence of SEQ ID NO: 2 or a partial sequence of SEQ ID NO: 2. And a polynucleotide comprising a sequence comprising the base sequence of SEQ ID NO: 3.
  • the second polynucleotide can be readily produced using standard molecular biology techniques (eg, chemical synthesis or recombinant methods).
  • colorectal cancer can be diagnosed based on the expression level of miR-31. That is, miR-31 can be used as a colorectal cancer marker.
  • miR-31 mimics are introduced into colon cancer cell lines, the cell invasion ability is increased, and high miR-31 expression is significantly higher than that of KRAS and BRAF mutations and proximal colon cancer by microRNA analysis of patient samples.
  • High expression of miR-31 is associated with poor prognosis.
  • colon cancer cells into which miR-31 or miR-31 mimics have been introduced can be used as model cells for colon cancer cells or proximal colon cancer in which at least one of the KRAS gene and BRAF gene has been mutated. Indicates that there is. That is, miR-31 or miR-31 mimetic is preferably used for the production of colon cancer model cells.
  • the present invention provides a composition (third composition) for producing colon cancer model cells, which contains miR-31 or miR-31 mimics.
  • a polynucleotide having a partially different sequence from miR-31 can be used in the present invention as a miR-31 mimetic as long as the function of miR-31 is maintained.
  • a miR-31 mimetic is one in which one or several bases are substituted or deleted in the base sequence of miR-31 and retains the function of miR-31. Can be easily constructed by those skilled in the art.
  • the total number of substitution or deletion bases described above is preferably 3 or less, more preferably 1 or 2.
  • miR-31 or miR-31 mimetic has activity to increase the cell invasion ability of colon cancer cells, and its base sequence is (i) a sequence consisting of the base sequence of SEQ ID NO: 2, (ii) sequence A sequence comprising a partial sequence of No. 2 and comprising the base sequence of SEQ ID No. 3, or (iii) a sequence obtained by substituting or deleting one or several bases in the sequence of (i) or (ii) above, It is preferable that it contains.
  • a miR-31 or miR-31 mimetic is also referred to as a third polynucleotide, and the third polynucleotide is the third composition in the form contained in the expression vector. It may be contained in the product.
  • miR-31 or miR-31 mimetic those produced using standard molecular biological techniques (for example, chemical synthesis methods or recombinant methods), or commercially available products can be used.
  • various suppliers for example, Ambion, Cosmo Bio, BizScience, etc.
  • receiving orders for the synthesis of miR-31 or miR-31 mimetics are well known, and those skilled in the art can recognize miR-31 or miR.
  • -31 mimetics are readily available.
  • a drug for treating colorectal cancer cells can be screened.
  • the drug obtained by such a screening method can be used for the treatment of colorectal cancer cells and proximal colon cancer in which at least one of the KRAS gene and the BRAF gene is mutated, and is resistant to treatment with an anti-EGFR antibody. It may be used to treat certain colon cancer cells or may be used in combination therapy with an anti-EGFR antibody.
  • the agent is preferably provided in the form of a composition or kit with the anti-EGFR antibody.
  • miR-31 mimics into colorectal cancer cell lines increased cell invasive capacity, and high miR-31 expression was significantly higher than KRAS and BRAF mutations and proximal colon cancer by microRNA analysis of patient samples That high expression of miR-31 is associated with a poor prognosis is that miR-31 in this cell is used in colon cancer cells in which at least one of the KRAS gene and BRAF gene is mutated. It is shown that a drug for treating intestinal cancer cells can be screened by using the change in the expression level of as an index.
  • colon cancer cells having a mutation in at least one of the KRAS gene and the BRAF gene are incubated with a candidate substance, and the expression level of miR-31 after incubation is lower than the expression level of miR-31 before incubation
  • a drug for treating colorectal cancer can be screened.
  • the drug obtained by such a screening method can also be used for the treatment of colon cancer cells or proximal colon cancer in which at least one of the KRAS gene and BRAF gene is mutated, and is resistant to treatment with an anti-EGFR antibody.
  • the agent is preferably provided in the form of a composition or kit with the anti-EGFR antibody.
  • U6 snRNA (RNU6B; Applied Biosystems) served as an endogenous control.
  • ⁇ Ct was calculated by subtracting the Ct value of U6 from the Ct value of the target gene.
  • RNA miR-31 expression was analyzed using TaqMan micro-RNA Assays (Applied Biosystems). Briefly, total RNA is amplified and detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using specific primers and TaqMan probe, then 5 ng of total RNA is reversed using specific stem-loop RT primer I copied it. PCR was performed 3 times using 7500 Fast Real-Time PCR System (Applied Biosystems), and SDS v1.4 software (Applied Biosystems) was used for comparative ⁇ Ct analysis. U6 was the endogenous control.
  • qRT-PCR quantitative reverse transcription polymerase chain reaction
  • Genomic DNA was extracted from FFPE tissue of colorectal cancer using QIAamp DNA FFPE Tissue Kit. Using the extracted genomic DNA, PCR and pyrosequencing targeting KRAS (codons 12 and 13), BRAF (codon 600) and PIK3CA (exons 9 and 20) were performed. MSI analysis was performed using 10 microsatellite markers (D2S123, D5S346, D17S250, BAT25, BAT26, BAT40, D18S55, D18S56, D18S67 and D18S487). MSI-high was defined as the instability at 30% or more of the marker, and MSI-low / microsatellite stability (MSS) was defined as the instability at less than 30% of the marker.
  • MSI-high was defined as the instability at 30% or more of the marker
  • MSI-low / microsatellite stability (MSS) was defined as the instability at less than 30% of the marker.
  • Nucleofector I electroporation device for DLD-1 (Lonza), HCT-116 for COLO-320-HSR and RKO or Lipofectamine 2000 (Invitrogen, by Life Technologies), HT-29, Lovo as described in the instruction manual
  • the cells were transformed with Cell Line Nucleofector Kit V (Lonza, Basel, Switzerland) using SW480.
  • miR-31 inhibitor and miR-31 mimic were mirVana (registered trademark) miRNA inhibitor (Assay ID: MH11465) and mirVana (registered trademark) miRNA mimic (Assay ID: MC11465) (both Applied Biosystems) Used in.
  • JMP JMP (Version 9) (SAS Institute, Cary, NC, USA) and theSAS software programs (Version 9.1) (SAS Institute) were used for statistical analysis.
  • a chi-square test was used to assess associations between categorical variables, and analysis of variance (ANOVA) was used to compare mean age or tumor size across quartiles of miR-31 expression levels.
  • ANOVA analysis of variance
  • survival distribution was evaluated using Kaplan-Meier method.
  • a log rank test was performed.
  • TNM stage I, IIA, IIB, IIIA, IIIB, IIIC, IV
  • miR-31 expression status gender involved (male vs female), age at diagnosis (continuous variable), tumor size (continuous variable), tumor site (proximal colon vs distal colon and Calculate the hazard rate (HR) due to rectal), tissue type (low differentiation vs. high differentiation), MSI status (MSI-high vs.
  • the high expression group of miR-31 is prominent in larger tumor size, proximal site, poorly differentiated adenocarcinoma, high stage, BRAF mutation, KRAS mutation, and microsatellite instability (all P ⁇ 0.0042) It was related.
  • miR-31 expression variables were constructed in which the Q4 case was defined as a “high expression case” and the Q1, Q2 and Q3 cases were defined as a “low expression case”.
  • the initial variables (potential predictions) included in the regression model are shown in the table, gender, age, year of diagnosis, histology, TNM stage, microsatellite stability, MLH1 methylation, tumor size, Variables of tumor site, KRAS mutation, BRAF mutation and PIK3CA mutation.
  • a variable reduction method with a threshold P 0.10 was used to select the variables of the final model.
  • the significance P value was adjusted to 0.0042 by Bonferroni correction for multiple temporary verifications.
  • CI indicates a confidence interval.
  • HCT-116 cells KRAS mutation; c.38G> A
  • RKO cells BRAF mutation
  • DLD-1 cells DLD-1 cells
  • SW480 cells SW480 cells
  • the present invention providing an excellent tool can be used in the fields of medicine and pharmacy, and can greatly contribute to the development of pharmaceuticals and biochemical reagents.

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Abstract

The present invention treats colorectal cancer by using a composition containing an miR-31 inhibitor. The present invention diagnoses on the basis of the amount of miR-31 expression in a biological sample derived from a colorectal cancer patient. The present invention prepares a colorectal-cancer cell containing miR-31 or an miR-31 mimic, and screens a drug for treating colorectal cancer by using this colorectal-cancer cell. As a result, the present invention provides a technique for treating and/or diagnosing colorectal cancer having KRAS or BRAF mutations.

Description

大腸癌を治療および／または診断するための組成物ならびにその利用Composition for treating and / or diagnosing colorectal cancer and use thereof
　本発明は、大腸癌を治療および／または診断するための技術に関するものであり、より詳細には、変異型ＫＲＡＳや変異型ＢＲＡＦを有する大腸癌を治療および／または診断するための技術に関するものである。 The present invention relates to a technique for treating and / or diagnosing colorectal cancer, and more particularly to a technique for treating and / or diagnosing colorectal cancer having mutant KRAS or mutant BRAF. is there.
　大腸癌は、欧米では癌死亡率の上位を占めており、生活の習慣の欧米化にともなって日本人にも多く発生するようになっている。大腸癌の患者の半数以上において、ＫＲＡＳ、ＢＲＡＦおよびＰＩＫ３ＣＡのいずれかの遺伝子に変異が認められる。ＫＲＡＳ、ＢＲＡＦおよびＰＩＫ３ＣＡはいずれも受容体チロシンキナーゼの下流のシグナル伝達経路における重要なエフェクタータンパク質であり、特に、ＫＲＡＳタンパク質をコードする遺伝子が野生型である転移性大腸癌（ＫＲＡＳ遺伝子野生型転移性大腸癌）の治療には、ＥＧＦレセプターに対する抗体（抗ＥＧＦＲ抗体）がよく用いられている。 Colorectal cancer occupies the top cancer mortality rate in Europe and the United States, and it is becoming more common among Japanese people due to the westernization of lifestyle habits. In more than half of patients with colorectal cancer, mutations are found in any of the KRAS, BRAF and PIK3CA genes. KRAS, BRAF and PIK3CA are all important effector proteins in the signal transduction pathway downstream of the receptor tyrosine kinase, and in particular, metastatic colon cancer (KRAS gene wild-type metastasis) in which the gene encoding the KRAS protein is a wild type. For the treatment of colorectal cancer, an antibody against the EGF receptor (anti-EGFR antibody) is often used.
　しかし、転移性大腸癌には、ＫＲＡＳ遺伝子に変異が生じているものもあり、ＫＲＡＳ遺伝子が変異型であると抗ＥＧＦＲ抗体による治療効果を期待することができない。また、ＥＧＦレセプターの下流のシグナル伝達経路における重要なエフェクタータンパク質であるＢＲＡＦやＰＩＫ３ＣＡの遺伝子に変異が生じている場合もまた、ＫＲＡＳ遺伝子が野生型であったとしても抗ＥＧＦＲ抗体による治療効果を期待することができない。 However, some metastatic colorectal cancers have mutations in the KRAS gene, and if the KRAS gene is mutated, the therapeutic effect of anti-EGFR antibody cannot be expected. In addition, when a mutation occurs in the BRAF or PIK3CA genes that are important effector proteins in the signal transduction pathway downstream of the EGF receptor, even if the KRAS gene is a wild type, anti-EGFR antibody is expected to have a therapeutic effect. Can not do it.
　ＢＲＡＦおよびＰＩＫ３ＣＡはいずれもＫＲＡＳの下流にて機能するエフェクタータンパク質であるが、ＫＲＡＳからＢＲＡＦを経るシグナル伝達経路と、ＫＲＡＳからＰＩＫ３ＣＡを経るシグナル伝達経路は互いに独立した経路である。そのために、治療標的をＥＧＦレセプターの下流のシグナル伝達経路に注目したとしても、ＫＲＡＳ、ＢＲＡＦおよびＰＩＫ３ＣＡにおける変異の有無に応じて多種多様の治療剤を開発しなければならない。そもそも、これらの遺伝子の変異を検出するには、個々にシークエンス解析を行うことが必要であるため、それぞれに応じた治療剤の開発は容易に実現し得ない。 BRAF and PIK3CA are both effector proteins that function downstream of KRAS, but the signal transduction pathway from KRAS through BRAF and the signal transduction pathway from KRAS through PIK3CA are independent from each other. Therefore, even if the therapeutic target is focused on the signal transduction pathway downstream of the EGF receptor, a wide variety of therapeutic agents must be developed depending on the presence or absence of mutations in KRAS, BRAF, and PIK3CA. In the first place, since it is necessary to perform individual sequence analysis in order to detect mutations in these genes, it is not possible to easily develop therapeutic agents for each.
　ノンコーディングＲＮＡの機能性ＲＮＡであるマイクロＲＮＡ（ｍｉＲＮＡ）は１６～２５塩基の短いＲＮＡであり、ヒトのｍｉＲＮＡだけでも千種類以上が登録されている。ｍｉＲＮＡは、相同性を有する多数の標的遺伝子のｍＲＮＡからタンパク質への翻訳を阻害することによって、種々の遺伝子の発現を抑制的に制御する。癌におけるｍｉＲＮＡの解析は、発癌のメカニズムを解明するためだけでなく、新しい癌診断法や癌治療法を開発するために注目されている。 Micro RNA (miRNA), which is a functional RNA of non-coding RNA, is a short RNA of 16 to 25 bases, and more than 1,000 types of human miRNA are registered. miRNA suppressively controls the expression of various genes by inhibiting the translation of mRNAs of many target genes having homology into proteins. Analysis of miRNA in cancer has attracted attention not only for elucidating the mechanism of carcinogenesis but also for developing new cancer diagnostic methods and cancer treatment methods.
　非特許文献１には、大腸癌細胞株における１５６個のｍｉＲＮＡの発現をリアルタイムＰＣＲによって調べ、大腸癌と非腫瘍組織との間で差示的に発現している１３個の成熟ｍｉＲＮＡを同定したことが記載されており、特に、ｍｉＲ－３１、ｍｉＲ－９６、ｍｉＲ－１３３ｂ、ｍｉＲ－１３５ｂ、ｍｉＲ－１４５、およびｍｉＲ－１８３の差異が顕著であったことや、ｍｉＲ－３１の発現レベルが大腸癌のステージＩＩとステージＩＶとの間で明らかに異なっていることが開示されている。また、非特許文献２には、大腸癌において、ＢＲＡＦ変異が抗ＥＧＦＲ抗体耐性に関連していることが記載されており、ＨＳＡ－ＭＩＲ３１－３Ｐが、野生型ＫＲＡＳ遺伝子を有する転移性大腸癌患者における抗ＥＧＦＲ抗体応答性を予測するためのツールであることが示唆されている。特許文献１、非特許文献３および４には、ｍｉＲ－３１が、乳癌および胃癌において癌抑制因子として機能することが開示されている。 In Non-Patent Document 1, the expression of 156 miRNAs in colorectal cancer cell lines was examined by real-time PCR, and 13 mature miRNAs that were differentially expressed between colorectal cancer and non-tumor tissues were identified. In particular, the difference between miR-31, miR-96, miR-133b, miR-135b, miR-145, and miR-183 was significant, and the expression level of miR-31 was It is disclosed that there is a clear difference between stage II and stage IV of colorectal cancer. Non-patent document 2 describes that BRAF mutation is associated with anti-EGFR antibody resistance in colorectal cancer, and HSA-MIR31-3P is a metastatic colorectal cancer patient having a wild-type KRAS gene. It has been suggested that it is a tool for predicting anti-EGFR antibody responsiveness. Patent Document 1 and Non-Patent Documents 3 and 4 disclose that miR-31 functions as a tumor suppressor in breast cancer and gastric cancer.
