WO2020054474A1 - Method for examining rheumatoid arthritis - Google Patents

Method for examining rheumatoid arthritis Download PDF

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WO2020054474A1
WO2020054474A1 PCT/JP2019/034364 JP2019034364W WO2020054474A1 WO 2020054474 A1 WO2020054474 A1 WO 2020054474A1 JP 2019034364 W JP2019034364 W JP 2019034364W WO 2020054474 A1 WO2020054474 A1 WO 2020054474A1
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mir
rheumatoid arthritis
biomarker
group
test
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PCT/JP2019/034364
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French (fr)
Japanese (ja)
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随象 岡田
悠一 前田
敏博 岸川
沙央里 坂上
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国立大学法人大阪大学
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Priority to JP2020545923A priority Critical patent/JPWO2020054474A1/en
Publication of WO2020054474A1 publication Critical patent/WO2020054474A1/en
Priority to JP2024031786A priority patent/JP2024051168A/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • the present invention relates to a method for testing rheumatoid arthritis, a test drug for rheumatoid arthritis, a test kit for rheumatoid arthritis, and the like.
  • Rheumatoid arthritis (Rheumatoid Arthritis: (RA ⁇ [MIM 180300])) is an autoimmune systemic inflammatory disease that affects approximately 1% of the world's population. It is characterized by joint destruction due to cartilage and bone destruction, and is an autoimmune disease of unknown cause, which causes a decrease in QOL and socioeconomic loss for patients.
  • MicroRNA is a small in vivo molecule composed of 20-25 bases, and is regarded as a promising biomarker for predicting disease onset.
  • attention has been paid to autoimmune diseases including rheumatoid arthritis and an invention relating to microRNA contributing to the diagnosis of rheumatoid arthritis has been reported (Patent Document 1).
  • the present expressor has previously developed an information analysis technology, MIGWAS (microRNAmicroenrichmentASanalysis in GWAS), which integrates the results of large-scale disease genome analysis and the microRNA-target gene network on a computer (Non-Patent Document 1). ).
  • MIGWAS microRNAmicroenrichmentASanalysis in GWAS
  • This time by integrating microRNA expression profile information measured for each cell tissue into the information analysis pipeline, it has become possible to screen for biomarker microRNAs taking into account cell tissue specificity.
  • Non-patent Document 2 a biomarker microRNA candidate contributing to the diagnosis of rheumatoid arthritis was successfully identified.
  • microRNAs were examined using expression information in microRNA fractions extracted from peripheral blood mononuclear cells collected from 30 independent rheumatoid arthritis patients and 33 control groups.
  • microRNAs miR-93-5p, miR-106b-5p, miR-301b-3p, and miR-762 having different expression levels from the control group were successfully identified. As a result of further research based on this finding, the present invention has been completed.
  • the present invention includes the following embodiments.
  • Item 1 A method for testing rheumatoid arthritis, (1) detecting at least one biomarker selected from the group consisting of miR-93-5p, miR-106b-5p, miR-301b-3p, and miR-762 in a body fluid collected from a subject Process, Inspection methods, including:
  • Item 1A (1) detecting at least one biomarker selected from the group consisting of miR-93-5p, miR-106b-5p, miR-301b-3p, and miR-762 in a body fluid collected from a subject Process, And a measuring method.
  • Item 1B A method to assist in the examination of rheumatoid arthritis, (1) detecting at least one biomarker selected from the group consisting of miR-93-5p, miR-106b-5p, miR-301b-3p, and miR-762 in a body fluid collected from a subject Process, Inspection methods, including:
  • the detected biomarkers include at least one biomarker (BM1) selected from the group consisting of miR-93-5p, miR-106b-5p, and miR-301b-3p, (2a) a step of determining that the subject is suffering from rheumatoid arthritis when the amount or concentration of the BM1 detected in the step (1) is equal to or less than a cutoff value; Item 1.
  • the inspection method according to Item 1, comprising:
  • Item 3 If the detected biomarkers include miR-762 (BM2), (2b) a step of determining that the subject is suffering from rheumatoid arthritis when the amount or concentration of the BM2 detected in the step (1) is not less than a cutoff value; Item 1.
  • Item 4 When the detected biomarkers include at least one biomarker (BM1) selected from the group consisting of miR-93-5p, miR-106b-5p, and miR-301b-3p, and miR-762 (BM2) ,further, (2c) When the amount or concentration of the BM1 detected in the step (1) is equal to or less than a cutoff value, and the amount or concentration of the BM2 detected in the step (1) is equal to or more than a cutoff value.
  • BM1 biomarker
  • BM2 miR-762
  • Item 5 The test method according to any one of Items 1 to 4, wherein the body fluid is at least one selected from the group consisting of whole blood, plasma, and serum.
  • Item 6. ⁇ The method according to any one of Items 1 to 5, wherein the subject is a human.
  • a test agent for rheumatoid arthritis comprising a detection agent for at least one biomarker selected from the group consisting of miR-93-5p, miR-106b-5p, miR-301b-3p, and miR-762.
  • Item 7A Agent for detecting at least one biomarker selected from the group consisting of miR-93-5p, miR-106b-5p, miR-301b-3p, and miR-762 for use as a test agent for rheumatoid arthritis .
  • Item 7B Use of an agent for detecting at least one biomarker selected from the group consisting of miR-93-5p, miR-106b-5p, miR-301b-3p, and miR-762 for testing rheumatoid arthritis.
  • Item 7C For the manufacture of a test agent for rheumatoid arthritis, miR-93-5p, miR-106b-5p, miR-301b-3p, and a detection agent for at least one biomarker selected from the group consisting of miR-762 use.
  • a test kit for rheumatoid arthritis comprising an agent for detecting at least one biomarker selected from the group consisting of miR-93-5p, miR-106b-5p, miR-301b-3p, and miR-762.
  • Item 9 At least one biomarker selected from the group consisting of miR-93-5p, miR-106b-5p, miR-301b-3p, and miR-762 in a body fluid collected from an animal treated with the test substance A method for screening an active ingredient of a prophylactic or therapeutic agent for rheumatoid arthritis, using the amount or concentration of the drug as an index.
  • Item 10 At least one biomarker selected from the group consisting of miR-93-5p, miR-106b-5p, miR-301b-3p, and miR-762 in a body fluid collected from an animal treated with the test substance For evaluating rheumatoid arthritis inducibility or malignancy using the amount or concentration of as an index.
  • a biomarker for rheumatoid arthritis can be provided.
  • rheumatoid arthritis can be examined, an active ingredient of a preventive or therapeutic agent for rheumatoid arthritis can be screened, and rheumatoid arthritis can be induced or aggravated.
  • FIG. 9 is a view showing a screening result of Test Example 2.
  • the plot shows each miRNA.
  • the horizontal axis shows the logarithmic value of the value obtained by dividing the expression level in the rheumatoid arthritis patient group by the expression level in the control group, and the vertical axis shows the false discovery rate when negating the null hypothesis that there is no difference in the expression level. , A logarithmic conversion with a base of 10 and a negative sign.
  • FIG. 8 shows a receiver operating characteristic (ROC) curve for early rheumatoid arthritis patients (30 patients) in the case of diagnosis with hsa-miR-93-5p in Test Example 3.
  • FIG. The vertical axis indicates sensitivity, and the horizontal axis indicates 1-specificity.
  • FIG. 8 shows a receiver operating characteristic (ROC) curve for an early rheumatoid arthritis patient (30 patients) in the case of diagnosis with hsa-miR-106b-5p in Test Example 3.
  • FIG. The vertical axis indicates sensitivity, and the horizontal axis indicates 1-specificity.
  • the figure also shows the area under the curve (Area Under the Curve, AUC) calculated based on the ROC curve.
  • FIG. 9 shows a receiver operating characteristic (ROC) curve for early rheumatoid arthritis patients (30 patients) in the case of diagnosis with hsa-miR-301b-3p in Test Example 3.
  • the vertical axis indicates sensitivity, and the horizontal axis indicates 1-specificity.
  • the figure also shows the area under the curve (Area Under the Curve, AUC) calculated based on the ROC curve.
  • FIG. 8 shows a receiver operating characteristic (ROC) curve for early rheumatoid arthritis patients (30 patients) in the case of diagnosis with hsa-miR-762 in Test Example 3.
  • FIG. The vertical axis indicates sensitivity, and the horizontal axis indicates 1-specificity.
  • the figure also shows the area under the curve (Area Under the Curve, AUC) calculated based on the ROC curve.
  • Fig. 4 shows receiver operating characteristic (ROC) curves for rheumatoid arthritis patients (30 patients).
  • the vertical axis indicates sensitivity, and the horizontal axis indicates 1-specificity.
  • the figure also shows the area under the curve (Area Under the Curve, AUC) calculated based on the ROC curve.
  • FIG. 9 shows a receiver operating characteristic (ROC) curve for anti-CCP antibody-negative rheumatoid arthritis patients (14 patients) in the case of diagnosis with hsa-miR-93-5p in Test Example 3.
  • FIG. The vertical axis indicates sensitivity, and the horizontal axis indicates 1-specificity. The figure also shows the area under the curve (Area Under the Curve, AUC) calculated based on the ROC curve.
  • FIG. 8 shows a receiver operating characteristic (ROC) curve for an anti-CCP antibody-negative rheumatoid arthritis patient (14 patients) in the case of diagnosis with hsa-miR-106b-5p in Test Example 3.
  • FIG. The vertical axis indicates sensitivity, and the horizontal axis indicates 1-specificity.
  • FIG. 9 shows a receiver operating characteristic (ROC) curve for an anti-CCP antibody-negative rheumatoid arthritis patient (14 patients) in the case of diagnosis with hsa-miR-301b-3p in Test Example 3.
  • ROC receiver operating characteristic
  • FIG. The vertical axis indicates sensitivity, and the horizontal axis indicates 1-specificity.
  • the figure also shows the area under the curve (Area Under the Curve, AUC) calculated based on the ROC curve.
  • FIG. 9 shows a receiver operating characteristic (ROC) curve for an anti-CCP antibody-negative rheumatoid arthritis patient (14 patients) in the case of diagnosis with hsa-miR-762 in Test Example 3.
  • FIG. The vertical axis indicates sensitivity, and the horizontal axis indicates 1-specificity.
  • the figure also shows the area under the curve (Area (Under the Curve, AUC) calculated based on the ROC curve.