日本国特許公開公表２００９－５３１０１９号（２００９（平成２１）年９月３日公表）Japan Patent Publication Announcement 2009-531019 (Announced September 3, 2009)
　変異型ＢＲＡＦを有する大腸癌は不良な予後と相関しており、抗ＥＧＦＲ抗体に抵抗性である。非特許文献１に記載されているｍｉＲ－３１に関する知見はあくまでも、大腸癌と非腫瘍組織との間での差示的な発現であり、大腸癌における野生型ＫＲＡＳと変異型ＫＲＡＳとの間の違いを反映した結果や、大腸癌における野生型ＢＲＡＦと変異型ＢＲＡＦとの間の違いを反映した結果ではない。また、非特許文献１に、ＫＲＡＳに変異を有する大腸癌細胞株にて過剰発現しているｍｉＲＮＡの１つであるｍｉＲ－９は、ＢＲＡＦ調節についてのｍｉＲＮＡであることが推測されるが、このｍｉＲＮＡは野生型ＢＲＡＦを有する大腸癌細胞株でのみ過剰発現している旨が記載されている。このことは、ｍｉＲ－９の過剰発現が、変異型ＫＲＡＳ／野生型ＢＲＡＦの指標となり得ることを示唆しているとともに、非特許文献１にて調べられたｍｉＲＮＡの中にはＢＲＡＦにおける変異の有無に相関するｍｉＲＮＡが見出されなかったことを教示している。 Colorectal cancer with mutant BRAF correlates with poor prognosis and is resistant to anti-EGFR antibodies. The knowledge about miR-31 described in Non-Patent Document 1 is only a differential expression between colorectal cancer and non-tumor tissue, and it is between wild-type KRAS and mutant KRAS in colorectal cancer. It is not a result reflecting a difference or a result reflecting a difference between wild type BRAF and mutant BRAF in colorectal cancer. Non-patent document 1 suggests that miR-9, which is one of miRNAs overexpressed in colorectal cancer cell lines having mutations in KRAS, is a miRNA for BRAF regulation. It is described that miRNA is overexpressed only in a colon cancer cell line having wild type BRAF. This suggests that miR-9 overexpression can be an indicator of mutant KRAS / wild-type BRAF, and among the miRNAs investigated in Non-Patent Document 1, there is a mutation in BRAF. Teaches that no miRNAs were found that correlate with.
　このように、変異型ＫＲＡＳや変異型ＢＲＡＦを有する大腸癌を検出および／または治療するための技術は未だ開発されていない。 Thus, a technique for detecting and / or treating colorectal cancer having mutant KRAS or mutant BRAF has not been developed yet.
　本発明者らは、大腸癌を含む７８１例のサンプルを用いたマイクロＲＮＡ解析によって、マイクロＲＮＡ３１（ｍｉＲ－３１）の高発現がＫＲＡＳ変異およびＢＲＡＦ変異と有意に相関していること、ｍｉＲ－３１の高発現が近位結腸癌や予後不良と関連していることを見出し、さらに、ｍｉＲ－３１模倣物を大腸癌細胞株へ導入すると細胞浸潤能が上昇し、ｍｉＲ－３１に対する阻害剤（以下、ｍｉＲ－３１阻害剤）を大腸癌細胞へ投与すると細胞の増殖が抑制されることを見出し、本発明を完成するに至った。 The inventors of the present invention confirmed that high expression of microRNA31 (miR-31) is significantly correlated with KRAS mutation and BRAF mutation by microRNA analysis using 781 samples including colon cancer, miR-31. Was found to be associated with proximal colon cancer and poor prognosis. Further, when miR-31 mimics were introduced into colon cancer cell lines, the cell invasion ability increased and an inhibitor against miR-31 (hereinafter referred to as miR-31) , MiR-31 inhibitor) was administered to colorectal cancer cells, and cell growth was suppressed, and the present invention was completed.
　すなわち、本発明の第１の組成物は、大腸癌を治療するためにｍｉＲ－３１阻害剤を含有していることを特徴としている。本発明の第１の組成物は、上記ｍｉＲ－３１阻害剤として、第１のポリヌクレオチドまたは第１のポリヌクレオチドを含んでいる発現ベクターを含んでいることが好ましい。本発明の第１の組成物は、ＫＲＡＳ遺伝子およびＢＲＡＦ遺伝子の少なくとも一方に変異が生じている大腸癌細胞の治療に用いられることが好ましく、抗ＥＧＦＲ抗体による治療に耐性である大腸癌細胞または転移性大腸癌の治療に用いられても、抗ＥＧＦＲ抗体との組合せ療法に用いられてもよい。抗ＥＧＦＲ抗体との組合せ療法に用いられる場合、本発明の第１の組成物は、抗ＥＧＦＲ抗体をさらに含有していてもよく、抗ＥＧＦＲ抗体とともにキットに備えられている構成であってもよい。 That is, the first composition of the present invention is characterized by containing a miR-31 inhibitor for treating colorectal cancer. The first composition of the present invention preferably contains the first polynucleotide or an expression vector containing the first polynucleotide as the miR-31 inhibitor. The first composition of the present invention is preferably used for the treatment of colorectal cancer cells in which at least one of the KRAS gene and the BRAF gene is mutated, and the colorectal cancer cells or metastasis resistant to treatment with an anti-EGFR antibody It may be used for the treatment of invasive colorectal cancer or may be used for combination therapy with an anti-EGFR antibody. When used in combination therapy with an anti-EGFR antibody, the first composition of the present invention may further contain an anti-EGFR antibody, and may be configured to be provided in a kit together with the anti-EGFR antibody. .
　乳癌および胃癌において腫瘍抑制因子として機能することが報告されているｍｉＲ－３１について、その阻害剤によって大腸癌細胞の増殖を抑制し得たことは当業者が予測し得たものでない。すなわち、本発明の組成物が、大腸癌細胞を治療し得ることは、本発明者らの独自の観点および創意工夫によって見出された新たな知見である。 It has not been predicted by those skilled in the art that miR-31, which has been reported to function as a tumor suppressor in breast cancer and gastric cancer, could suppress colon cancer cell growth by its inhibitor. That is, the fact that the composition of the present invention can treat colorectal cancer cells is a new finding discovered by the inventors' original viewpoint and inventive idea.
　本発明の大腸癌を診断する方法は、大腸癌患者由来の生物学的サンプルにおいて、ｍｉＲ－３１の発現量を測定する工程、および、測定した上記発現量を対照値と比較する工程を包含することを特徴としている。本方法は、大腸癌の診断を補助する方法として、大腸癌を診断するためのデータを取得する方法でもあり得る。なお、上記ｍｉＲ－３１は、配列番号１～１６のいずれか１つからなるポリヌクレオチドであることが好ましい。 The method for diagnosing colorectal cancer of the present invention comprises the steps of measuring the expression level of miR-31 in a biological sample derived from a colorectal cancer patient, and comparing the measured expression level with a control value. It is characterized by that. This method can also be a method of acquiring data for diagnosing colorectal cancer as a method for assisting in the diagnosis of colorectal cancer. The miR-31 is preferably a polynucleotide consisting of any one of SEQ ID NOs: 1 to 16.
　また、本発明の方法に用いるに好適なツールとして、本発明の第２の組成物は、大腸癌を診断するために、第２のポリヌクレオチドを含有していることを特徴としている。 In addition, as a tool suitable for use in the method of the present invention, the second composition of the present invention is characterized by containing a second polynucleotide in order to diagnose colorectal cancer.
　非特許文献２には、野生型ＫＲＡＳを有する患者におけるｍｉｒ－３１－３ｐ発現が抗ＥＧＦ応答の予想因子であることや、ｍｉｒ－３１－３ｐ発現レベル、年齢、性別およびＢＲＡＦ変異状態を考慮して確立したノモグラムが、抗ＥＧＦＲ治療のために野生型ＫＲＡＳを有する患者を選択するためのツールであることが記載されている。このような知見は、ＫＲＡＳ遺伝子およびＢＲＡＦ遺伝子の少なくとも一方に変異が生じている大腸癌細胞に対してｍｉＲ－３１阻害剤が有効であることを教示も示唆もしていない。そもそも、ヘアピン構造を有する前駆体マイクロＲＮＡ分子から切り出された二本鎖のうちの一方が分解されて、他方が成熟型マイクロＲＮＡ分子として機能する。非特許文献２には、野生型ＫＲＡＳを有する患者においてｍｉｒ－３１－３ｐが機能性の成熟型マイクロＲＮＡ分子であることが強く教示されている。非特許文献２は、ｍｉｒ－３１－５ｐの関与を記載も示唆もしていないばかりか、上記教示によってｍｉｒ－３１－５ｐの関与に想到する動機付けを妨げる。すなわち、ｍｉｒ－３１－５ｐを用いることによって、大腸癌患者が、ＫＲＡＳ遺伝子およびＢＲＡＦ遺伝子の少なくとも一方に変異が生じている大腸癌細胞を有しているか否かを知ることができるということは、当業者が非特許文献２から予測し得ることでない。このように、本発明の第２の組成物が、大腸癌を診断し得ることは、本発明者らの独自の観点および創意工夫によって見出された新たな知見である。 Non-Patent Document 2 considers that mir-31-3p expression in patients with wild-type KRAS is a predictor of anti-EGF response, mir-31-3p expression level, age, sex, and BRAF mutation status The established nomogram is described as a tool for selecting patients with wild-type KRAS for anti-EGFR therapy. Such findings do not teach or suggest that miR-31 inhibitors are effective against colon cancer cells in which at least one of the KRAS gene and BRAF gene has been mutated. In the first place, one of the double strands cut out from the precursor microRNA molecule having a hairpin structure is degraded, and the other functions as a mature microRNA molecule. Non-Patent Document 2 strongly teaches that mir-31-3p is a functional mature microRNA molecule in patients with wild-type KRAS. Non-Patent Document 2 not only describes or suggests the involvement of mir-31-5p, but also prevents the motivation to arrive at the involvement of mir-31-5p by the above teaching. That is, by using mir-31-5p, it is possible to know whether a colon cancer patient has colon cancer cells in which at least one of the KRAS gene and the BRAF gene has a mutation. It cannot be predicted by those skilled in the art from Non-Patent Document 2. Thus, the fact that the second composition of the present invention can diagnose colorectal cancer is a new finding discovered by the inventors' original viewpoint and inventive idea.
　本発明の方法を用いれば、大腸癌患者に対する抗ＥＧＦＲ抗体治療についてのコンパニオン診断、または大腸癌患者の予後の判定が可能である。すなわち、本発明の方法において、上記診断は、大腸癌患者に対する抗ＥＧＦＲ抗体治療についてのコンパニオン診断と、大腸癌患者の予後の判定との少なくとも一方であることが好ましい。 Using the method of the present invention, it is possible to make a companion diagnosis for anti-EGFR antibody treatment for colorectal cancer patients or to determine the prognosis of colorectal cancer patients. That is, in the method of the present invention, the diagnosis is preferably at least one of a companion diagnosis for anti-EGFR antibody treatment for colorectal cancer patients and a prognosis determination for colorectal cancer patients.
　本発明の方法に用いられる上記対照値は、健常者由来の生物学的サンプル中のｍｉＲ－３１の発現量、または、診断の対象である大腸癌患者から一定期間前に採取した生物学的サンプル中のｍｉＲ－３１の発現量、あるいは、診断対象である大腸癌患者からの大腸癌組織の採取と同時期に当該大腸癌患者から採取した正常な大腸組織中のｍｉＲ－３１の発現量、または健常者から採取した正常な大腸組織中のｍｉＲ－３１の発現量であり得る。 The control value used in the method of the present invention is the expression level of miR-31 in a biological sample derived from a healthy subject, or a biological sample collected from a colon cancer patient to be diagnosed for a certain period of time before Expression level of miR-31 in the normal colon tissue collected from the colon cancer patient at the same time as the colon cancer tissue collection from the colon cancer patient to be diagnosed, or It can be the expression level of miR-31 in normal colon tissue collected from healthy individuals.
　本発明のスクリーニング方法は、抗ＥＧＦＲ抗体による治療に耐性である大腸癌を治療するための薬剤をスクリーニングするために、ＫＲＡＳ遺伝子およびＢＲＡＦ遺伝子の少なくとも一方に変異が生じている大腸癌細胞を候補物質とともにインキュベートする工程、および、インキュベートした後のｍｉＲ－３１の発現量をインキュベートする前のｍｉＲ－３１の発現量よりも低下させた候補物質を選択する工程を包含することを特徴としている。スクリーニングされた薬剤は、抗ＥＧＦＲ抗体との組合せ療法に用いられてもよく、転移性大腸癌が治療対象であってもよい。抗ＥＧＦＲ抗体との組合せ療法に用いられる場合、上記薬剤は、抗ＥＧＦＲ抗体とともに組成物またはキットの形態で提供されることが好ましい。 The screening method of the present invention uses a colon cancer cell having a mutation in at least one of the KRAS gene and BRAF gene as a candidate substance in order to screen a drug for treating colon cancer that is resistant to treatment with an anti-EGFR antibody. And a step of selecting a candidate substance whose miR-31 expression level after incubation is lower than the miR-31 expression level before incubation. The screened drug may be used for combination therapy with an anti-EGFR antibody, and metastatic colorectal cancer may be the subject of treatment. When used in combination therapy with an anti-EGFR antibody, the agent is preferably provided in the form of a composition or kit with the anti-EGFR antibody.
　マイクロＲＮＡは、細胞生理学の基礎研究だけでなく、疾患診断または治療予後のバイオマーカーとしても重要である。本発明を用いれば、大腸癌のステージを知ることができるだけでなく、大腸癌患者に対する抗ＥＧＦＲ抗体治療についてのコンパニオン診断が可能になる。また、ｍｉＲ－３１を導入した大腸癌細胞は、細胞浸潤能が上昇したｍｉＲ－３１高発現細胞として機能するため、本発明は、ｍｉＲ－３１高発現細胞に対する治療剤のスクリーニングに利用可能である。さらに、ｍｉＲ－３１の発現量に基づいて、ＥＧＦレセプターの下流のシグナル伝達経路におけるタンパク質の変異の有無を容易に予測することができる。 Micro RNA is important not only for basic research of cell physiology but also as a biomarker for disease diagnosis or prognosis of treatment. By using the present invention, it is possible not only to know the stage of colorectal cancer, but also to make a companion diagnosis for anti-EGFR antibody treatment for colorectal cancer patients. In addition, since colon cancer cells into which miR-31 has been introduced function as miR-31 high-expressing cells with enhanced cell invasion ability, the present invention can be used for screening therapeutic agents for miR-31 high-expressing cells. . Furthermore, based on the expression level of miR-31, the presence or absence of a protein mutation in the signal transduction pathway downstream of the EGF receptor can be easily predicted.
大腸癌におけるｍｉＲ－３１発現レベルに従う癌特異的生存（左パネル）および全体的な生存（右パネル）についてのカプランマイヤー（Kaplan-Meier）曲線を示す。ｍｉＲ－３１の高発現のケース（ケースＱ４）は、癌特異的生存（ログランクＰ＝０．００１３）および全体的な生存（ログランクＰ＝０．００２６）のいずれにおいても低発現のケースよりも顕著に高い死亡率を示した。Shown are Kaplan-Meier curves for cancer specific survival (left panel) and overall survival (right panel) according to miR-31 expression levels in colorectal cancer. The case of high expression of miR-31 (case Q4) is lower than the case of low expression in both cancer-specific survival (log rank P = 0.0014) and overall survival (log rank P = 0.026). Also showed a significantly higher mortality rate. 大腸癌細胞株へｍｉＲ－３１模倣物またはｍｉＲ－３１阻害剤をトランスフェクトした結果を示す。大腸癌細胞株（ＤＬＤ－１（ＫＲＡＳ変異；ｃ．３８Ｇ＞Ａ）およびＳＷ４８０（ＫＲＡＳ変異；ｃ．３５Ｇ＞Ｔ））にｍｉＲ－３１模倣物をトランスフェクトし、これらの細胞においてｍｉＲ－３１の発現が上方制御されることを確認した（図中Ａ）。大腸癌細胞株（ＨＴ－２９（ＢＲＡＦ変異；ｃ．１７９９Ｔ＞Ａ）、ＨＣＴ－１１６（ＫＲＡＳ変異；ｃ．３８Ｇ＞Ａ）、ＲＫＯ（ＢＲＡＦ変異；ｃ．１７９９Ｔ＞Ａ）、ＤＬＤ－１およびＳＷ４８０）にｍｉＲ－３１阻害剤をトランスフェクトし、これらの細胞においてｍｉＲ－３１の発現が下方制御されることを確認した（図中Ｂ）。The results of transfecting colon cancer cell lines with miR-31 mimetics or miR-31 inhibitors are shown. Colon cancer cell lines (DLD-1 (KRAS mutation; c.38G> A) and SW480 (KRAS mutation; c.35G> T)) were transfected with miR-31 mimics and miR-31 It was confirmed that expression was up-regulated (A in the figure). Colon cancer cell lines (HT-29 (BRAF mutation; c.1799T> A), HCT-116 (KRAS mutation; c.38G> A), RKO (BRAF mutation; c.1799T> A), DLD-1 and SW480 ) Was transfected with a miR-31 inhibitor, and it was confirmed that the expression of miR-31 was down-regulated in these cells (B in the figure). （Ａ）は、ＭＴＴ増殖アッセイにおいて、ｍｉＲ－３１阻害剤が、ＨＣＴ－１１６細胞における細胞増殖を顕著に低下させたことを示す。（Ｂ）は、マトリゲル浸潤アッセイにおいて、ＤＬＤ－１細胞における浸潤が、ｍｉＲ－３１がこの細胞株にトランスフェクトされた後に８．５倍増加したことを示す。（Ｃ）は、ウエスタンブロット解析において、ｍｉＲ－３１阻害剤によってＢＲＡＦタンパク質の発現が低下したことを示す。（Ｂ）は、大腸癌細胞株におけるｍｉＲ－３１―３ｐの発現量を示す図である。(A) shows that in the MTT proliferation assay, miR-31 inhibitors significantly reduced cell proliferation in HCT-116 cells. (B) shows that in the Matrigel invasion assay, invasion in DLD-1 cells increased 8.5-fold after miR-31 was transfected into this cell line. (C) shows that expression of BRAF protein was reduced by miR-31 inhibitor in Western blot analysis. (B) shows the expression level of miR-31-3p in colorectal cancer cell lines.