  • AUC Under the Curve
  • ROC receiver operating characteristic
  • the present invention relates to a method for testing rheumatoid arthritis, comprising: (1) miR-93-5p, miR-106b-5p, miR- in a body fluid collected from a subject; A step of detecting at least one biomarker selected from the group consisting of 301b-3p and miR-762, including a step of detecting the biomarker (herein, also referred to as "the rheumatoid arthritis test method of the present invention”. There is.) Hereinafter, this will be described.
  • the type of rheumatoid arthritis to be tested is not particularly limited. Rheumatoid arthritis of all classes, grades and stages in various classification criteria for rheumatoid arthritis is to be tested. Rheumatoid arthritis also includes anti-CCP antibody negative rheumatoid arthritis.
  • the subject is the target organism of the test method of the present invention, and the species is not particularly limited.
  • the species of the subject include various mammals such as humans, monkeys, mice, rats, dogs, cats and rabbits, and humans are preferred.
  • the state of the subject is not particularly limited.
  • a subject for example, a sample that is unknown whether it is suffering from rheumatoid arthritis, a sample that has already been determined to be suffering from rheumatoid arthritis, a method that is already suffering from rheumatoid arthritis by another method
  • Examples include a sample that has been determined and a sample that is undergoing treatment for rheumatoid arthritis.
  • the body fluid is not particularly limited.
  • the body fluid include whole blood, serum, plasma, cerebrospinal fluid, saliva, synovial fluid, urine, tissue fluid (including bronchoalveolar lavage fluid), sweat, tears, sputum, nasal discharge, and the like, and preferably whole blood, serum , Plasma and cerebrospinal fluid, more preferably whole blood, serum and plasma.
  • the body fluid is preferably a body fluid containing peripheral blood mononuclear cells. The body fluid may be used alone or in combination of two or more.
  • Body fluid can be collected from a subject by a method known to those skilled in the art.
  • whole blood can be collected by blood collection using a syringe or the like.
  • Serum is a portion of whole blood from which blood cells and specific blood clotting factors have been removed, and can be obtained, for example, as a supernatant after coagulating whole blood.
  • Plasma is a portion of whole blood from which blood cells have been removed, and can be obtained, for example, as a supernatant when subjected to centrifugation under conditions that do not allow whole blood to coagulate.
  • the detection target in step (1) is at least one biomarker selected from the group consisting of miR-93-5p, miR-106b-5p, miR-301b-3p, and miR-762 (in the present specification, These are sometimes collectively referred to as “target biomarkers.”).
  • the target biomarker is a biomarker whose expression level is changed in rheumatoid arthritis, and using this as an index, rheumatoid arthritis can be distinguished.
  • At least one biomarker (BM1) selected from the group consisting of miR-93-5p, miR-106b-5p, and miR-301b-3p has an amount in a rheumatoid arthritis sample in a healthy sample.
  • the target biomarker is lower than the amount.
  • miR-762 is a target biomarker whose amount in rheumatoid arthritis samples is higher than that in healthy samples.
  • the base sequence of the target biomarker can be specified in a known database (for example, miRBase: http://www.mirbase.org/).
  • its base sequence is as follows. > hsa-miR-93-5p MIMAT0000093 CAAAGUGCUGUUCGUGCAGGUAG (SEQ ID NO: 1) > hsa-miR-106b-5p MIMAT0000680 UAAAGUGCUGACAGUGCAGAU (SEQ ID NO: 2) > hsa-miR-301b-3p MIMAT0004958 CAGUGCAAUGAUAUUGUCAAAGC (SEQ ID NO: 3) > hsa-miR-762 MIMAT0010313 GGGGCUGGGGCCGGGGCCGAGC (SEQ ID NO: 4).
  • the target biomarker is preferably a mature miRNA, but may be a precursor (eg, pri-miRNA, pre-miRNA, etc.).
  • the number of target biomarkers in step (1) may be only one, but may be two or more, three or more, or a combination of four. By combining more target biomarkers, it becomes possible to more accurately perform an examination of rheumatoid arthritis and the like.
  • the target biomarker in the step (1) preferably contains at least one selected from the group consisting of miR-106b-5p, miR-301b-3p, and miR-762.
  • Detection is usually performed by measuring the amount or concentration of the target biomarker.
  • concentration is not limited to the absolute concentration, but may be a relative concentration, a weight per unit volume, or raw data measured to know the absolute concentration.
  • the method for detecting the target biomarker is not particularly limited as long as it can specifically detect a part or all of the target biomarker.
  • Specific examples of the detection method include, for example, RNA-seq analysis, RT-PCR, nucleic acid chip analysis, and Northern blot.
  • RNA-seq analysis method specifically, cDNA is prepared from RNA from the subject in accordance with a conventional method, sequence analysis is performed using a next-generation sequencer, etc., and based on the obtained data, A method of obtaining expression level information by performing mapping, gene expression analysis, expression level analysis, and the like.
  • a cDNA is prepared from RNA derived from a subject according to a conventional method, and a pair of primers (the cDNA (- A method for detecting the amplified double-stranded DNA obtained by hybridizing the positive strand binding to the strand) and the reverse strand binding to the + strand) and performing PCR according to a conventional method can be exemplified.
  • the detection of the amplified double-stranded DNA was performed by detecting the labeled double-stranded DNA produced by performing the above PCR using primers that had been labeled with RI or a fluorescent substance in advance.
  • a method in which double-stranded DNA is transferred to a nylon membrane or the like according to a conventional method, hybridized with a labeled probe and detected, or the like can be used.
  • nucleic acid chip analysis When using nucleic acid chip analysis, prepare a nucleic acid chip to which a nucleic acid probe (single-stranded or double-stranded) is attached, and hybridize it with RNA derived from a subject or a nucleic acid prepared from the RNA by a conventional method. There can be mentioned a method of detecting a double strand formed by soybean.
  • the above-described expression system in which the probe is labeled with a radioisotope (eg, 32P, 33P: RI) or a fluorescent substance and then transferred to a nylon membrane or the like according to a conventional method.
  • a radioisotope eg, 32P, 33P: RI
  • the duplex of the formed diagnostic agent and the mRNA derived from the subject's sample is converted into a signal derived from the probe label (a labeling substance such as RI or a fluorescent substance).
  • a labeling substance such as RI or a fluorescent substance
  • the test method of the present invention including the step (1), it is possible to provide the amount and / or concentration of the target biomarker, which is a detection index for rheumatoid arthritis, and thereby assist in detecting rheumatoid arthritis and the like. it can.
  • test results of the test method of the present invention including the step (1) are used to determine the therapeutic effect, elucidate the pathology of rheumatoid arthritis, predict the prognosis of rheumatoid arthritis, stratify patients, select a treatment method (individualized medicine, treatment responsiveness) And so on.
  • the detected biomarker is at least one biomarker (BM1) selected from the group consisting of miR-93-5p, miR-106b-5p, and miR-301b-3p. If you include (2a) a step of determining that the subject is suffering from rheumatoid arthritis when the amount or concentration of the BM1 detected in the step (1) is equal to or less than a cutoff value; It is preferable to include According to the test method of the present invention including the step 2a, rheumatoid arthritis can be determined.
  • BM1 biomarker
  • the test method of the present invention further comprises, when the detected biomarker includes miR-762 (BM2), (2b) a step of determining that the subject is suffering from rheumatoid arthritis when the amount or concentration of the BM2 detected in the step (1) is not less than a cutoff value; It is preferable to include According to the test method of the present invention including the step 2b, it is possible to determine rheumatoid arthritis.
  • BM2 miR-762
  • the detected biomarker is at least one biomarker (BM1) selected from the group consisting of miR-93-5p, miR-106b-5p, and miR-301b-3p. , And miR-762 (BM2), (2c)
  • BM1 biomarker
  • BM2 miR-762
  • the amount or concentration of the BM1 detected in the step (1) is equal to or less than a cutoff value
  • the amount or concentration of the BM2 detected in the step (1) is equal to or more than a cutoff value.
  • the step of determining that the subject is suffering from rheumatoid arthritis It is preferable to include According to the test method of the present invention including the step 2c, rheumatoid arthritis can be determined.
  • Cut-off value, sensitivity, specificity, positive predictive value can be appropriately set by those skilled in the art from the viewpoint of negative predictive value, for example, in a body fluid collected from a subject not suffering from rheumatoid arthritis
  • a predetermined value or a predetermined value can be used.
  • the cut-off value is, for example, the amount and / or concentration of the target biomarker in a body fluid collected from a subject not suffering from rheumatoid arthritis (for a plurality of subjects, an average value, a median, etc.), for example,
  • the value can be 0.5 to 1.5 times.
  • the cutoff value is determined based on, for example, the amount and / or concentration of the target biomarker in a past sample of the same sample. By setting these values, the therapeutic effect can be determined.
  • the test method of the present invention is further added to the rheumatoid arthritis doctor.
  • the test method of the present invention can more accurately detect rheumatoid arthritis, by combining the above-described steps with the test method of the present invention, it is possible to more efficiently and more accurately diagnose "affected by rheumatoid arthritis". .
  • test method of the present invention is further applied, or the above-mentioned “2. If it is diagnosed as having rheumatoid arthritis as described in “Diagnosis with Higher Accuracy”, the combination of the test method of the present invention and the step of applying a diagnosis by a physician, By performing a step of treating the disease for a subject determined or diagnosed as having rheumatism, the disease of the subject can be treated.
  • Step 3 is performed for the test method of the present invention or for a combination of the test method of the present invention and a step of applying a diagnosis by a doctor.
  • a subject suffering from rheumatoid arthritis can be treated more efficiently and more reliably.
  • the method of treating rheumatoid arthritis is not particularly limited, but typically includes medication.
  • the drug used for the drug treatment is not particularly limited, and examples thereof include antirheumatic drugs, biological preparations (biopharmaceuticals), non-steroidal anti-inflammatory drugs, and steroids (corticosteroids).
  • antirheumatic drugs include antirheumatic drugs, biological preparations (biopharmaceuticals), non-steroidal anti-inflammatory drugs, and steroids (corticosteroids).
  • One, two, or three or more pharmaceuticals can be used in combination.
  • test agent for rheumatoid arthritis provides at least one biomarker selected from the group consisting of miR-93-5p, miR-106b-5p, miR-301b-3p, and miR-762.