　〔１．ｍｉＲ－３１〕
　ｍｉＲＮＡは、ｓｉＲＮＡと同様に短い一本鎖のＲＮＡである。標的遺伝子ｍＲＮＡの翻訳領域と結合するｓｉＲＮＡと異なり、ｍｉＲＮＡは、標的遺伝子ｍＲＮＡの３’非翻訳領域（ＵＴＲ）と結合し、これによって発現または翻訳の抑制、あるいは標的ｍＲＮＡの分解を引き起こす。また、ｓｉＲＮＡは標的遺伝子ｍＲＮＡと完全に結合して遺伝子発現を抑制するが、ｍｉＲＮＡは標的遺伝子ｍＲＮＡと部分的にしか結合しないので、標的遺伝子ｍＲＮＡに対するミスマッチを含む不完全な相補鎖であったとしても翻訳阻害を引き起こすことができる。 [1. miR-31]
miRNA is short single-stranded RNA like siRNA. Unlike siRNAs that bind to the translation region of the target gene mRNA, miRNAs bind to the 3 ′ untranslated region (UTR) of the target gene mRNA, thereby causing suppression of expression or translation or degradation of the target mRNA. In addition, siRNA completely binds to the target gene mRNA and suppresses gene expression, but miRNA binds only partially to the target gene mRNA, so that it is an incomplete complementary strand containing a mismatch to the target gene mRNA. Can also cause translational inhibition.
　本明細書中にて使用される場合、「マイクロＲＮＡ３１（ｍｉＲ－３１）」は、前駆体マイクロＲＮＡ分子（ｐｒｅ－ｍｉＲＮＡ）であっても成熟型マイクロＲＮＡ分子であってもよい。遺伝子からｐｒｉ－ｍｉＲＮＡが転写され、次いで、ｐｒｉ－ｍｉＲＮＡからヘアピン構造を有するｐｒｅ－ｍｉＲＮＡが生成され、最終的に、Ｄｉｃｅｒ介在プロセシングによってｐｒｅ－ｍｉＲＮＡから一本鎖ＲＮＡとして成熟型ｍｉＲＮＡが生成される。成熟型ｍｉＲＮＡ分子は主に一本鎖として存在するが、ｐｒｅ－ｍｉＲＮＡ分子は二本鎖構造を形成し得る、少なくとも部分的に自己相補的な構造である。本明細書中にて使用される場合、用語「相補的」は、一方のヌクレオチド配列の塩基と他方のヌクレオチド配列の塩基とが互いに結合して塩基対を形成し得る状態が意図される。 As used herein, “microRNA 31 (miR-31)” may be a precursor microRNA molecule (pre-miRNA) or a mature microRNA molecule. The pri-miRNA is transcribed from the gene, then a pre-miRNA having a hairpin structure is generated from the pri-miRNA, and finally a mature miRNA is generated from the pre-miRNA as a single-stranded RNA by Dicer-mediated processing. . While mature miRNA molecules exist primarily as single strands, pre-miRNA molecules are at least partially self-complementary structures that can form double-stranded structures. As used herein, the term “complementary” intends a condition in which the base of one nucleotide sequence and the base of the other nucleotide sequence can bind to each other to form a base pair.
　ｍｉＲ－３１の前駆体分子（ｐｒｅ－ｍｉＲＮＡ３１）の塩基配列は、「ＧＧＡＧＡＧＧＡＧＧＣＡＡＧＡＵＧＣＵＧＧＣＡＵＡＧＣＵＧＵＵＧＡＡＣＵＧＧＧＡＡＣＣＵＧＣＵＡＵＧＣＣＡＡＣＡＵＡＵＵＧＣＣＡＵＣＵＵＵＣＣ（配列番号１）」である。本明細書中にて使用される場合、特に言及されない限り、成熟型ｍｉＲＮＡ３１はｍｉＲ－３１－５ｐが意図され、その塩基配列は、「ＧＡＧＧＣＡＡＧＡＵＧＣＵＧＧＣＡＵＡＧＣＵＧＵＵ（配列番号２）」またはそのフラグメントでありかつ「ＧＧＣＡＡＧＡＵＧＣＵＧＧＣＡＵ（配列番号３）」を含むものが意図され、「ＡＧＧＣＡＡＧＡＵＧＣＵＧＧＣＡＵＡＧＣＵ（配列番号７）」を含むものが好ましい。成熟型ｍｉＲＮＡ３１としては、例えば、「ＧＡＧＧＣＡＡＧＡＵＧＣＵＧＧＣＡＵＡＧＣＵ（配列番号４）」、「ＡＧＧＣＡＡＧＡＵＧＣＵＧＧＣＡＵＡＧＣＵＧ（配列番号５）」、「ＡＧＧＣＡＡＧＡＵＧＣＵＧＧＣＡＵＡＧＣＵＧＵ（配列番号６）」、「ＡＧＧＣＡＡＧＡＵＧＣＵＧＧＣＡＵＡＧＣＵ（配列番号７）」、「ＡＧＧＣＡＡＧＡＵＧＣＵＧＧＣＡＵＡＧＣ（配列番号８）」、「ＡＧＧＣＡＡＧＡＵＧＣＵＧＧＣＡＵＡＧ（配列番号９）」、「ＡＧＧＣＡＡＧＡＵＧＣＵＧＧＣＡＵＡＧＣＵＧＵＵ（配列番号１０）」、「ＡＧＧＣＡＡＧＡＵＧＣＵＧＧＣＡＵ（配列番号１１）」、「ＡＧＧＣＡＡＧＡＵＧＣＵＧＧＣＡＵＡ（配列番号１２）」、「ＧＧＣＡＡＧＡＵＧＣＵＧＧＣＡＵＡＧＣＵＧ（配列番号１３）」、「ＧＧＣＡＡＧＡＵＧＣＵＧＧＣＡＵＡＧＣＵ（配列番号１４）」、「ＧＧＣＡＡＧＡＵＧＣＵＧＧＣＡＵＡＧＣＵＧＵＵ（配列番号１５）」、「ＧＧＣＡＡＧＡＵＧＣＵＧＧＣＡＵＡＧＣＵＧＵ（配列番号１６）」等が挙げられ、配列番号２、４～７および９からなる群より選択されるものが好ましい。 The base sequence of the precursor molecule (pre-miRNA31) of miR-31 is “GGAGAGGAGGCAAGAUUGCUGGCAAUAGCUGUUGAACUGGGGAACCUGCUAUUGCCAACAUAUUGCCAUCUUCC (SEQ ID NO: 1)”. As used herein, unless otherwise stated, mature miRNA 31 is intended to be miR-31-5p, the base sequence thereof is “GAGGCAAGAUUGCUGGCAAUAGCUGUU (SEQ ID NO: 2)” or a fragment thereof and “GGCAAGAUUGCUGCGCAU” (SEQ ID NO: 3) "is intended, and those containing" AGGCAAGAUUGCUGGCAAUAGCU (SEQ ID NO: 7) "are preferred. Examples of the mature miRNA 31 include “GAGGCAAGAUUGCUGGCAAUAGCU (SEQ ID NO: 4)”, “AGGCAAGAUUGCUGGCAAUAGCUUG (SEQ ID NO: 5)”, “AGGCAAGUGCUGGCAUGAGUGUG (SEQ ID NO: 6)”, “AGGCAAGUGUG sequence”. ) "," AGGGCAAGAUUGCUGGCAAUAG (SEQ ID NO: 9) "," AGGGCAAGAUUGCUGGCAAUAGCUGUU (SEQ ID NO: 10) "," AGGCAAGAUGCUGGCAAU (SEQ ID NO: 11) "," AGGCAAGAUGCUGGCAAU (Guide No. 13 CA, G AAGAUGCUGGCAUAGCU (SEQ ID NO: 14), "" GGCAAGAUGCUGGCAUAGCUGUU (SEQ ID NO: 15) ", include such as" GGCAAGAUGCUGGCAUAGCUGU (SEQ ID NO: 16) ", those selected from the group consisting of SEQ ID NO: 2, 4 to 7 and 9 are preferred.
　〔２．ｍｉＲ－３１阻害剤〕
　本発明中にて使用される場合、用語「マイクロＲＮＡ３１（ｍｉＲ－３１）阻害剤」は、細胞内にてｍｉＲ－３１の細胞内での発現または活性を低減させることによってｍｉＲ－３１の機能を阻害する物質が意図され、より詳細には、ｍｉＲ－３１に直接的に作用してｍｉＲ－３１の発現レベルまたはその活性を低減させる物質、あるいはｍｉＲ－３１の上流の調節因子に作用して、ｍｉＲ－３１の発現を転写レベルで低減させるか、発現されたｍｉＲ－３１の分解を増加させるか、あるいはｍｉＲ－３１の活性を抑制することによって、ｍｉＲ－３１の発現レベルまたはその活性を間接的に低減させる物質が意図される。ｍｉＲ－３１阻害剤の標的は、好ましくはｐｒｅ－ｍｉＲ－３１および成熟型ｍｉＲ－３１であり、より好ましくは成熟型ｍｉＲ－３１である。なお、ｍｉＲ－３１阻害剤の合成を受注する種々の供給業者（例えば、Ａｍｂｉｏｎ社、コスモ・バイオ社、ＢｉｚＳｃｉｅｎｃｅ社等）がよく知られており、当業者はｍｉＲ－３１阻害剤を容易に入手することができる。 [2. miR-31 inhibitor]
As used herein, the term “microRNA31 (miR-31) inhibitor” refers to miR-31 function by reducing the intracellular expression or activity of miR-31 in the cell. A substance that inhibits is intended, more specifically, a substance that acts directly on miR-31 to reduce the expression level of miR-31 or its activity, or acts on a regulator upstream of miR-31, Indirectly miR-31 expression level or its activity by reducing miR-31 expression at the transcriptional level, increasing degradation of expressed miR-31, or suppressing the activity of miR-31 It is intended to reduce substances. The target of the miR-31 inhibitor is preferably pre-miR-31 and mature miR-31, more preferably mature miR-31. In addition, various suppliers (for example, Ambion, Cosmo Bio, BizScience, etc.) receiving orders for the synthesis of miR-31 inhibitors are well known, and those skilled in the art can easily obtain miR-31 inhibitors. can do.
　本発明に利用可能なｍｉＲ－３１阻害剤は、ｍｉＲ－３１の機能を阻害する活性を有する物質であれば特に限定されないが、生体分子（例えば、核酸またはタンパク質）、化合物、動植物からの抽出物であってもよく、好ましくは、ｍｉＲ－３１の塩基配列に相補的な配列を有するアンチセンス核酸分子（ｍｉＲ－３１のアンチセンス核酸分子）、ｍｉＲ－３１に特異的なｓｉＲＮＡ、ＲＮＡアプタマーおよびリボザイムであり得る。 The miR-31 inhibitor that can be used in the present invention is not particularly limited as long as it is a substance having an activity that inhibits the function of miR-31, but it is a biomolecule (eg, nucleic acid or protein), compound, or extract from animals or plants. Preferably, an antisense nucleic acid molecule having a sequence complementary to the base sequence of miR-31 (an antisense nucleic acid molecule of miR-31), a siRNA specific to miR-31, an RNA aptamer and a ribozyme It can be.
　一実施形態において、ｍｉＲ－３１阻害剤はｍｉＲ－３１のアンチセンス核酸分子である。本発明に利用可能なアンチセンス核酸分子は、ＲＮＡ－ＲＮＡ、ＲＮＡ－ＤＮＡまたはＲＮＡＰＮＡ（タンパク質核酸）の相互作用によってｍｉＲ－３１に結合してこれらの活性を調節する非酵素的な核酸化合物である。ｍｉＲ－３１のアンチセンス核酸分子は、ｐｒｅ－ｍｉＲ－３１の一本鎖断片に相補的であるか、あるいは成熟型ｍｉＲ－３１に相補的である。 In one embodiment, the miR-31 inhibitor is a miR-31 antisense nucleic acid molecule. Antisense nucleic acid molecules that can be used in the present invention are non-enzymatic nucleic acid compounds that bind to miR-31 through the interaction of RNA-RNA, RNA-DNA, or RNAPNA (protein nucleic acid) and modulate these activities. . The antisense nucleic acid molecule of miR-31 is complementary to a single stranded fragment of pre-miR-31 or is complementary to mature miR-31.
　また、ｍｉＲ－３１のアンチセンス核酸分子は、アンチオリゴヌクレオチド形態であることが好ましく、これは、細胞に導入された際にｍｉＲ－３１の発現を阻害させることができ、内因性ヌクレアーゼ（例えば、エキソヌクレアーゼおよびエンドヌクレアーゼ）に耐性であり、生体内にて安定的であり、必要に応じて変形されたオリゴヌクレオチドである。変形されたオリゴヌクレオチドとしては、標的に対するオリゴヌクレオチドの親和性を増加させ、標的配列のミスマッチに対する耐性を付与するように変形されたオリゴマー模倣物（例えばペプチド核酸（ＰＮＡ）および固定核酸（ＬＮＡ））が挙げられるがこれに限定されない。なお、用語「導入」は、当該分野において公知の種々の手法を用いて外来遺伝子を細胞内へ入れることが意図され、これにより、外来遺伝子を導入された細胞は形質転換される。 Also, the miR-31 antisense nucleic acid molecule is preferably in the form of an anti-oligonucleotide, which can inhibit the expression of miR-31 when introduced into a cell, and is capable of inhibiting endogenous nucleases (eg, Exonuclease and endonuclease), stable in vivo, and optionally modified oligonucleotides. Modified oligonucleotides include oligomeric mimics (eg, peptide nucleic acids (PNA) and immobilized nucleic acids (LNA)) that have been modified to increase the affinity of the oligonucleotide for the target and confer resistance to target sequence mismatches. However, it is not limited to this. Note that the term “introduction” is intended to introduce a foreign gene into a cell using various techniques known in the art, and thus the cell into which the foreign gene has been introduced is transformed.
　ｍｉＲ－３１のアンチセンス核酸分子は、標準的な分子生物学的手法（例えば化学合成法または組換え法）を用いて生成したもの、あるいは市販のものを使用することができる。ｍｉＲ－３１のアンチセンス核酸分子は、１５～４０塩基長であることが好ましく、配列番号１～１６のいずれか１つの相補配列を含む１６～４０塩基長のポリヌクレオチドであってもよい。 As the antisense nucleic acid molecule of miR-31, one produced using a standard molecular biological method (for example, a chemical synthesis method or a recombinant method) or a commercially available one can be used. The antisense nucleic acid molecule of miR-31 is preferably 15 to 40 bases in length, and may be a polynucleotide of 16 to 40 bases in length containing the complementary sequence of any one of SEQ ID NOs: 1 to 16.
　ｍｉＲ－３１のアンチセンス核酸分子は、細胞内にて発現させるための発現ベクターに含まれて提供されてもよい。このような発現ベクターは、当該分野において周知の種々の手法を用いて細胞に導入され得る。 MiR-31 antisense nucleic acid molecules may be provided in an expression vector for expression in cells. Such expression vectors can be introduced into cells using various techniques well known in the art.
　〔３．大腸癌の治療〕
　ｍｉＲ－３１阻害剤を大腸癌細胞へ投与すると細胞の増殖が抑制された。すなわち、ｍｉＲ－３１阻害剤は大腸癌の治療に好適に用いられる。 [3. Treatment of colorectal cancer
When a miR-31 inhibitor was administered to colon cancer cells, cell proliferation was suppressed. That is, the miR-31 inhibitor is suitably used for the treatment of colorectal cancer.