  • a test agent for rheumatoid arthritis containing a detection agent herein, sometimes referred to as “test agent of the present invention”.
  • test agent of the present invention a detection agent
  • test agent of the present invention a detection agent of the present invention
  • MiR-93-5p, miR-106b-5p, miR-301b-3p, miR-762, rheumatoid arthritis and the like are the same as defined in the above “1. Method for testing rheumatoid arthritis”.
  • the detection agent of the present invention is not particularly limited as long as it can specifically detect the target biomarker.
  • Examples of the detection agent include primers and probes for the target biomarker.
  • the detection agent of the present invention may be modified as long as its function is not significantly impaired.
  • modifications include addition of a label, for example, a fluorescent dye, an enzyme, a protein, a radioisotope, a chemiluminescent substance, biotin, and the like.
  • the fluorescent dye used in the present invention generally used are those which label nucleotides and are used for the detection and quantification of nucleic acids.
  • HEX 4,7,2 ', 4', 5 ', 7 '-hexachloro-6-carboxylfluorescein, green fluorescent dye
  • fluorescein fluorescein
  • NED trade name, manufactured by Applied Biosystems, yellow fluorescent dye
  • 6-FAM trade name, manufactured by Applied Biosystems, yellow
  • Green fluorescent dye rhodamine or a derivative thereof (eg, tetramethylrhodamine (TMR)), but is not limited thereto.
  • a method for labeling nucleotides with a fluorescent dye an appropriate one of known labeling methods can be used [see Nature Biotechnology, ⁇ 14, ⁇ 303-308 ⁇ (1996)].
  • a commercially available fluorescent labeling kit can be used (for example, oligonucleotide ECL '3'-oligolabeling system, manufactured by Amersham Pharmacia).
  • test agent of the present invention can also be used by immobilizing it on any solid phase. Therefore, the test agent of the present invention can be provided in the form of a substrate on which a detection agent is immobilized (for example, a microarray chip on which a probe is immobilized).
  • the solid phase used for immobilization is not particularly limited as long as it can immobilize a polynucleotide or the like, and examples thereof include a glass plate, a nylon membrane, microbeads, a silicon chip, a capillary, and other substrates. Can be.
  • Immobilization of the detection agent on the solid phase is not particularly limited.
  • the immobilization method is well known in the art depending on the type of the immobilized probe, for example, using a commercially available spotter (for example, Amersham) if it is a microarray [for example, photolithographic technology (Affymetrix), In-situ synthesis of oligonucleotides by inkjet technology (Rosetta Inpharmatics).
  • the primer and probe are not particularly limited as long as they selectively (specifically) recognize the target biomarker and the nucleic acid derived therefrom.
  • “selectively (specifically) recognizes” means, for example, that a target biomarker can be specifically detected in Northern blotting, and a target biomarker or a nucleic acid derived therefrom in RT-PCR. (CDNA, etc.) is specifically amplified, but is not limited thereto, provided that a person skilled in the art can determine that the above-mentioned detected substance or amplified substance is derived from the target biomarker. Good.
  • primers and probes include the polynucleotide described in (a) below and the polynucleotide described in (b) below: (A) a polynucleotide having at least 15 consecutive nucleotides in the base sequence of the target biomarker and / or a polynucleotide complementary to the polynucleotide; and (b) a base sequence of the target biomarker or a base sequence complementary thereto. At least one selected from the group consisting of polynucleotides having at least 15 bases that hybridize under stringent conditions is included.
  • a complementary polynucleotide or a complementary nucleotide sequence refers to a full-length sequence of a polynucleotide comprising a nucleotide sequence of a target biomarker, or a nucleotide sequence of at least 15 nucleotides continuous in the nucleotide sequence.
  • a polynucleotide or base having a base complementary relationship to its partial sequence (for convenience, these are also referred to as “positive chains”) based on base pairing such as A: T and G: C. Means an array.
  • such a complementary strand is not limited to a case where it forms a completely complementary sequence with the base sequence of the target positive strand, but has a complementary relationship that allows hybridization with the target positive strand under stringent conditions.
  • the stringent conditions bind the complex or probe as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques in Enzymology, Vol. 152, Academic Press, San Diego CA). It can be determined based on the melting temperature (Tm) of the nucleic acid. For example, as washing conditions after hybridization, there can be usually mentioned conditions of about “1 ⁇ SSC, 0.1% SDS, 37 ° C.”.
  • the complementary strand is preferably one that maintains a hybridized state with the target positive strand even when washed under such conditions.
  • the more stringent hybridization conditions are about 0.5 ⁇ SSC, 0.1% SDS, 42 ° C.
  • the more stringent hybridization conditions are about 0.1 ⁇ SSC, 0.1% SDS, 65 ° C.
  • a complementary strand a strand consisting of a nucleotide sequence completely complementary to the nucleotide sequence of the target positive strand, and at least 90%, preferably 95%, more preferably A chain consisting of a base sequence having 98% or more, more preferably 99% or more identity can be exemplified.
  • Primers and probes can be designed based on the base sequence of the target biomarker, for example, using various design programs. Specifically, a primer or probe candidate sequence obtained by subjecting the base sequence of the target biomarker to a design program, or a sequence partially containing at least the sequence can be used as the primer or probe.
  • the base length of the primer or probe is not particularly limited as long as it has a length of at least 15 consecutive bases as described above, and can be appropriately set according to the application.
  • the base length is, for example, 15 to 35 bases when used as a primer, and for example, 15 to 35 bases when used as a probe.
  • the test agent of the present invention may contain other detection agents (eg, a probe for detecting a nucleic acid such as another miRNA, an antibody, etc.) other than the detection agent of the present invention.
  • the test agent of the present invention may be a test agent capable of testing other diseases and conditions in addition to rheumatoid arthritis.
  • the detection agent of the present invention is included as a detection agent for a rheumatoid arthritis test.
  • the test agent of the present invention is, in one aspect thereof, a test agent for rheumatoid arthritis, which contains a detection agent for rheumatoid arthritis test comprising the detection agent of the present invention.
  • the test agent of the present invention may be in the form of a composition.
  • the composition may contain other components as necessary.
  • Other components include, for example, bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, fragrances And chelating agents.
  • the test agent of the present invention may be in the form of a kit.
  • the kit may contain, besides the above-mentioned detection agent or the above-mentioned composition containing the same, a kit that can be used for detection of a target biomarker in a body fluid of a subject.
  • a kit that can be used for detection of a target biomarker in a body fluid of a subject.
  • Specific examples of such a substance include various reagents (for example, a buffer solution or the like) and instruments (for example, instruments for purifying and separating body fluids).
  • the present invention relates to, in one embodiment, miR-93-5p, miR-106b-5p, miR in a body fluid collected from an animal treated with a test substance. -301b-3p, and an amount or concentration of at least one biomarker selected from the group consisting of miR-762 as an index, a method for screening an active ingredient of a preventive or therapeutic agent for rheumatoid arthritis (herein, It may also be referred to as “the active ingredient screening method of the present invention.” Hereinafter, this will be described.
  • miR-93-5p For body fluid, miR-93-5p, miR-106b-5p, miR-301b-3p, miR-762, rheumatoid arthritis, measurement of the amount or concentration of a target biomarker, etc., see “1. Is the same as the definition in
  • Animal species are not particularly limited. Examples of animal species include various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits.
  • any of naturally occurring compounds or artificially produced compounds can be widely used.
  • a purified compound but also a composition in which various kinds of compounds are mixed, and an extract of animals and plants can be used.
  • the compound is not limited to a low molecular compound, but also includes a high molecular compound such as a protein, a nucleic acid, and a polysaccharide.
  • the active ingredient screening method of the present invention the biomarker as an indicator, at least one selected from the group consisting of miR-93-5p, miR-106b-5p, and miR-301b-3p If the value of the above index is higher than the amount or concentration (control value) of the corresponding biomarker in a body fluid collected from an animal not treated with the test substance, Selecting a test substance as an active ingredient of a prophylactic or therapeutic agent for rheumatoid arthritis (or a candidate substance as an active ingredient of a prophylactic or therapeutic agent for rheumatoid arthritis).
  • the biomarker serving as an index is miR-762 (BM2)
  • the value of the index is collected from an animal that has not been treated with the test substance.
  • the test substance is used as an active ingredient of a prophylactic or therapeutic agent for rheumatoid arthritis (or a candidate for an active ingredient of a preventive or therapeutic agent for rheumatoid arthritis).
  • a prophylactic or therapeutic agent for rheumatoid arthritis or a candidate for an active ingredient of a preventive or therapeutic agent for rheumatoid arthritis.
  • the corresponding biomarker means the same miRNA as the target biomarker used as an index.
  • “High” means that, for example, the index value is twice, five times, ten times, twenty times, fifty times, and one hundred times the control value.
  • Low means, for example, that the index value is 1/2, 1/5, 1/10, 1/20, 1/50, 1/100 of the control value.
  • the present invention in one embodiment, in a body fluid collected from an animal treated with a test substance, miR-93-5p, miR-106b-5p, miR-301b -3p, and a method for evaluating the induction or malignancy of rheumatoid arthritis, using as an index the amount or concentration of at least one biomarker selected from the group consisting of miR-762 (in the present specification, Toxicity evaluation method ").
  • miR-762 in the present specification, Toxicity evaluation method "
  • Body fluid miR-93-5p, miR-106b-5p, miR-301b-3p, miR-762, rheumatoid arthritis, measurement of the amount or concentration of the target biomarker, animal species, test substance, etc. This is the same as the definition in “1. Method for testing rheumatoid arthritis” and “6. Method for screening active ingredient of preventive or therapeutic agent for rheumatoid arthritis”.
  • the toxicity evaluation method of the present invention more specifically, the biomarker as an index, miR-93-5p, miR-106b-5p, and at least one selected from the group consisting of miR-301b-3p
  • the biomarker as an index, miR-93-5p, miR-106b-5p, and at least one selected from the group consisting of miR-301b-3p
  • BM1 a biomarker
  • the value of the above index is lower than the amount or concentration (control value) of the corresponding biomarker in a body fluid collected from an animal not treated with the test substance, Determining the substance to be rheumatoid arthritis-induced or malignant.
  • the value of the index is obtained from an animal not treated with the test substance. If the amount or concentration of the corresponding biomarker in the body fluid is higher than the control value, the test substance is determined to be rheumatoid arthritis-inducing or malignant.