　本発明は、ｍｉＲ－３１阻害剤を含有している、大腸癌を治療するための組成物（第１の組成物）を提供する。ｍｉＲ－３１の高発現とＫＲＡＳおよびＢＲＡＦの変異型を有する大腸癌との間に相関性が示された。このことは、ｍｉＲ－３１の発現抑制は、ＫＲＡＳ遺伝子およびＢＲＡＦ遺伝子の少なくとも一方に変異が生じている大腸癌細胞の治療に有効であることを強く示唆する。すなわち、第１の組成物は、ＫＲＡＳ遺伝子およびＢＲＡＦ遺伝子の少なくとも一方に変異が生じている大腸癌細胞の治療に用いられることが好ましく、抗ＥＧＦＲ抗体による治療に耐性である大腸癌細胞の治療に用いられても、抗ＥＧＦＲ抗体との組合せ療法に用いられてもよい。抗ＥＧＦＲ抗体との組合せ療法に用いられる場合、本発明の第１の組成物は、抗ＥＧＦＲ抗体をさらに含有していてもよく、抗ＥＧＦＲ抗体とともにキットに備えられている構成であってもよい。 The present invention provides a composition (first composition) for treating colorectal cancer, which contains a miR-31 inhibitor. A correlation was shown between high expression of miR-31 and colorectal cancer with KRAS and BRAF variants. This strongly suggests that miR-31 expression suppression is effective in the treatment of colorectal cancer cells in which at least one of the KRAS gene and BRAF gene is mutated. That is, the first composition is preferably used for the treatment of colorectal cancer cells in which at least one of the KRAS gene and the BRAF gene is mutated, and for the treatment of colorectal cancer cells that are resistant to treatment with an anti-EGFR antibody. Or may be used in combination therapy with anti-EGFR antibodies. When used in combination therapy with an anti-EGFR antibody, the first composition of the present invention may further contain an anti-EGFR antibody, and may be configured to be provided in a kit together with the anti-EGFR antibody. .
　本明細書中にて使用される場合、「組成物」は各種成分が一物質中に含有されている形態であることが意図される。また、本明細書中にて使用される場合、「キット」は各種成分の少なくとも１つが別物質中に含有されている形態であることが意図される。 As used herein, “composition” is intended to be a form in which various components are contained in one substance. Further, as used herein, a “kit” is intended to be a form in which at least one of various components is contained in another substance.
　一般に、組成物は「二種以上の成分が全体として均質に存在し、一物質として把握されるもの」が意図され、例えば、物質Ａを主成分として含有する単一物、主成分としての物質Ａと物質Ｂとを含有する単一物であり得る。このような組成物は、物質Ａおよび物質Ｂ以外に他の成分（例えば、薬学的に受容可能なキャリア）を含有してもよい。組成物は、有効成分を物質Ａとして含有していることを特徴としており、単独で使用されても、他の物質または組成物と併用されてもよい。この場合、目的の単一の組成物中に、併用されるべき他の物質または他の組成物が提供されてもよく、目的の単一の組成物と、併用されるべき他の物質または他の組成物とが別々に単一のキットとして提供されてもよい。 In general, a composition is intended to be "a substance in which two or more components are present homogeneously as a whole and grasped as one substance", for example, a single substance containing substance A as a main component, a substance as a main component It may be a single substance containing A and substance B. Such a composition may contain other components (for example, a pharmaceutically acceptable carrier) in addition to the substances A and B. The composition is characterized by containing an active ingredient as substance A, and may be used alone or in combination with other substances or compositions. In this case, another substance or other composition to be used in combination may be provided in the single composition of interest, and the other substance or other to be used in combination with the single composition of interest. May be provided separately as a single kit.
　本明細書中にて使用される場合、用語「キット」は、特定の材料を内包する容器（例えば、ボトル、プレート、チューブ、ディッシュなど）を備えた包装が意図される。好ましくは各材料を使用するための指示書を備える。本明細書中にてキットの局面において使用される場合、「備えた（備えている）」は、キットを構成する個々の容器のいずれかの中に内包されている状態が意図される。また、キットは、複数の異なる組成物を１つに梱包した包装であり得、ここで、組成物の形態は上述したような形態であり得、溶液形態の場合は容器中に内包されていてもよい。キットは、物質Ａおよび物質Ｂを同一の容器に混合して備えていても別々の容器に備えていてもよい。「指示書」には、キット中の各構成を、治療および／または診断に適用する手順が示されている。なお、「指示書」は、紙またはその他の媒体に書かれていても印刷されていてもよく、あるいは磁気テープ、コンピューター読み取り可能ディスクまたはテープ、ＣＤ－ＲＯＭなどのような電子媒体に付されてもよい。キットはまた、希釈剤、溶媒、洗浄液またはその他の試薬を内包した容器を備え得る。さらに、キットは、治療および／または診断に適用するために必要な器具をあわせて備えていてもよい。 DETAILED DESCRIPTION As used herein, the term “kit” is intended to include packaging with containers (eg, bottles, plates, tubes, dishes, etc.) containing specific materials. Preferably, instructions for using each material are provided. As used herein in the context of a kit, “comprising” is intended to mean being contained within any of the individual containers that make up the kit. In addition, the kit may be a package in which a plurality of different compositions are packed together, where the form of the composition may be a form as described above, and in the case of a solution form, it is contained in a container. Also good. The kit may comprise substance A and substance B mixed in the same container or in separate containers. “Instructions” indicate procedures for applying each component in the kit to treatment and / or diagnosis. The “instructions” may be written or printed on paper or other media, or attached to an electronic medium such as magnetic tape, computer-readable disk or tape, CD-ROM, etc. Also good. The kit may also comprise a container containing a diluent, solvent, washing solution or other reagent. Furthermore, the kit may also be equipped with the necessary equipment for application to therapy and / or diagnosis.
　一実施形態において、上記ｍｉＲ－３１阻害剤は、ｍｉＲ－３１のアンチセンス核酸分子（第１のポリヌクレオチド）または第１のポリヌクレオチドを含んでいる発現ベクターであることが好ましい。第１のポリヌクレオチドは、ｍｉＲ－３１と細胞内環境下にてハイブリダイズするポリヌクレオチドであり、大腸癌細胞の増殖を抑制する活性を有し、その塩基配列が、ｍｉＲ－３１の塩基配列の相補配列からなるか、または当該相補配列において１または数個の塩基が置換または欠失してなる配列からなることが好ましく、ｍｉＲ－３１の塩基配列は、配列番号２の塩基配列からなる配列であるか、または配列番号２の部分配列でありかつ配列番号３の塩基配列を含む配列であることが好ましい。上述した置換または欠失の塩基数は、合計が３個以下であることが好ましく、１個または２個であることがより好ましい。 In one embodiment, the miR-31 inhibitor is preferably an miR-31 antisense nucleic acid molecule (first polynucleotide) or an expression vector containing a first polynucleotide. The first polynucleotide is a polynucleotide that hybridizes with miR-31 in an intracellular environment, has an activity of suppressing the growth of colon cancer cells, and its base sequence is that of the base sequence of miR-31. Preferably, it consists of a complementary sequence or a sequence in which one or several bases are substituted or deleted in the complementary sequence, and the base sequence of miR-31 is a sequence consisting of the base sequence of SEQ ID NO: 2. Preferably, it is a partial sequence of SEQ ID NO: 2 and a sequence containing the base sequence of SEQ ID NO: 3. The total number of substitution or deletion bases described above is preferably 3 or less, more preferably 1 or 2.
　大腸癌を治療するために患者へ導入する組成物の形態としては、例えばリポソーム製剤が挙げられる。本発明の組成物は、患者の癌組織へ直接局所投与することができる。また、癌組織の周囲へｍｉＲ－３１阻害剤を存在しやすくするために、徐放性製剤を癌組織近傍へ配置することも可能である。あるいは、ポンプ等を用いて癌組織へ連続的に徐々に投与することも可能である。さらには、発現ベクターを直接癌患者の癌組織に導入する方法であっても、標的細胞である大腸癌細胞を患者から取り出し、患者の体外にて発現ベクターを該細胞に導入し、その細胞を体内へ戻す方法であってもよい。
　このように、大腸癌の治療の観点から、ｍｉＲ－３１阻害剤または第１の組成物を患者へ投与する工程を包含する大腸癌または大腸癌細胞を治療する方法もまた本発明の範囲であるといえる。この場合、上記投与する工程は、ｍｉＲ－３１阻害剤または第１の組成物を患者の癌組織へ直接局所することや、徐放性製剤を癌組織近傍へ配置することによって実現されてもよく、上述したポンプ等または発現ベクターを用いて実現されてもよい。 Examples of the form of the composition to be introduced into a patient to treat colorectal cancer include a liposome preparation. The compositions of the present invention can be administered topically directly to the cancerous tissue of the patient. In order to facilitate the presence of the miR-31 inhibitor around the cancer tissue, it is also possible to place a sustained-release preparation near the cancer tissue. Alternatively, it can be gradually and gradually administered to a cancer tissue using a pump or the like. Furthermore, even in a method of directly introducing an expression vector into cancer tissue of a cancer patient, a colon cancer cell that is a target cell is removed from the patient, the expression vector is introduced into the cell outside the patient's body, and the cell is It may be a method of returning to the body.
Thus, from the viewpoint of treatment of colorectal cancer, a method for treating colorectal cancer or colorectal cancer cells comprising the step of administering a miR-31 inhibitor or the first composition to a patient is also within the scope of the present invention. It can be said. In this case, the administration step may be realized by directly localizing the miR-31 inhibitor or the first composition to the cancer tissue of the patient, or by arranging a sustained-release preparation in the vicinity of the cancer tissue. The above-described pump or the like or an expression vector may be used.
　〔４．大腸癌の診断〕
　大腸の鋸歯状病変は、腺管が鋸歯状の管腔構造を呈する病変である。病理組織学的に、過形成性ポリープ（hyperplastic polyp；ＨＰ）、sessile serrated adenoma/polyp（ＳＳＡ／Ｐ）および鋸歯状腺腫（traditional serrated adenoma；ＴＳＡ）の３つのカテゴリーに分類されている。 [4. Diagnosis of colorectal cancer)
A serrated lesion of the large intestine is a lesion in which the gland duct has a serrated luminal structure. Histopathologically, it is classified into three categories: hyperplastic polyp (HP), sessile serrated adenoma / polyp (SSA / P) and traditional serrated adenoma (TSA).
　ＨＰは、左側結腸～直腸に認められることが多く、大きさ５ｍｍ以下の白色調広基性ポリープ病変である。ＨＰは、基本的に大腸の正常陰窩と類似した組織構築および細胞動態が保たれているとみなされる病変である。ＳＳＡ／Ｐは、右側結腸に多く認められる。ＨＰと異なり、多くが５ｍｍを超える大きさを呈し、１０ｍｍを超えるものもみられる。典型的には、やや境界不明瞭な平坦な白色調広基性ポリープ病変として認められるが、よりpolypoidな形態を呈する場合もある。鋸歯状陰窩からなるが、種々の構造異常を呈し、また、細胞の増殖や成熟異常が認められる。ＴＳＡは、通常型の腺腫と類似した腫瘍性異型を呈する鋸歯状腺腫をＳＳＡ／Ｐと区別して称され、左側大腸に好発し、５ｍｍ～１０ｍｍ大の、有茎性を含んだ***の強いポリープ病変を呈するものが多い。ＨＰ、ＳＳＡ／ＰおよびＴＳＡは、同一病変内に併存することがあり、その場合は混合ポリープ（mixed polyp）と称される。 HP is often found in the left colon to rectum and is a white-toned basic polyp lesion of 5 mm or less in size. HP is a lesion that is considered to maintain tissue organization and cell dynamics that are basically similar to the normal crypt of the large intestine. SSA / P is frequently found in the right colon. Unlike HP, many exhibit sizes greater than 5 mm, and some exceed 10 mm. Typically, it is recognized as a flat white-toning basic polyp lesion with slightly unclear borders, but may also have a more polypoid form. Although it consists of serrated crypts, it exhibits various structural abnormalities, and abnormal cell proliferation and maturation. TSA is a serrated adenoma that exhibits a tumorous type similar to that of a normal type of adenoma and is referred to as SSA / P. It is commonly found in the left large intestine. Many exhibit lesions. HP, SSA / P and TSA may coexist within the same lesion, in which case they are referred to as a mixed polyp.
　従来、大腸の鋸歯状腺管構造を有するポリープは、病理組織学的にＨＰと診断され、非腫瘍性病変で癌化の危険性はない病変であると考えられていた。しかし、近年、ＨＰやＳＳＡ／Ｐを介した新しい大腸癌の発癌経路が提唱されるようになり、特にＳＳＡ／Ｐ病変は右側結腸に発生する遺伝子マイクロサテライト不安定性（ＭＳＩ）陽性の大腸癌の前駆病変とされ注目されている。 Conventionally, a polyp having a serrated gland duct structure of the large intestine was diagnosed as HP histopathologically and was considered to be a non-neoplastic lesion with no risk of canceration. However, in recent years, a new carcinogenic pathway for colorectal cancer via HP and SSA / P has been proposed, and in particular, SSA / P lesion is a gene microsatellite instability (MSI) positive colon cancer that occurs in the right colon. It has been noted as a precursor lesion.
　ｍｉＲ－３１の高発現と大腸の組織型との間に、継続した顕著な関連性が示された。すなわち、ｍｉＲ－３１阻害剤は大腸癌の診断に好適に用いられる。 There was a continuous and significant relationship between the high expression of miR-31 and the colon histology. That is, the miR-31 inhibitor is suitably used for the diagnosis of colorectal cancer.
　本発明は、大腸癌を診断する方法（あるいは大腸癌の診断を補助する方法）を提供する。本発明の方法は、大腸癌患者由来の生物学的サンプルにおいて、ｍｉＲ－３１の発現量を測定する工程、および、測定した上記発現量を対照値と比較する工程を包含することを特徴としている。 The present invention provides a method for diagnosing colorectal cancer (or a method for assisting colorectal cancer diagnosis). The method of the present invention comprises the steps of measuring the expression level of miR-31 in a biological sample derived from a colon cancer patient, and comparing the measured expression level with a control value. .
　大腸癌患者由来の生物学的サンプルは、大腸癌患者から採取した大腸癌組織またはその細胞が意図され、例えば手術中に摘出される大腸癌の組織またはその細胞が挙げられる。対照として用いられる生物学的サンプルは、健常者から採取した正常な大腸組織またはその細胞、あるいは、診断の対象である大腸癌患者由来の正常な大腸組織またはその細胞が意図される。また、生物学的サンプルとしては、近位結腸から採取したサンプルが用いられてもよい。 A biological sample derived from a colon cancer patient is intended to be a colon cancer tissue or a cell thereof collected from a colon cancer patient, and includes, for example, a colon cancer tissue or a cell removed during surgery. The biological sample used as a control is intended to be normal colon tissue or cells thereof collected from a healthy person, or normal colon tissue or cells thereof derived from a colon cancer patient to be diagnosed. In addition, a sample collected from the proximal colon may be used as the biological sample.
　後述する実施例にて、ｍｉＲ－３１の高発現が近位結腸癌や予後不良と関連していることを示している。このように、ｍｉＲ－３１発現の上昇を検出することにより、大腸癌患者の予後の判定を行うことができる。大腸癌患者由来の生物学的サンプル中のｍｉＲ－３１の発現量を、健常者由来の生物学的サンプル中のｍｉＲ－３１の発現量、あるいは、上記診断の対象である大腸癌患者から一定期間前に採取した生物学的サンプル中のｍｉＲ－３１の発現量、あるいは、診断対象である大腸癌患者からの大腸癌組織の採取と同時期に当該大腸癌患者から採取した正常な大腸組織中のｍｉＲ－３１の発現量、または健常者から採取した正常な大腸組織中のｍｉＲ－３１の発現量と比較する。比較することによって、大腸癌患者由来の生物学的サンプル中のｍｉＲ－３１の発現量が有意に上昇している場合に、診断対象の患者が予後不良であると判定することができる。例えば、予後不良な大腸癌患者由来の生物学的サンプル中のｍｉＲ－３１の発現量は、健常者由来の生物学的サンプル中のｍｉＲ－３１の発現量よりも５０％以上、好ましくは８０％以上上昇している。 In the examples described below, it is shown that high expression of miR-31 is associated with proximal colon cancer and poor prognosis. Thus, by detecting an increase in miR-31 expression, prognosis of colorectal cancer patients can be determined. The expression level of miR-31 in a biological sample derived from a colorectal cancer patient, the expression level of miR-31 in a biological sample derived from a healthy subject, or a certain period of time from the colorectal cancer patient that is the subject of the diagnosis The expression level of miR-31 in the biological sample collected before, or in the normal colon tissue collected from the colon cancer patient at the same time as the collection of the colon cancer tissue from the colon cancer patient to be diagnosed The expression level of miR-31 or the expression level of miR-31 in normal colon tissue collected from healthy subjects is compared. By comparison, when the expression level of miR-31 in a biological sample derived from a colon cancer patient is significantly increased, it can be determined that the patient to be diagnosed has a poor prognosis. For example, the expression level of miR-31 in a biological sample derived from a colon cancer patient with a poor prognosis is 50% or more, preferably 80%, than the expression level of miR-31 in a biological sample derived from a healthy subject. More than that.
　本発明の方法によれば、大腸癌患者の治療後（例えば、手術後）の予後が良好か否か、再発がないか否かを、簡単にかつ高い信頼性をもって判断することができる。さらに、治療前（例えば、手術前）の大腸癌患者由来の生物学的サンプルに対して本発明の方法を適用することで、手術を含む治療そのものの方針、手術後の治療方針の決定に対する重要な情報が提供される。 According to the method of the present invention, it is possible to easily and reliably determine whether the prognosis after treatment (for example, after surgery) of a colorectal cancer patient is good or not. Furthermore, by applying the method of the present invention to a biological sample derived from a colorectal cancer patient before treatment (for example, before surgery), it is important to determine the treatment policy including surgery and the treatment policy after surgery. Information is provided.
　また、後述する実施例にて、ｍｉＲ－３１の高発現がＫＲＡＳ変異およびＢＲＡＦ変異と有意に相関していることを示している。このように、ｍｉＲ－３１発現の上昇を検出することにより、ＫＲＡＳおよびＢＲＡＦの機能的変異を一度に検出することができ、これにより抗ＥＧＦＲ抗体治療の効果を予測するコンパニオン診断を行うことができる。 In the examples described later, it is shown that high expression of miR-31 is significantly correlated with KRAS mutation and BRAF mutation. Thus, by detecting an increase in miR-31 expression, functional mutations in KRAS and BRAF can be detected at a time, thereby making it possible to perform a companion diagnosis that predicts the effect of anti-EGFR antibody treatment. .
　ポリヌクレオチドの発現量は、当該分野における周知の手法を用いて測定されればよく、例えば、ＤＮＡアレイ法、ノーザンブロット法、ＲＴ－ＰＣＲ法、リアルタイムＲＴ－ＰＣＲ法、ＲＴ－ＬＡＭＰ法およびｉｎ　ｓｉｔｕハイブリダイゼーション法からなる群より選択される方法によって測定される。 The expression level of the polynucleotide may be measured using a well-known technique in the art, for example, DNA array method, Northern blot method, RT-PCR method, real-time RT-PCR method, RT-LAMP method and in situ. It is measured by a method selected from the group consisting of hybridization methods.
　ハイブリダイゼーション法が採用される場合、ｍｉＲ－３１に対するハイブリダイゼーションプローブ（第２のポリヌクレオチド）が利用可能である。すなわち、本発明は、上記方法に用いるに好適なツールとして、第２のポリヌクレオチドを含有している、大腸癌を診断するための組成物（第２の組成物）を提供する。第２のポリヌクレオチドは、ｍｉＲ－３１の相補配列からなり、ここで、ｍｉＲ－３１は、配列番号２の塩基配列からなる配列からなるポリヌクレオチドであるか、または配列番号２の部分配列でありかつ配列番号３の塩基配列を含む配列からなるポリヌクレオチドである。第２のポリヌクレオチドは、標準的な分子生物学的手法（例えば化学合成法または組換え法）を用いて容易に生成することができる。 When the hybridization method is employed, a hybridization probe (second polynucleotide) for miR-31 can be used. That is, this invention provides the composition (2nd composition) for diagnosing colon cancer containing the 2nd polynucleotide as a tool suitable for using for the said method. The second polynucleotide consists of a complementary sequence of miR-31, where miR-31 is a polynucleotide consisting of a sequence consisting of the base sequence of SEQ ID NO: 2 or a partial sequence of SEQ ID NO: 2. And a polynucleotide comprising a sequence comprising the base sequence of SEQ ID NO: 3. The second polynucleotide can be readily produced using standard molecular biology techniques (eg, chemical synthesis or recombinant methods).
　〔５．ｍｉＲ－３１の利用〕
　上述したように、ｍｉＲ－３１の発現量に基づいて大腸癌を診断することができる。すなわち、ｍｉＲ－３１は大腸癌マーカーとして利用可能である。 [5. Use of miR-31]
As described above, colorectal cancer can be diagnosed based on the expression level of miR-31. That is, miR-31 can be used as a colorectal cancer marker.
　また、ｍｉＲ－３１模倣物を大腸癌細胞株へ導入すると細胞浸潤能が上昇し、患者サンプルのマイクロＲＮＡ解析によって、ｍｉＲ－３１の高発現がＫＲＡＳ変異およびＢＲＡＦ変異、ならびに近位結腸癌と有意に相関していること、ｍｉＲ－３１の高発現が予後不良と関連している。このことは、ｍｉＲ－３１またはｍｉＲ－３１模倣物を導入した大腸癌細胞が、ＫＲＡＳ遺伝子およびＢＲＡＦ遺伝子の少なくとも一方に変異が生じている大腸癌細胞や近位結腸癌のモデル細胞として利用可能であることを示す。すなわち、ｍｉＲ－３１またはｍｉＲ－３１模倣物は大腸癌モデル細胞の作製に好適に用いられる。 In addition, when miR-31 mimics are introduced into colon cancer cell lines, the cell invasion ability is increased, and high miR-31 expression is significantly higher than that of KRAS and BRAF mutations and proximal colon cancer by microRNA analysis of patient samples. , High expression of miR-31 is associated with poor prognosis. This means that colon cancer cells into which miR-31 or miR-31 mimics have been introduced can be used as model cells for colon cancer cells or proximal colon cancer in which at least one of the KRAS gene and BRAF gene has been mutated. Indicates that there is. That is, miR-31 or miR-31 mimetic is preferably used for the production of colon cancer model cells.
　本発明は、ｍｉＲ－３１またはｍｉＲ－３１模倣物を含有している、大腸癌モデル細胞を作製するための組成物（第３の組成物）を提供する。 The present invention provides a composition (third composition) for producing colon cancer model cells, which contains miR-31 or miR-31 mimics.
　ｍｉＲ－３１と配列が一部異なるポリヌクレオチドであっても、ｍｉＲ－３１の機能を維持している限りｍｉＲ－３１模倣物として本発明に利用可能である。本明細書中にて使用される場合、ｍｉＲ－３１模倣物は、ｍｉＲ－３１の塩基配列において１または数個の塩基が置換または欠失されたものでありかつｍｉＲ－３１の機能を保持しているものとして当業者は容易に構築し得る。上述した置換または欠失の塩基数は、合計が３個以下であることが好ましく、１個または２個であることがより好ましい。すなわち、ｍｉＲ－３１またはｍｉＲ－３１模倣物は、大腸癌細胞の細胞浸潤能を上昇させる活性を有し、その塩基配列が、（ｉ）配列番号２の塩基配列からなる配列、（ｉｉ）配列番号２の部分配列でありかつ配列番号３の塩基配列を含む配列、あるいは、（ｉｉｉ）上記（ｉ）または（ｉｉ）の配列において１または数個の塩基が置換または欠失してなる配列、を含んでいることが好ましい。本明細書中にて使用される場合、ｍｉＲ－３１またはｍｉＲ－３１模倣物を、第３のポリヌクレオチドともいい、発現ベクターに含まれている形態にて第３のポリヌクレオチドが第３の組成物中に含有されていてもよい。 Even a polynucleotide having a partially different sequence from miR-31 can be used in the present invention as a miR-31 mimetic as long as the function of miR-31 is maintained. As used herein, a miR-31 mimetic is one in which one or several bases are substituted or deleted in the base sequence of miR-31 and retains the function of miR-31. Can be easily constructed by those skilled in the art. The total number of substitution or deletion bases described above is preferably 3 or less, more preferably 1 or 2. That is, miR-31 or miR-31 mimetic has activity to increase the cell invasion ability of colon cancer cells, and its base sequence is (i) a sequence consisting of the base sequence of SEQ ID NO: 2, (ii) sequence A sequence comprising a partial sequence of No. 2 and comprising the base sequence of SEQ ID No. 3, or (iii) a sequence obtained by substituting or deleting one or several bases in the sequence of (i) or (ii) above, It is preferable that it contains. As used herein, a miR-31 or miR-31 mimetic is also referred to as a third polynucleotide, and the third polynucleotide is the third composition in the form contained in the expression vector. It may be contained in the product.
　ｍｉＲ－３１またはｍｉＲ－３１模倣物は、標準的な分子生物学的手法（例えば化学合成法または組換え法）を用いて生成したもの、あるいは市販のものを使用することができる。なお、ｍｉＲ－３１またはｍｉＲ－３１模倣物の合成を受注する種々の供給業者（例えば、Ａｍｂｉｏｎ社、コスモ・バイオ社、ＢｉｚＳｃｉｅｎｃｅ社等）がよく知られており、当業者はｍｉＲ－３１またはｍｉＲ－３１模倣物を容易に入手することができる。 As the miR-31 or miR-31 mimetic, those produced using standard molecular biological techniques (for example, chemical synthesis methods or recombinant methods), or commercially available products can be used. In addition, various suppliers (for example, Ambion, Cosmo Bio, BizScience, etc.) receiving orders for the synthesis of miR-31 or miR-31 mimetics are well known, and those skilled in the art can recognize miR-31 or miR. -31 mimetics are readily available.
　第３のポリヌクレオチドまたは第３のポリヌクレオチドを含んでいる発現ベクターを導入した大腸癌モデル細胞に、得られた形質転換細胞を候補物質とともにインキュベートし、インキュベートした後の細胞浸潤能をインキュベートする前の細胞浸潤能よりも低下させた候補物質を選択することによって、大腸癌細胞を治療するための薬剤をスクリーニングすることができる。このようなスクリーニング方法によって得られた薬剤は、ＫＲＡＳ遺伝子およびＢＲＡＦ遺伝子の少なくとも一方に変異が生じている大腸癌細胞や近位結腸癌の治療に利用可能であり、抗ＥＧＦＲ抗体による治療に耐性である大腸癌細胞の治療に用いられても、抗ＥＧＦＲ抗体との組合せ療法に用いられてもよい。抗ＥＧＦＲ抗体との組合せ療法に用いられる場合、上記薬剤は、抗ＥＧＦＲ抗体とともに組成物またはキットの形態で提供されることが好ましい。 Before incubating the cell invasion ability after incubating the obtained transformed cell with a candidate substance in a colon cancer model cell into which the third polynucleotide or an expression vector containing the third polynucleotide has been introduced. By selecting a candidate substance having a decreased cell invasive ability, a drug for treating colorectal cancer cells can be screened. The drug obtained by such a screening method can be used for the treatment of colorectal cancer cells and proximal colon cancer in which at least one of the KRAS gene and the BRAF gene is mutated, and is resistant to treatment with an anti-EGFR antibody. It may be used to treat certain colon cancer cells or may be used in combination therapy with an anti-EGFR antibody. When used in combination therapy with an anti-EGFR antibody, the agent is preferably provided in the form of a composition or kit with the anti-EGFR antibody.
　さらに、ｍｉＲ－３１模倣物を大腸癌細胞株へ導入すると細胞浸潤能が上昇し、患者サンプルのマイクロＲＮＡ解析によって、ｍｉＲ－３１の高発現がＫＲＡＳ変異およびＢＲＡＦ変異、ならびに近位結腸癌と有意に相関していること、ｍｉＲ－３１の高発現が予後不良と関連していることは、ＫＲＡＳ遺伝子およびＢＲＡＦ遺伝子の少なくとも一方に変異が生じている大腸癌細胞を用いてこの細胞におけるｍｉＲ－３１の発現量の変動を指標にすれば、腸癌細胞を治療するための薬剤をスクリーニングすることができることを示す。すなわち、ＫＲＡＳ遺伝子およびＢＲＡＦ遺伝子の少なくとも一方に変異が生じている大腸癌細胞を候補物質とともにインキュベートし、インキュベートした後のｍｉＲ－３１の発現量をインキュベートする前のｍｉＲ－３１の発現量よりも低下させた候補物質を選択することによって、大腸癌を治療するための薬剤をスクリーニングすることができる。このようなスクリーニング方法によって得られた薬剤もまた、ＫＲＡＳ遺伝子およびＢＲＡＦ遺伝子の少なくとも一方に変異が生じている大腸癌細胞や近位結腸癌の治療に利用可能であり、抗ＥＧＦＲ抗体による治療に耐性である大腸癌細胞の治療に用いられても、抗ＥＧＦＲ抗体との組合せ療法に用いられてもよい。抗ＥＧＦＲ抗体との組合せ療法に用いられる場合、上記薬剤は、抗ＥＧＦＲ抗体とともに組成物またはキットの形態で提供されることが好ましい。 Furthermore, introduction of miR-31 mimics into colorectal cancer cell lines increased cell invasive capacity, and high miR-31 expression was significantly higher than KRAS and BRAF mutations and proximal colon cancer by microRNA analysis of patient samples That high expression of miR-31 is associated with a poor prognosis is that miR-31 in this cell is used in colon cancer cells in which at least one of the KRAS gene and BRAF gene is mutated. It is shown that a drug for treating intestinal cancer cells can be screened by using the change in the expression level of as an index. That is, colon cancer cells having a mutation in at least one of the KRAS gene and the BRAF gene are incubated with a candidate substance, and the expression level of miR-31 after incubation is lower than the expression level of miR-31 before incubation By selecting the candidate substance, a drug for treating colorectal cancer can be screened. The drug obtained by such a screening method can also be used for the treatment of colon cancer cells or proximal colon cancer in which at least one of the KRAS gene and BRAF gene is mutated, and is resistant to treatment with an anti-EGFR antibody. Or may be used for combination therapy with anti-EGFR antibody. When used in combination therapy with an anti-EGFR antibody, the agent is preferably provided in the form of a composition or kit with the anti-EGFR antibody.
　〔１．材料および方法〕
　〔１ａ．大腸癌患者および組織試料〕
　１９９７年４月から２０１２年６月の間に、札幌医科大学、恵佑会札幌病院およびＪＲ札幌病院において内視鏡的切除術または外科的処置を受けた、７２１症例のＣＲＣ（Colorectal cancer（大腸癌））（ステージＩ－ＩＶ）、３８１症例の鋸歯状病変、および２５１症例の通常腺腫の、フォルマリン固定パラフィン包埋（ＦＦＰＥ）組織を収集した。悪性細胞の粘膜筋板を超える浸潤を、ＣＲＣ診断の基準とした。粘膜内悪性腫瘍および元の位置の悪性腫瘍を、腺腫に分類した。遺伝性非腺腫性ＣＲＣまたは家族性大腸腺腫症の基準を満たす患者はいなかった。大腸癌部位を、近位結腸（盲腸、上行結腸および横行結腸）、遠位結腸（下行結腸およびＳ字結腸）、および直腸に分類した。患者を、死亡するまで、または２０１２年１２月までのいずれか早い方まで経過観察した。試料の収集前に、全ての患者からインフォームドコンセントを得た。さらに、本実験は、各施設内倫理委員会によって承認された。腫瘍および一対の正常結腸直腸組織を、２人の病理医によって診断した。 [1. Materials and methods〕
[1a. (Colon cancer patients and tissue samples)
From April 1997 to June 2012, 721 CRC (Colorectal cancer) patients who underwent endoscopic resection or surgery at Sapporo Medical University, Keikeikai Sapporo Hospital and JR Sapporo Hospital. )) (Stage I-IV), 381 cases of serrated lesions, and 251 cases of normal adenomas were collected in formalin-fixed paraffin-embedded (FFPE) tissue. Infiltration beyond the mucosal muscle plate of malignant cells was used as a criterion for CRC diagnosis. Intramucosal and in situ malignant tumors were classified as adenomas. None of the patients met hereditary non-adenomatous CRC or familial colorectal adenomatous criteria. Colon cancer sites were classified into proximal colon (cecum, ascending and transverse colon), distal colon (descending and sigmoid colon), and rectum. Patients were followed up until death or until December 2012, whichever comes first. Informed consent was obtained from all patients prior to sample collection. In addition, this experiment was approved by each institutional ethics committee. The tumor and a pair of normal colorectal tissues were diagnosed by two pathologists.
　〔１ｂ．鋸歯状病変および通常腺腫の組織病理学評価〕
　全ての試料における組織病理学的所見を、臨床情報および分子情報が通知されていない病理学者が評価した。通常腺腫を標準的な基準を用いて診断した。鋸歯状の病変（ＨＰ、ＳＳＡ／Ｐ、およびＴＳＡ）を、以前に報告された基準に基づいて分類した。ＨＰを、より管腔状の位置の鋸歯が変異している腺窩の伸長、および、狭い基底部の腺窩の伸長によって特徴付けた。基底部近傍における過剰な鋸歯状に沿って、基底部において腺窩が湾曲および膨張しており、突出した鋸歯状であり、かつ、細胞異形症を有さない、腺窩の伸長によって、ＳＳＡ／Ｐを特徴付けた。ＴＳＡを、細胞異形症を伴うＳＳＡ／Ｐ、全体として鋸歯構造の通常腺腫、または、複合体全体および異形成と特徴づけられた細胞型を示す細胞の絨毛型によって特徴づけられた病変、として定義付けた。 [1b. Histopathological evaluation of serrated lesions and normal adenomas)
Histopathological findings in all samples were evaluated by pathologists who were not informed of clinical and molecular information. Usually adenoma was diagnosed using standard criteria. Serrated lesions (HP, SSA / P, and TSA) were classified based on previously reported criteria. HP was characterized by an extension of crypts with more luminal serrations and an extension of narrow basal crypts. Along with the excessive serrations near the base, the crypts are curved and dilated in the base, protruding serrated, and without cell dysplasia, by extension of the crypts, SSA / P was characterized. TSA is defined as SSA / P with cellular dysplasia, generally serrated normal adenomas, or lesions characterized by the overall complex and cell villous type exhibiting a cell type characterized as dysplasia I attached.