  • the corresponding biomarker means the same miRNA as the target biomarker used as an index.
  • “High” means that, for example, the index value is twice, five times, ten times, twenty times, fifty times, and one hundred times the control value.
  • Low means, for example, that the index value is 1/2, 1/5, 1/10, 1/20, 1/50, 1/100 of the control value.
  • Test example 1 Screening for rheumatoid arthritis biomarkers 1
  • a list of target genes predicted using a plurality of algorithms is created for microRNAs registered in the database.
  • microRNA expression profile information measured for each cell tissue registered in the database is obtained, the microRNA expression information is normalized for each tissue, and a microRNA having a high expression level is identified in a tissue-specific manner.
  • the MlGWAS analysis pipeline was applied to the results of a large-scale genome-wide association analysis of rheumatoid arthritis (19243 patients and 61565 controls), and 48 microRNAs were identified as biomarker candidates.
  • Test example 2 Screening for rheumatoid arthritis biomarkers 2 The expression information in microRNA fractions extracted from peripheral blood mononuclear cells collected from 30 independent rheumatoid arthritis patients and 33 control groups was examined. When detailed expression information was obtained using the next-generation sequencer HiSeq2500, microRNAs whose expression levels in rheumatoid arthritis were significantly different from those of healthy individuals, that is, 94 microRNAs were identified as biomarker candidates.
  • Test example 3 Analysis of Screening Results
  • Four miRNAs overlapping the candidate miRNA obtained in Test Example 1 and the candidate miRNA obtained in Test Example 2 were extracted as highly reliable biomarkers (hsa-miR-93- 5p, hsa-miR-106b-5p, hsa-miR-301b-3p, hsa-miR-762, Q value after FDR correction ⁇ 0.05).
  • FIG. 1 shows a Volcano plot in which the expression levels of each miRNA in rheumatoid arthritis patients and healthy subjects were evaluated.
  • microRNAs hsa-miR-93-5p, hsa-miR-106b-5p and hsa-miR-301b-3p
  • the expression level was significantly lower in the patient group
  • one microRNA hsa-miR-93-5p, hsa-miR-106b-5p
  • miR-762 miR-762
  • the microRNAs included in the present invention were compared with those of a rheumatoid arthritis patient group and a control group.
  • the ratios (odds ratio) are 0.696-fold, 0.570-fold, 0.516-fold, and 2.23-fold, respectively.
  • Table 1 The detailed data is shown in Table 1.
  • the above analysis was also performed on anti-CCP antibody negative rheumatoid arthritis patients.
  • the microRNA hsa-miR-93-5p, hsa-miR-106b-5p, hsa-miR-301b-3p, hsa-miR-762 included in the present invention is a group of patients with anti-CCP antibody negative rheumatoid arthritis and It has different expression levels with the control group, the ratios (odds ratio) are 0.733, 0.641, 0.567 and 2.65, respectively. Table 2 shows the detailed data.

Abstract

The present invention addresses the problem of providing a novel biomarker for rheumatoid arthritis and a method for using the same. This problem is solved by a method for examining rheumatoid arthritis, said method comprising (1) a step for detecting at least one kind of biomarker selected from the group consisting of miR-93-5p, miR-106b-5p, miR-301b-3p and miR-762 in a body fluid sampled from a subject.

Description

関節リウマチを検査する方法How to test for rheumatoid arthritis
　本発明は、関節リウマチを検査する方法、関節リウマチの検査薬、関節リウマチの検査キット等に関する。 (4) The present invention relates to a method for testing rheumatoid arthritis, a test drug for rheumatoid arthritis, a test kit for rheumatoid arthritis, and the like.
　関節リウマチ（Rheumatoid Arthritis ：（RA [ MIM 180300 ]）は、自己免疫性の全身炎症性疾患であり、罹患者は全世界の人口の約1％である。関節リウマチは、滑膜の炎症とそれに伴う軟骨・骨破壊による関節破壊を特徴とする。原因不明の自己免疫疾患であり、患者のQOL低下と社会経済的損失を生じている。 Rheumatoid arthritis (Rheumatoid Arthritis: (RA 自己 [MIM 180300])) is an autoimmune systemic inflammatory disease that affects approximately 1% of the world's population. It is characterized by joint destruction due to cartilage and bone destruction, and is an autoimmune disease of unknown cause, which causes a decrease in QOL and socioeconomic loss for patients.
　マイクロRNAは20-25塩基で構成される生体内小分子であり、疾患発症を予測するバイオマーカーとして有望視されている。特に、関節リウマチを含む自己免疫疾患において注目を集めており、関節リウマチの診断に寄与するマイクロRNAに関する発明は報告が認められる（特許文献１）。 MicroRNA is a small in vivo molecule composed of 20-25 bases, and is regarded as a promising biomarker for predicting disease onset. In particular, attention has been paid to autoimmune diseases including rheumatoid arthritis, and an invention relating to microRNA contributing to the diagnosis of rheumatoid arthritis has been reported (Patent Document 1).