　〔１ｃ．ＲＮＡ抽出物およびマイクロＲＮＡアレイ解析〕
　ＲＮＡを、miRNeasy FFPE Kit（Qiagen, Valencia, CA, USA）を用いて、ＦＦＰＥ組織から抽出した。TaqMan Array Human MicroRNA A+B Cards Set v3.0（Applied Biosystems, Foster City, CA, USA）を、microfluidic PCR platformにおいて、７６０症例のｍｉＲＮＡの発現を同時に測定するために用いた。簡単に説明すると、特定のプライマーおよびＴａｑＭａｎプローブを用いたＰＣＲによってｍｉＲＮＡを増幅および検出した後、総ＲＮＡ　１μｇを、Megaplex Pools kit（Applied Biosystems）を用いて逆転写した。ＰＣＲを7900HT Fast Real-Time PCR system（Applied Biosystems）にて行い、比較ΔＣｔ解析にSDS2.2.2 software（Applied Biosystems）を用いた。Ｕ６　ｓｎＲＮＡ（ＲＮＵ６Ｂ；Applied Biosystems）を内因性コントロールとした。目的とする遺伝子のＣｔ値からＵ６のＣｔ値を差し引くことによって、ΔＣｔを算出した。腫瘍サンプルにおける各ｍｉＲＮＡの発現を、２－ΔＣｔ（但し、ΔＣｔ＝（Ｃｔ　ｍｉＲＮＡ－Ｃｔ　Ｕ６））によって算出した。 [1c. RNA extract and microRNA array analysis]
RNA was extracted from FFPE tissue using the miRNeasy FFPE Kit (Qiagen, Valencia, CA, USA). TaqMan Array Human MicroRNA A + B Cards Set v3.0 (Applied Biosystems, Foster City, CA, USA) was used to simultaneously measure the expression of miRNA in 760 cases on a microfluidic PCR platform. Briefly, after miRNA was amplified and detected by PCR using specific primers and TaqMan probe, 1 μg of total RNA was reverse transcribed using Megaplex Pools kit (Applied Biosystems). PCR was performed with 7900HT Fast Real-Time PCR system (Applied Biosystems), and SDS2.2.2 software (Applied Biosystems) was used for comparative ΔCt analysis. U6 snRNA (RNU6B; Applied Biosystems) served as an endogenous control. ΔCt was calculated by subtracting the Ct value of U6 from the Ct value of the target gene. The expression of each miRNA in the tumor sample was calculated by 2 −ΔCt (where ΔCt = (Ct miRNA−Ct U6)).
　〔１ｄ．マイクロＲＮＡの定量的ＲＴ－ＰＣＲ〕
　ｍｉＲ－３１発現を、TaqMan micro-RNA Assays（Applied Biosystems）を用いて解析した。簡単に説明すると、特定のプライマーおよびＴａｑＭａｎプローブを用いた定量的逆転写ポリメラーゼ連鎖反応（ｑＲＴ－ＰＣＲ）によって総ＲＮＡを増幅および検出した後、specific stem-loop RT プライマーを用いて総ＲＮＡ　５ｎｇを逆転写した。7500 Fast Real-Time PCR System（Applied Biosystems）を用いてＰＣＲを３回行い、比較ΔＣｔ解析にSDS v1.4 software（Applied Biosystems）を用いた。Ｕ６を内因性コントロールとした。 [1d. Quantitative RT-PCR of microRNA]
miR-31 expression was analyzed using TaqMan micro-RNA Assays (Applied Biosystems). Briefly, total RNA is amplified and detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using specific primers and TaqMan probe, then 5 ng of total RNA is reversed using specific stem-loop RT primer I copied it. PCR was performed 3 times using 7500 Fast Real-Time PCR System (Applied Biosystems), and SDS v1.4 software (Applied Biosystems) was used for comparative ΔCt analysis. U6 was the endogenous control.
　〔１ｅ．ＫＲＡＳ、ＢＲＡＦおよびＰＩＫ３ＣＡのＤＮＡ抽出およびパイロシークエンス、ならびにマイクロサテライト不安定性（ＭＳＩ）解析〕
　QIAamp DNA FFPE Tissue Kitを用いて、大腸癌のＦＦＰＥ組織からゲノムＤＮＡを抽出した。抽出したゲノムＤＮＡを用いて、ＰＣＲ、ならびに、ＫＲＡＳ（コドン１２および１３）、ＢＲＡＦ（コドン６００）およびＰＩＫ３ＣＡ（エクソン９および２０）を標的としたパイロシークエンスを行った。ＭＳＩ解析を１０個のマイクロサテライトマーカー（Ｄ２Ｓ１２３、Ｄ５Ｓ３４６、Ｄ１７Ｓ２５０、ＢＡＴ２５、ＢＡＴ２６、ＢＡＴ４０、Ｄ１８Ｓ５５、Ｄ１８Ｓ５６、Ｄ１８Ｓ６７およびＤ１８Ｓ４８７）を用いて行った。ＭＳＩ－ｈｉｇｈを、マーカーの３０％以上における不安定性として定義付け、ＭＳＩ－ｌｏｗ／マイクロサテライト安定性（ＭＳＳ）を、マーカーの３０％未満における不安定性として定義付けた。 [1e. DNA extraction and pyrosequencing of KRAS, BRAF and PIK3CA, and microsatellite instability (MSI) analysis]
Genomic DNA was extracted from FFPE tissue of colorectal cancer using QIAamp DNA FFPE Tissue Kit. Using the extracted genomic DNA, PCR and pyrosequencing targeting KRAS (codons 12 and 13), BRAF (codon 600) and PIK3CA (exons 9 and 20) were performed. MSI analysis was performed using 10 microsatellite markers (D2S123, D5S346, D17S250, BAT25, BAT26, BAT40, D18S55, D18S56, D18S67 and D18S487). MSI-high was defined as the instability at 30% or more of the marker, and MSI-low / microsatellite stability (MSS) was defined as the instability at less than 30% of the marker.
　〔１ｆ．ＭＬＨ１のプロモーターメチル化を測定するためのバイサルファイト処理およびパイロシークエンス〕
　ゲノムＤＮＡのバイサルファイトを、BisulFlash DNA modification kit（Epigentek, Brooklyn, NY）を用いて行った。ＭＬＨ１メチル化のためのバイサルファイトパイロシークエンスを、the PyroMark kit（Qiagen）を用いて行った。本発明者らは、８％以上のＭＬＨ１メチル化の症例を高メチル化腫瘍と定義付けた。 [1f. Bisulfite treatment and pyrosequence for measuring promoter methylation of MLH1]
Bisulphite of genomic DNA, BisulFlash   DNA modification kit (Epigentek, Brooklyn, NY) was used. Bisulfite pyrosequencing for MLH1 methylation was performed using the PyroMark kit (Qiagen). We defined 8% or more cases of MLH1 methylation as hypermethylated tumors.
　〔１ｇ．大腸癌細胞株およびマイクロＲＮＡ一過性トランスフェクション〕
　表１に示した７つの癌細胞株（ＣＯＬＯ－３２０－ＨＳＲ、ＤＬＤ－１、ＨＣＴ－１１６、ＨＴ－２９、Ｌｏｖｏ、ＲＫＯ、ＳＷ４８０）を用いた。TRIzol Reagent（Invitrogen by Life Technologies, Carlsbad, CA, USA）を用いて、細胞ペレットからＲＮＡを抽出した。ｍｉＲ－３１模倣物、ｍｉＲ－３１阻害剤、またはネガティブコントロール（Ambion, Austin, TX, USA）を、大腸癌細胞株にトランスフェクトした。取扱説明書の記載に従って、ＤＬＤ－１のためのNucleofector I electroporation device（Lonza）、ＣＯＬＯ－３２０－ＨＳＲのためのＨＣＴ－１１６およびＲＫＯもしくはLipofectamine 2000（Invitrogen, by Life Technologies）、ＨＴ－２９、Ｌｏｖｏ、ならびにＳＷ４８０を用いて、Cell Line Nucleofector Kit V（Lonza, Basel, Switzerland）によって、細胞をトランスフォームした。
　ｍｉＲ－３１阻害剤およびｍｉＲ－３１模倣物としてそれぞれ、mirVana（登録商標） miRNA inhibitor（Assay ID: MH11465）およびmirVana（登録商標） miRNA mimic （Assay ID:MC11465）（いずれもApplied Biosystems）を実施例にて用いた。 [1 g. Colorectal cancer cell lines and microRNA transient transfection]
Seven cancer cell lines shown in Table 1 (COLO-320-HSR, DLD-1, HCT-116, HT-29, Lovo, RKO, SW480) were used. RNA was extracted from the cell pellet using TRIzol Reagent (Invitrogen by Life Technologies, Carlsbad, CA, USA). miR-31 mimetics, miR-31 inhibitors, or negative controls (Ambion, Austin, TX, USA) were transfected into colon cancer cell lines. Nucleofector I electroporation device for DLD-1 (Lonza), HCT-116 for COLO-320-HSR and RKO or Lipofectamine 2000 (Invitrogen, by Life Technologies), HT-29, Lovo as described in the instruction manual The cells were transformed with Cell Line Nucleofector Kit V (Lonza, Basel, Switzerland) using SW480.
Examples of miR-31 inhibitor and miR-31 mimic were mirVana (registered trademark) miRNA inhibitor (Assay ID: MH11465) and mirVana (registered trademark) miRNA mimic (Assay ID: MC11465) (both Applied Biosystems) Used in.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　表１において、大腸癌細胞におけるｍｉＲ－３１の発現を、２－ΔＣｔ（但し、ΔＣｔ＝（Ｃｔ　ｍｉＲＮＡ－Ｃｔ　Ｕ６））を用いて算出した。 In Table 1, miR-31 expression in colorectal cancer cells was calculated using 2 −ΔCt (where ΔCt = (Ct miRNA−Ct U6)).
　〔１ｈ．増殖アッセイおよび浸潤アッセイ〕
　３－（４，５－ジメチルチアゾル－２－イル）－２,５－ジフェニルテトラゾリウムブロマイド（ＭＴＴ）アッセイにおいて、トリチウム化チミジン取り込み量を測定することによって、ｍｉＲＮＡトランスフェクタントの増殖を解析した。簡単に説明すると、トランスフェクトされた細胞を、９６ウェルプレートに１ウェルあたり５×１０３個の細胞濃度で播種した。０ｈ、２４ｈ、４８ｈおよび７２ｈのインキュベーションの後、Cell Counting kit-8（Dojindo, Tokyo, Japan）を用いて、取扱説明書に従ってＭＴＴアッセイを行った。 [1h. Proliferation and invasion assays
The growth of miRNA transfectants was analyzed by measuring tritiated thymidine incorporation in a 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) assay. . Briefly, transfected cells were seeded in 96 well plates at a concentration of 5 × 10 3 cells per well. After incubation for 0h, 24h, 48h and 72h, MTT assay was performed using Cell Counting kit-8 (Dojindo, Tokyo, Japan) according to the instruction manual.
　細胞浸潤を、Ｍａｔｒｉｇｅｌ浸潤アッセイを用いて評価した。２４時間のインキュベーションの後、予めリン酸緩衝生理食塩水（ＰＢＳ）を付与したBD BioCoat Matrigel Invasion Chambers（BD Biosciences, Bedford, MA, USA）の上方に、５００μＬの無血清培地に懸濁した１×１０６個のトランスフェクト細胞を添加した。そして、７５０μＬの１０％ウシ胎仔血清（ＦＢＳ）を添加した培地を、プレートの下方のウェルに添加した。２４時間のインキュベーションの後、浸潤された細胞を固定および染色し、そして、顕微鏡下にて、膜毎に５つの任意の領域における細胞数を計数することによって、解析した。 Cell invasion was assessed using the Matrigel invasion assay. After incubation for 24 hours, 1 × suspended in 500 μL of serum-free medium above BD BioCoat Matrigel Invasion Chambers (BD Biosciences, Bedford, MA, USA) pre-applied with phosphate buffered saline (PBS). 10 6 transfected cells were added. Then, medium supplemented with 750 μL of 10% fetal bovine serum (FBS) was added to the lower well of the plate. After 24 hours of incubation, infiltrated cells were fixed and stained and analyzed by counting the number of cells in any of the five regions per membrane under the microscope.
　〔１ｉ．ウエスタンブロット解析〕
　タンパク質発現を抗ＫＲＡＳ抗体および抗ＢＲＡＦ抗体を用いた標準的なイムノブロット法によって解析した。全ての一次抗体をSanta Cruz Biotechnology（Santa Cruz, CA, USA）から入手した。抗β－アクチンモノクローナル抗体（Oncogene Research Products, La Jolla, CA, USA）を、ローディングコントロールとして用いた。β－アクチンに対して正規化した後に、the Quantity One software（Bio-Rad Laboratories, Hercules, CA, USA）を用いてそれぞれのバンドブロットの光学濃度を測定することによって、発現タンパク質を定量化した。 [1i. Western blot analysis
Protein expression was analyzed by standard immunoblotting using anti-KRAS and anti-BRAF antibodies. All primary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-β-actin monoclonal antibody (Oncogene Research Products, La Jolla, CA, USA) was used as a loading control. After normalization to β-actin, the expressed protein was quantified by measuring the optical density of each band blot using the Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).
　〔１ｊ．統計分析〕
　統計分析のために、ＪＭＰ（Version 9）（SAS Institute, Cary, NC, USA）およびtheSAS software programs（Version 9.1）（SAS Institute）を用いた。χ二乗検定を、分類変数間の関連性を評価するために用い、ｍｉＲ－３１の発現レベルの四分位にわたって平均年齢または腫瘍サイズを比較するために分散分析（ＡＮＯＶＡ）を用いた。生存期間解析のために、カプランマイヤー法を用いて生存期間分布を評価した。また、ログランク検定を行った。死亡率に対するｍｉＲ－３１の発現レベルの独立した効果を評価するために、残余交絡および過剰適合を避けるためのSAS“proc phreg”commandの”stera” oprionを用いたコックスモデルにおける層化（マッチング）変数として、ＴＮＭ病期（Ｉ、ＩＩＡ、ＩＩＢ、ＩＩＩＡ、ＩＩＩＢ、ＩＩＩＣ、ＩＶ）を用いた。本発明者らは、ｍｉＲ－３１発現ステータス、含まれる性別（男性対女性）、診断時の年齢（連続型変数）、腫瘍サイズ（連続型変数）、腫瘍部位（近位結腸　対　遠位結腸および直腸）、組織型（低分化型対高分化型）、ＭＳＩ状態（ＭＳＩ－ｈｉｇｈ　対　ＭＳＳ／ＭＳＩ－ｌｏｗ）、ＭＬＨ１メチル化、ならびにＢＲＡＦ、ＫＲＡＳおよびＰＩＫ３ＣＡの変異、によるハザード率（ＨＲ）を計算するための、多変量段階層化Ｃｏｘ比例ハザードモデル（multivariate, stage-stratified Cox proportional hazard model）を構築した。最終モデルの変数を選択するために、閾値Ｐ＝０．１０の変数増減法を用いた。分類された変数（組織型（１．７％）、ＭＳＩの状態（１．９％）、ＭＬＨ１メチル化（４．９％）、ＢＲＡＦ（０．１％）、ＫＲＡＳ（１．５％）およびＰＩＫ３ＣＡ（０．１％）の変異）の何れかに不明情報がある場合は、モデルの選択および最終モデルのための完全主部法（complete-subject method）を用いた。選択が完了した後、最終モデルに不明情報がある場合のために、分離欠測指示子（separate missing indicator variables）を割り当てた。 [1j. Statistical analysis)
JMP (Version 9) (SAS Institute, Cary, NC, USA) and theSAS software programs (Version 9.1) (SAS Institute) were used for statistical analysis. A chi-square test was used to assess associations between categorical variables, and analysis of variance (ANOVA) was used to compare mean age or tumor size across quartiles of miR-31 expression levels. For survival analysis, survival distribution was evaluated using Kaplan-Meier method. In addition, a log rank test was performed. To evaluate the independent effect of miR-31 expression level on mortality, stratification in Cox model using “stera” oprion of SAS “proc phreg” command to avoid residual confounding and overfitting As a variable, TNM stage (I, IIA, IIB, IIIA, IIIB, IIIC, IV) was used. We have included miR-31 expression status, gender involved (male vs female), age at diagnosis (continuous variable), tumor size (continuous variable), tumor site (proximal colon vs distal colon and Calculate the hazard rate (HR) due to rectal), tissue type (low differentiation vs. high differentiation), MSI status (MSI-high vs. MSS / MSI-low), MLH1 methylation, and mutations in BRAF, KRAS and PIK3CA A multivariate, stage-stratified Cox proportional hazard model was constructed. In order to select the variables of the final model, a variable increase / decrease method with a threshold P = 0.10 was used. Classified variables (histotype (1.7%), MSI status (1.9%), MLH1 methylation (4.9%), BRAF (0.1%), KRAS (1.5%) and If any of the PIK3CA (0.1% mutation) had unknown information, the model selection and the complete-subject method for the final model was used. After selection was completed, separate missing indicator variables were assigned in case there was unknown information in the final model.
　〔２．結果〕
　〔２ａ．ＢＲＡＦ変異型大腸癌におけるマイクロＲＮＡ３１の高レベル発現のマイクロＲＮＡアレイ解析による検出〕
　ＢＲＡＦ変異ＣＲＣにおけるｍｉＲＮＡ発現を試験するために、７２１症例のＣＲＣから２９症例のＣＲＣ（７症例のＢＲＡＦ変異型大腸癌および２２症例のＢＲＡＦ野生型大腸癌）を任意で選択し、ｍｉＲＮＡアレイ解析を行った。２９名のＣＲＣ患者の臨床的、病理的または分子的な特徴を、表２に示す。２－ΔＣｔ（但し、ΔＣｔ＝（Ｃｔ　ｍｉＲＮＡ－Ｃｔ　Ｕ６））を用いて、７６０症例のｍｉＲＮＡの発現を算出し、ＢＲＡＦ野生型グループにおける発現と共に、ＢＲＡＦ変異グループにおけるそれぞれの発現の平均と比較した。 [2. result〕
[2a. Detection of microRNA31 high level expression in BRAF mutant colorectal cancer by microRNA array analysis]
To test miRNA expression in BRAF mutant CRC, 721 CRCs to 29 CRCs (7 BRAF mutant colorectal cancers and 22 BRAF wild type colon cancers) were arbitrarily selected, and miRNA array analysis was performed. went. The clinical, pathological or molecular characteristics of 29 CRC patients are shown in Table 2. 2 −ΔCt (where ΔCt = (Ct miRNA−Ct U6)) was used to calculate the expression of miRNA in 760 cases and compared with the expression in the BRAF wild type group as well as the average of each expression in the BRAF mutant group .
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　表２において、Ｐ値は、年齢についてはＡＮＯＶＡ（変数解析）によって、他の変数全てについてはχ二乗検定によって算出した。 In Table 2, P values were calculated by ANOVA (variable analysis) for age and by chi-square test for all other variables.
　ｍｉＲＮＡアレイデータは、３３症例の個々のｍｉＲＮＡにおいて、２つのグループ間の発現に差異があることを示した（表３）。３３症例のｍｉＲＮＡの全てが、ＢＲＡＦ野生型グループよりもＢＲＡＦ変異グループにおいて高い発現レベルを示した。また、ｍｉＲ－３１は、７６０症例のｍｉＲＮＡの内で最も上方制御されていた（３３４．９倍率変化＝２９９２４．６（ＢＲＡＦ変異グループ）／８９．３（ＢＲＡＦ野生型グループ）,Ｐ＝０．００９）。 MiRNA array data showed that there were differences in expression between the two groups in 33 individual miRNAs (Table 3). All 33 cases of miRNA showed higher expression levels in the BRAF mutant group than in the BRAF wild type group. In addition, miR-31 was most up-regulated among 760 cases of miRNA (334.9-fold change = 29924.6 (BRAF mutation group) /89.3 (BRAF wild-type group), P = 0. 009).
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　表３において、各マイクロＲＮＡの発現を、２－ΔＣｔ（但し、ΔＣｔ＝（Ｃｔ　ｍｉＲＮＡ－Ｃｔ　Ｕ６））を用いて算出した。Ｐ値を、マン・ホイットニーのＵ検定によって解析し、Ｐ＜０．０５を示すｍｉＲＮＡを示した。 In Table 3, the expression of each microRNA was calculated using 2 −ΔCt (where ΔCt = (Ct miRNA−Ct U6)). P values were analyzed by Mann-Whitney U test and showed miRNAs showing P <0.05.
　〔２ｂ．大腸癌（Ｎ＝７２１）におけるｍｉＲＮＡ発現レベルの分布、ならびに臨床的、病理的および分子的な特徴とｍｉＲ－３１の発現レベルとの関係〕
　ＣＲＣ試料および一対の正常粘膜試料におけるｍｉＲ－３１の発現レベルを定量化し、７２１症例の有効な結果を得た。ｍｉＲ－３１の発現を２－ΔＣｔ（但し、ΔＣｔ＝（Ｃｔ　ｍｉＲＮＡ－Ｃｔ　Ｕ６））を用いて算出した。ＣＲＣおよび正常粘膜におけるｍｉＲ－３１の発現分布は、それぞれ以下の通りであった：平均±標準偏差（ＳＤ），０．１８±０．７３；中央値，０．０２７、および、平均±ＳＤ，０．０４６±０．２３；中央値，０．００４５。各ＣＲＣにおけるｍｉＲ－３１の相対的な発現を算出するために、一対の正常組織の２－ΔＣｔによって、癌組織の２－ΔＣｔを除した。７２１症例のＣＲＣにおける相対的なｍｉＲ－３１発現レベルの分布は、以下の通りであった：平均，４１．９；中央値，６．３；ＳＤ，１７６．２；範囲，０．０４～２１０８；四分位範囲，２．０～２３．４。さらなる解析のために、ｍｉＲ－３１発現レベルを四分位に分割した（Ｑ１（＜２．０,ｎ＝１８０），Ｑ２（２．０－６．２，ｎ＝１８０），Ｑ３（６.３－２３．３，ｎ＝１８１）,およびＱ４（≧２３．４，ｎ＝１８０））。表４に、ＣＲＣにおけるｍｉＲ－３１発現による臨床的、病理的および分子的な特徴を示す。ｍｉＲ－３１の高発現グループは、より大きな腫瘍サイズ、近位部位、低分化腺癌、高い病期、ＢＲＡＦ変異、ＫＲＡＳ変異、および、マイクロサテライト不安定性（全てＰ≦０．００４２）に顕著に関連していた。 [2b. Distribution of miRNA expression level in colorectal cancer (N = 721) and relationship between clinical, pathological and molecular characteristics and miR-31 expression level]
The expression level of miR-31 in the CRC sample and a pair of normal mucosa samples was quantified, and valid results for 721 cases were obtained. The expression of miR-31 was calculated using 2 −ΔCt (where ΔCt = (Ct miRNA−Ct U6)). The expression distribution of miR-31 in CRC and normal mucosa was as follows: mean ± standard deviation (SD), 0.18 ± 0.73; median, 0.027, and mean ± SD, 0.046 ± 0.23; median, 0.0045. To calculate the relative expression of miR-31 in each CRC, 2 -ΔCt in cancer tissue was divided by 2 -ΔCt in a pair of normal tissues. The distribution of relative miR-31 expression levels in CRC of 721 cases was as follows: mean, 41.9; median, 6.3; SD, 176.2; range, 0.04 to 2108 The interquartile range, 2.0-23.4. For further analysis, miR-31 expression levels were divided into quartiles (Q1 (<2.0, n = 180), Q2 (2.0-6.2, n = 180), Q3 (6. 3-23.3, n = 181), and Q4 (≧ 23.4, n = 180)). Table 4 shows the clinical, pathological and molecular features of miR-31 expression in CRC. The high expression group of miR-31 is prominent in larger tumor size, proximal site, poorly differentiated adenocarcinoma, high stage, BRAF mutation, KRAS mutation, and microsatellite instability (all P ≦ 0.0042) It was related.
　　
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-I000005
　　
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-I000005
　表４において、比率（％）は、ｍｉＲ－３１発現の与えられた四分位カテゴリー（Ｑ１、Ｑ２、Ｑ３およびＱ４）間において、特異的な臨床的、病理的および分子的特徴を有するケースの割合を示す。Ｐ値は、年齢および腫瘍サイズについてＡＮＯＶＡ（変数解析）によって、他の全ての変数についてχ二乗検定によって算出した。複数の仮設検証のために、有意性のＰ値をボンフェローニの補正によって０．００４２に調整した。ＭＳＩはマイクロサテライト不安定性を、ＭＳＳはマイクロサテライト安定性を、ＳＤは標準偏差をそれぞれ示している。 In Table 4, the percentages are for cases with specific clinical, pathological and molecular characteristics among the given quartile categories of miR-31 expression (Q1, Q2, Q3 and Q4). Indicates the percentage. P values were calculated by ANOVA (variable analysis) for age and tumor size and by chi-square test for all other variables. The significance P value was adjusted to 0.0042 by Bonferroni correction for multiple temporary verifications. MSI indicates microsatellite instability, MSS indicates microsatellite stability, and SD indicates standard deviation.
　〔２ｃ．ｍｉＲ－３１の高い発現レベルおよび患者の生存〕
　ＣＲＣ患者における臨床結果に対する、ｍｉＲ－３１の発現が高いことの影響を評価した。生存解析に適した６８８名の患者の経過観察中に、総数１４９名が死亡した。このうち、１１５名の死亡がＣＲＣに起因することが確認された。分類変数（例えば、第1四分位ケース（Ｑ１）、第２四分位ケース（Ｑ２）、第３四分位ケース（Ｑ３）および第４四分位ケース（Ｑ４））を用いて、カプランメイヤー解析を行った。そして、ｍｉＲ－３１の発現レベルが低いものよりもｍｉＲ－３１の発現レベルが高いものの方が、癌特異的生存率（ログランクＰ＝０．００１３）および全ての生存率（ログランクＰ＝０．００２６）において、顕著に高い死亡率に直面することを見出した（図１）。単変量Ｃｏｘ回帰解析において、Ｑ１ケースと比較して、Ｑ２ケース（ハザード率（ＨＲ）：１．９６；９５％信頼区間（ＣＩ）：１．０６－３．７７；Ｐ＝０．０３１）、Ｑ３ケース（ＨＲ：２．１６；９５％ＣＩ：１．１８－４．１３；Ｐ＝０．０１２）、およびＱ４ケース（ＨＲ：３．１０；９５％ＣＩ：１．７６－５．７８；Ｐ＜０．０００１）は、それぞれ、顕著に高い死亡率に直面した（表５）。同様に、Ｑ１ケースと比較して、Ｑ４ケースは、癌特異的生存率のためのステージマッチ解析（ＨＲ：２．４９；９５％ＣＩ：１．４０－４．６７；Ｐ＝０．００１６）および多変量（臨床的、病理的および分子的な特徴のための調整）解析（ＨＲ：２．９１；９５％ＣＩ：１．６０－５．５７；Ｐ＝０．０００４）の両方において、より短い予後に独立して関連していた。一方、Ｑ２ケースおよびＱ３ケースにおいては、ステージマッチ解析（Ｑ２；Ｐ＝０．１１，Ｑ３；Ｐ＝０．２３）および多変量解析（Ｑ２；Ｐ＝０．１４，Ｑ３；Ｐ＝０．１８）において、Ｑ１ケースと比較して、顕著ではないがわずかに高い死亡率に直面した（表５）。同様の結果が、全ての生存率についてのステージマッチ解析および多変量解析において観察された（データ示さず）。よって、Ｑ４ケースを「高発現ケース」として、また、Ｑ１、Ｑ２およびＱ３ケースを組み合わせて「低発現ケース」として定義付けたｍｉＲ－３１発現変数を構築した。ｍｉＲ－３１の低発現ケースと比較した多変量解析において、ｍｉＲ－３１高発現ケースは、癌特異的解析の顕著に高い死亡率（ＨＲ：２．０６；９５％ＣＩ：１．３６－３．０９；Ｐ＝０．０００８）に直面した（表５）。 [2c. High expression levels of miR-31 and patient survival]
The impact of high miR-31 expression on clinical outcome in CRC patients was evaluated. A total of 149 died during follow-up of 688 patients suitable for survival analysis. Of these, it was confirmed that 115 deaths were attributed to CRC. Kaplan using classification variables (eg, 1st quartile case (Q1), 2nd quartile case (Q2), 3rd quartile case (Q3) and 4th quartile case (Q4)) A Mayer analysis was performed. Those having a higher expression level of miR-31 than those having a low expression level of miR-31 have a cancer-specific survival rate (log rank P = 0.0013) and all survival rates (log rank P = 0). 0026), we found a significantly higher mortality rate (FIG. 1). In univariate Cox regression analysis, compared to the Q1 case, the Q2 case (hazard rate (HR): 1.96; 95% confidence interval (CI): 1.06-3.77; P = 0.031), Q3 case (HR: 2.16; 95% CI: 1.18-4.13; P = 0.012) and Q4 case (HR: 3.10; 95% CI: 1.76-5.78; Each P <0.0001) faced significantly higher mortality (Table 5). Similarly, compared to the Q1 case, the Q4 case is a stage match analysis for cancer-specific survival (HR: 2.49; 95% CI: 1.40-4.67; P = 0.016) And multivariate (adjustment for clinical, pathological and molecular features) analysis (HR: 2.91; 95% CI: 1.60-5.57; P = 0.004) and more Short prognosis was independently related. On the other hand, in the case of Q2 and Q3, stage match analysis (Q2; P = 0.11, Q3; P = 0.23) and multivariate analysis (Q2; P = 0.14, Q3; P = 0.18) ) Faced slightly less mortality compared to the Q1 case (Table 5). Similar results were observed in stage match analysis and multivariate analysis for all survival rates (data not shown). Therefore, miR-31 expression variables were constructed in which the Q4 case was defined as a “high expression case” and the Q1, Q2 and Q3 cases were defined as a “low expression case”. In the multivariate analysis compared to the low expression case of miR-31, the high expression case of miR-31 was significantly higher in the cancer-specific analysis (HR: 2.06; 95% CI: 1.36-3. 09; P = 0.0008) (Table 5).
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
　表５において、ｍｉＲ－３１発現変数を含む多変量ステージマッチ（層化）Ｃｏｘモデルを、性別、年齢、腫瘍サイズ、診断年時、腫瘍部位、組織型、マイクロサテライト不安定性、ＭＬＨ１メチル化、ならびにＢＲＡＦ、ＫＲＡＳおよびＰＩＫ３ＣＡの変異によって層化した。閾値Ｐ＝０．１０の変数増減法を、最終モデルの変数を選択するために用いた。病期の調整（Ｉ、ＩＩＡ、ＩＩＢ、ＩＩＩＡ、ＩＩＩＢ、ＩＩＩＣ、ＩＶ）をSAS “proc phreg” commandの“strata” optionを用いて行った。ＣＩは信頼区間を、ＨＲはハザード率をそれぞれ示す。 In Table 5, multivariate stage-matched (stratified) Cox models including miR-31 expression variables are shown for gender, age, tumor size, year of diagnosis, tumor site, histology, microsatellite instability, MLH1 methylation, and Stratified by mutations in BRAF, KRAS and PIK3CA. A variable increment / decrement method with a threshold P = 0.10 was used to select variables for the final model. Stage adjustment (I, IIA, IIB, IIIA, IIIB, IIIC, IV) was performed using the “strata” option of SAS proc phreg command. CI indicates a confidence interval, and HR indicates a hazard rate.
　〔２ｄ．ｍｉＲ－３１高発現ケースの多変量ロジスティック回帰分析〕
　臨床的、病理的または分子的な変数のいずれかが、ｍｉＲ－３１高発現（Ｑ４ケース）を独立して予測できるか否かについて評価するために、多変量ロジスティック回帰分析もまた行った（表６）。結果は、高レベルのｍｉＲ－３１発現がＢＲＡＦ変異大腸癌（オッズ比（ＯＲ）：６．０２；９５％ＣＩ：２．４８－１５．０；Ｐ＜０．０００１）、ＫＲＡＳ変異大腸癌（ＯＲ：２．６３；９５％ＣＩ：１．７６－３．９３；Ｐ＜０．０００１）および近位結腸大腸癌（ＯＲ：２．１６；９５％ＣＩ：１．４６－３．２２；Ｐ＝０．０００１）に顕著に関連することを示した。 [2d. Multivariate logistic regression analysis of miR-31 high expression cases]
Multivariate logistic regression analysis was also performed to assess whether any clinical, pathological, or molecular variables could independently predict miR-31 high expression (Q4 case) (Table 6). The results showed that high levels of miR-31 expression were found in BRAF mutant colorectal cancer (odds ratio (OR): 6.02; 95% CI: 2.48-15.0; P <0.0001), KRAS mutant colorectal cancer ( OR: 2.63; 95% CI: 1.76-3.93; P <0.0001) and proximal colon colorectal cancer (OR: 2.16; 95% CI: 1.46-3.22; P = 0.0001).
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
　表６において、回帰モデルに含まれる初期の変数（潜在的予測）は、表に示す、性別、年齢、診断年時、組織型、ＴＮＭ病期、マイクロサテライト安定性、ＭＬＨ１メチル化、腫瘍サイズ、腫瘍部位、ＫＲＡＳ変異、ＢＲＡＦ変異およびＰＩＫ３ＣＡ変異の変数である。閾値Ｐ＝０．１０の変数減数法を、最終モデルの変数を選択するために用いた。複数の仮設検証のために、有意性のＰ値をボンフェローニの補正によって０．００４２に調整した。ＣＩは信頼区間を示す。 In Table 6, the initial variables (potential predictions) included in the regression model are shown in the table, gender, age, year of diagnosis, histology, TNM stage, microsatellite stability, MLH1 methylation, tumor size, Variables of tumor site, KRAS mutation, BRAF mutation and PIK3CA mutation. A variable reduction method with a threshold P = 0.10 was used to select the variables of the final model. The significance P value was adjusted to 0.0042 by Bonferroni correction for multiple temporary verifications. CI indicates a confidence interval.
　〔２ｅ．結腸直腸の鋸歯状病変および通常腺腫における、ｍｉＲ－３１と臨床的、病理的および分子的な特徴との関連性〕
　３８１症例の結腸直腸の鋸歯状病変および２５１症例の通常腺腫における、ｍｉＲ－３１発現レベルの定量を行った。切除するサンプルが小さいため、結腸直腸の鋸歯状病変および通常腺腫から、顕微切除によって一対の正常組織のｍｉＲＮＡを得ることは非常に困難である。それゆえに、ＣＲＣの一対の正常組織におけるｍｉＲ－３１発現の中央値（２－ΔＣｔ＝０．００４５）を利用した。相対的ｍｉＲ－３１発現の分布は、下記の通りであった：（平均±ＳＤ；中央値）：ＨＰ（１４．６±２６．５；３．６），ＳＳＡ／Ｐ（２９．８±３６．８；１９．０），ＴＳＡ（３４．３±４０．１；１７．２）、および通常腺腫（２７．０±５６．５；４．９）。 [2e. Association of miR-31 with clinical, pathological and molecular features in colorectal serrated lesions and normal adenomas]
Quantification of miR-31 expression levels in 381 colorectal serrated lesions and 251 normal adenomas was performed. Due to the small size of the sample to be excised, it is very difficult to obtain a pair of normal tissue miRNAs by microscopic excision from serrated lesions of the colorectum and usually adenomas. Therefore, the median miR-31 expression (2 −ΔCt = 0.0045) in a pair of normal CRC tissues was utilized. The distribution of relative miR-31 expression was as follows: (mean ± SD; median): HP (14.6 ± 26.5; 3.6), SSA / P (29.8 ± 36) .8; 19.0), TSA (34.3 ± 40.1; 17.2), and normal adenoma (27.0 ± 56.5; 4.9).
　鋸歯状病変および通常腺腫における相対的ｍｉＲ－３１発現を含む、臨床的、病理的および分子的な特徴を、表７に示した。 The clinical, pathological, and molecular features including relative miR-31 expression in serrated lesions and normal adenomas are shown in Table 7.
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
　表７において、Ｐ値は、年齢および腫瘍サイズについてはＡＮＯＶＡ（変数解析）によって、他の全ての変数をχ二乗検定によって算出した。ＨＰは過形成性ポリープを、ＴＳＡは鋸歯状腺腫を、ＭＳＩはマイクロサテライト不安定性をそれぞれ示す。 In Table 7, P values were calculated by ANOVA (variable analysis) for age and tumor size, and χ square test for all other variables. HP indicates hyperplastic polyp, TSA indicates serrated adenoma, and MSI indicates microsatellite instability.
　ｍｉＲ－３１高発現（ＣＲＣのＱ４ケース；発現レベル≧２３．４）は、ＳＳＡ／Ｐ（４３％（５５／１２８））およびＴＳＡ（４５％（５１／１１３））のケースにおいて、ＨＰ（１９％（２６／１４０））および通常腺腫（２５％（６３／２５１））のケースにおいて、より高く検出された（Ｐ＜０．０００１）。ＳＳＡ／ＰおよびＴＳＡにおいてのｍｉＲ－３１高発現が臨床的および分子的な特徴に高い割合で寄与するため、多変量ロジスティック回帰分析におけるＢＲＡＦ変異およびＫＲＡＳ変異、腫瘍部位、ならびに腫瘍サイズのステータスを調整した。結果は、ｍｉＲ－３１の高発現と組織型との間の継続した顕著な関連性を示した（ＳＳＡ／Ｐｓ，Ｐ＝０．００９３，ＴＳＡｓ，Ｐ＜０．０００１（ＨＰを対象として））。 miR-31 high expression (CRC Q4 case; expression level ≧ 23.4) is higher in HP (19%) in SSA / P (43% (55/128)) and TSA (45% (51/113)) cases. % (26/140)) and normal adenomas (25% (63/251)) were detected higher (P <0.0001). Adjusting BRAF and KRAS mutations, tumor site, and tumor size status in multivariate logistic regression analysis because high miR-31 expression in SSA / P and TSA contributes to clinical and molecular features at a high rate did. The results showed a sustained significant association between high expression of miR-31 and tissue type (SSA / Ps, P = 0.003, TSAs, P <0.0001 (for HP)) .
　〔２ｆ．大腸癌細胞株におけるｍｉＲ－３１発現による増殖アッセイおよび浸潤アッセイ〕
　表１に示す大腸癌細胞株にｍｉＲ－３１模倣物（図中ｍｉｍｉｃ）またはｍｉＲ－３１阻害剤（図中Ｉｎｈ）をトランスフェクトした。大腸癌細胞におけるｍｉＲ－３１の発現を、式２－ΔＣｔ（但し、ΔＣｔ＝（Ｃｔ　ｍｉＲＮＡ－Ｃｔ　Ｕ６））を用いて算出した。ＤＬＤ－１細胞（ＫＲＡＳ変異；ｃ．３８Ｇ＞Ａ）およびＳＷ４８０細胞（ＫＲＡＳ変異；ｃ．３５Ｇ＞Ｔ）においてｍｉＲ－３１発現の上方制御を確認し（図２Ａ）、ＨＴ－２９（ＢＲＡＦ変異）、ＨＣＴ－１１６細胞（ＫＲＡＳ変異；ｃ．３８Ｇ＞Ａ）、ＲＫＯ細胞（ＢＲＡＦ変異）、ＤＬＤ－１細胞およびＳＷ４８０細胞においてｍｉＲ－３１発現の下方制御をそれぞれ確認した（図２Ｂ）。 [2f. Proliferation assay and invasion assay by miR-31 expression in colorectal cancer cell lines]
The colon cancer cell lines shown in Table 1 were transfected with miR-31 mimics (mimic in the figure) or miR-31 inhibitors (Inh in the figure). MiR-31 expression in colorectal cancer cells was calculated using the formula 2 −ΔCt (where ΔCt = (Ct miRNA−Ct U6)). Confirmed upregulation of miR-31 expression in DLD-1 cells (KRAS mutation; c.38G> A) and SW480 cells (KRAS mutation; c.35G> T) (FIG. 2A), HT-29 (BRAF mutation) , Down-regulation of miR-31 expression was confirmed in HCT-116 cells (KRAS mutation; c.38G> A), RKO cells (BRAF mutation), DLD-1 cells and SW480 cells, respectively (FIG. 2B).
　ｍｉＲ－３１の機能を決定するために、大腸癌細胞株を用いた増殖アッセイおよび浸潤アッセイを行った。ＭＴＴ増殖アッセイにおいて、ｍｉＲ－３１阻害剤がＨＣＴ－１１６（Ｐ＝０．０００１）における細胞増殖を顕著に減少したことを見出した（図３Ａ）。一方、ｍｉＲ－３１模倣物を癌細胞株にトランスフェクトした後、ｍｉＲ－３１模倣物がＤＬＤ－１細胞の浸潤能を高めること（Ｐ＝０．００４５）を、Ｍａｔｒｉｇｅｌ浸潤アッセイによって明らかにした（図３Ｂ）。 In order to determine the function of miR-31, proliferation and invasion assays using colorectal cancer cell lines were performed. In the MTT proliferation assay, it was found that miR-31 inhibitors significantly reduced cell proliferation in HCT-116 (P = 0.0001) (FIG. 3A). On the other hand, it was revealed by Matrigel invasion assay that miR-31 mimics enhance the invasive ability of DLD-1 cells after transfection of miR-31 mimics into cancer cell lines (P = 0.0045) ( FIG. 3B).
　〔２ｇ．大腸癌細胞株におけるＢＲＡＦおよびＫＲＡＳのウエスタンブロット解析〕
　ｍｉＲ－３１がＢＲＡＦタンパク質およびＫＲＡＳタンパク質を標的とするか否かを試験するために、ｍｉＲ－３１阻害剤を細胞株にトランスフェクトする前およびトランスフェクトした後（７２時間後）のこれらのタンパク質の発現を比較した。ウエスタンブロット解析によって、ｍｉＲ－３１阻害剤のトランスフェクト後の細胞において、ＫＲＡＳタンパク質の発現量に変化がない一方でＢＲＡＦタンパク質の発現量が減少したことを見出した（図３Ｃ）。 [2 g. Western blot analysis of BRAF and KRAS in colorectal cancer cell lines]
To test whether miR-31 targets BRAF and KRAS proteins, before and after (72 hours after) transfection of miR-31 inhibitors into cell lines Expression was compared. Western blot analysis revealed that in the cells transfected with the miR-31 inhibitor, there was no change in the expression level of the KRAS protein while the expression level of the BRAF protein decreased (FIG. 3C).
　本発明は上述した各実施形態に限定されるものではなく、請求項に示した範囲で種々の変更が可能であり、異なる実施形態にそれぞれ開示された技術的手段を適宜組み合わせて得られる実施形態についても本発明の技術的範囲に含まれる。 The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims, and embodiments obtained by appropriately combining technical means disclosed in different embodiments. Is also included in the technical scope of the present invention.
　また、本明細書中に記載された学術文献および特許文献の全てが、本明細書中において参考として援用される。 In addition, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.
　優れたツールを提供する本発明は、医学、薬学の分野において利用可能であり、医薬品、生化学試薬の開発に大いに寄与することができる。 The present invention providing an excellent tool can be used in the fields of medicine and pharmacy, and can greatly contribute to the development of pharmaceuticals and biochemical reagents.

Claims (13)

  1. 　ｍｉＲ－３１阻害剤を含有していることを特徴とする大腸癌を治療するための組成物。 A composition for treating colorectal cancer, comprising a miR-31 inhibitor.
  2. 　前記ｍｉＲ－３１阻害剤が、ｍｉＲ－３１のアンチセンス核酸分子、あるいは、ｍｉＲ－３１に特異的なｓｉＲＮＡ、ＲＮＡアプタマーまたはリボザイムであるであることを特徴とする請求項１に記載の組成物。 The composition according to claim 1, wherein the miR-31 inhibitor is a miR-31 antisense nucleic acid molecule, or a miR-31-specific siRNA, RNA aptamer or ribozyme.
  3. 　前記ｍｉＲ－３１阻害剤が、第１のポリヌクレオチドまたは第１のポリヌクレオチドを含んでいる発現ベクターであり、
    　第１のポリヌクレオチドは、大腸癌細胞の増殖を抑制する活性を有し、ｍｉＲ－３１の塩基配列の相補配列からなるか、または当該相補配列において１または数個の塩基が置換または欠失してなる配列からなり、
    　上記ｍｉＲ－３１の塩基配列は、配列番号２の塩基配列からなる配列であるか、または配列番号２の部分配列でありかつ配列番号３の塩基配列を含む配列であることを特徴とする請求項１または２に記載の組成物。     The miR-31 inhibitor is a first polynucleotide or an expression vector comprising the first polynucleotide;
    The first polynucleotide has an activity of suppressing the growth of colorectal cancer cells, and is composed of a complementary sequence of the base sequence of miR-31, or one or several bases are substituted or deleted in the complementary sequence. Consisting of
    The base sequence of miR-31 is a sequence consisting of the base sequence of SEQ ID NO: 2, or a partial sequence of SEQ ID NO: 2 and a sequence comprising the base sequence of SEQ ID NO: 3. The composition according to 1 or 2.
  4. 　ＫＲＡＳ遺伝子およびＢＲＡＦ遺伝子の少なくとも一方に変異が生じている大腸癌細胞の治療に用いられることを特徴とする請求項１～３のいずれか１項に記載の組成物。 The composition according to any one of claims 1 to 3, which is used for treatment of colorectal cancer cells in which a mutation has occurred in at least one of the KRAS gene and the BRAF gene.
  5. 　抗ＥＧＦＲ抗体との組合せ療法に用いられることを特徴とする請求項１～４のいずれか１項に記載の組成物。 The composition according to any one of claims 1 to 4, which is used for combination therapy with an anti-EGFR antibody.
  6. 　抗ＥＧＦＲ抗体をさらに含有していることを特徴とする請求項５に記載の組成物。 The composition according to claim 5, further comprising an anti-EGFR antibody.
  7. 　請求項５に記載の組成物と抗ＥＧＦＲ抗体とを備えていることを特徴とする大腸癌を治療するためのキット。 A kit for treating colorectal cancer, comprising the composition according to claim 5 and an anti-EGFR antibody.
  8. 　大腸癌患者由来の生物学的サンプルにおいてｍｉＲ－３１の発現量を測定する工程、および
    　測定した上記発現量を対照値と比較する工程
    を包含することを特徴とする大腸癌の診断を補助する方法。     A method for assisting diagnosis of colorectal cancer, comprising a step of measuring the expression level of miR-31 in a biological sample derived from a colorectal cancer patient, and a step of comparing the measured expression level with a control value. .
  9. 　前記診断が、大腸癌患者に対する抗ＥＧＦＲ抗体治療についてのコンパニオン診断と、大腸癌患者の予後の判定との少なくとも一方であることを特徴とする請求項８に記載の方法。 The method according to claim 8, wherein the diagnosis is at least one of a companion diagnosis for anti-EGFR antibody treatment for a colorectal cancer patient and a prognosis determination for the colorectal cancer patient.
  10. 　前記対照値が、
    　健常者由来の生物学的サンプル中のｍｉＲ－３１の発現量、
    　診断の対象である大腸癌患者から一定期間前に採取した生物学的サンプル中のｍｉＲ－３１の発現量、
    　診断対象である大腸癌患者からの大腸癌組織の採取と同時期に当該大腸癌患者から採取した正常な大腸組織中のｍｉＲ－３１の発現量、または
    　健常者から採取した正常な大腸組織中のｍｉＲ－３１の発現量
    であることを特徴とする請求項８または９に記載の方法。     The control value is
    MiR-31 expression level in a biological sample from a healthy person,
    Expression level of miR-31 in a biological sample collected from a colon cancer patient to be diagnosed for a certain period of time,
    The expression level of miR-31 in normal colon tissue collected from the colon cancer patient at the same time as the collection of colorectal cancer tissue from the colorectal cancer patient to be diagnosed, or normal colon tissue collected from a healthy person The method according to claim 8 or 9, wherein the expression level is miR-31.
  11. 　前記ｍｉＲ－３１が、配列番号２の塩基配列からなる配列からなるポリヌクレオチドであるか、または配列番号２の部分配列でありかつ配列番号３の塩基配列を含む配列からなるポリヌクレオチドであり、
    　上記ポリヌクレオチドの相補配列からなるか、あるいは当該相補配列において１または数個の塩基が置換または欠失してなる配列からなる第２のポリヌクレオチドを含有する
    ことを特徴とする請求項８～１０のいずれか１項に記載の方法に用いて大腸癌を診断するための組成物。     The miR-31 is a polynucleotide consisting of a sequence consisting of the base sequence of SEQ ID NO: 2 or a polynucleotide consisting of a partial sequence of SEQ ID NO: 2 and a sequence containing the base sequence of SEQ ID NO: 3,
    A second polynucleotide comprising a complementary sequence of the polynucleotide, or a sequence obtained by substituting or deleting one or several bases in the complementary sequence. A composition for diagnosing colorectal cancer using the method according to any one of the above.
  12. 　ＫＲＡＳ遺伝子およびＢＲＡＦ遺伝子の少なくとも一方に変異が生じている大腸癌細胞を候補物質とともにインキュベートする工程、および
    　インキュベートした後のｍｉＲ－３１の発現量をインキュベートする前のｍｉＲ－３１の発現量よりも低下させた候補物質を選択する工程
    を包含することを特徴とする抗ＥＧＦＲ抗体による治療に耐性である大腸癌を治療するための薬剤をスクリーニングする方法。     Incubating colon cancer cells in which at least one of KRAS gene and BRAF gene is mutated with a candidate substance, and miR-31 expression level after incubation is lower than miR-31 expression level before incubation A method for screening a drug for treating colorectal cancer that is resistant to treatment with an anti-EGFR antibody, comprising a step of selecting a candidate substance.
  13. 　前記薬剤が、抗ＥＧＦＲ抗体との組合せ療法に用いられることを特徴とする請求項１２に記載の方法。 The method according to claim 12, wherein the drug is used in combination therapy with an anti-EGFR antibody.
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