　診断に寄与するマイクロRNAのスクリーニングは、これまで数サンプル～数十サンプルという比較的少数のサンプルを対象に、マイクロアレイ技術等を用いて、患者群および対照群におけるマイクロRNAの生体内発現量を実験的に測定することで実施されていた。しかし、これらの手法では、予め設計された数百程度のマイクロRNAのみがスクジーニング対象となっていた。マイクロRNAは生体内に数千種類存在すると報告されており、より広範囲に生体内マイクロRNAをスクリーニングする技術の開発と適応が必要とされていた。また、ゲノムワイド関連解析を代表とする大規模疾患ゲノム解析が実地され、数万人から数十万人というサンプルにおける疾患ゲノム情報が公開されている。これらの大規模サンプルにおける疾患ゲノム情報を活用した生体内マイクロRNAスクリーニング技術の開発も必要とされていた。 Screening of microRNAs that contribute to diagnosis has been performed on a relatively small number of samples, ranging from several to several tens of samples, using microarray technology to test the in vivo expression levels of microRNAs in patient and control groups. It was carried out by measuring it. However, in these methods, only about several hundred microRNAs designed in advance are targeted for squeezing. It has been reported that there are several thousand types of microRNAs in living organisms, and the development and adaptation of techniques for screening microRNAs in vivo more widely have been required. In addition, large-scale disease genome analysis represented by genome-wide association analysis has been practiced, and disease genome information in samples of tens to hundreds of thousands of people has been disclosed. Development of in vivo microRNA screening technology utilizing disease genome information in these large-scale samples was also required.
国際公開第2004/034685号International Publication No. 2004/034685
　本発明は、関節リウマチの新規バイオマーカー及びその利用方法を提供することを課題とする。 。 It is an object of the present invention to provide a novel biomarker for rheumatoid arthritis and a method for using the same.
　本発現者は以前、大規模疾患ゲノム解析結果と、マイクロRNA－標的遺伝子ネットワークをコンピューター上で統合する、情報解析技術、MIGWAS（microRNA enrichment analysis in GWAS）を開発してしいる（非特許文献１）。今回新規に、細胞組織別に測定されたマイクロRNA発現プロファイル情報を情報解析パイプラインに統合することで、細胞組織特異性を考慮した、バイオマーカーマイクロRNAのスクリーニングが可能となった。関節リウマチの大規模ゲノムワイド関連解析結果（非特許文献２）に当該パイプラインを適用することで、関節リウマチの診断に寄与するバイオマーカーマイクロRNA候補を同定することに成功した。さらに、これらの候補マイクロRNAを、独立した関節リウマチ患者群30名および対照群33名から採取された末梢血単核球より抽出したマイクロRNA分画における発現情報を用いて検討し、患者群と対照群とで異なった発現レベルを有する4つのマイクロRNA（miR-93-5p、miR-106b-5p、miR-301b-3p、及びmiR-762）を同定することに成功した。この知見に基づいて、さらに研究を進めた結果、本発明が完成した。 The present expressor has previously developed an information analysis technology, MIGWAS (microRNAmicroenrichmentASanalysis in GWAS), which integrates the results of large-scale disease genome analysis and the microRNA-target gene network on a computer (Non-Patent Document 1). ). This time, by integrating microRNA expression profile information measured for each cell tissue into the information analysis pipeline, it has become possible to screen for biomarker microRNAs taking into account cell tissue specificity. By applying the pipeline to the results of a large-scale genome-wide association analysis of rheumatoid arthritis (Non-patent Document 2), a biomarker microRNA candidate contributing to the diagnosis of rheumatoid arthritis was successfully identified. In addition, these candidate microRNAs were examined using expression information in microRNA fractions extracted from peripheral blood mononuclear cells collected from 30 independent rheumatoid arthritis patients and 33 control groups. Four microRNAs (miR-93-5p, miR-106b-5p, miR-301b-3p, and miR-762) having different expression levels from the control group were successfully identified. As a result of further research based on this finding, the present invention has been completed.
　即ち、本発明は、下記の態様を包含する。 That is, the present invention includes the following embodiments.
　項１．　関節リウマチを検査する方法であって、
（1）被検体から採取された体液における、miR-93-5p、miR-106b-5p、miR-301b-3p、及びmiR-762からなる群より選択される少なくとも1種のバイオマーカーを検出する工程、
を含む、検査方法。 Item 1. A method for testing rheumatoid arthritis,
(1) detecting at least one biomarker selected from the group consisting of miR-93-5p, miR-106b-5p, miR-301b-3p, and miR-762 in a body fluid collected from a subject Process,
Inspection methods, including:
　　項１Ａ．（1）被検体から採取された体液における、miR-93-5p、miR-106b-5p、miR-301b-3p、及びmiR-762からなる群より選択される少なくとも1種のバイオマーカーを検出する工程、
を含む、測定方法。 Item 1A. (1) detecting at least one biomarker selected from the group consisting of miR-93-5p, miR-106b-5p, miR-301b-3p, and miR-762 in a body fluid collected from a subject Process,
And a measuring method.
　　項１Ｂ．　関節リウマチの検査を補助する方法であって、
（1）被検体から採取された体液における、miR-93-5p、miR-106b-5p、miR-301b-3p、及びmiR-762からなる群より選択される少なくとも1種のバイオマーカーを検出する工程、
を含む、検査方法。 Item 1B. A method to assist in the examination of rheumatoid arthritis,
(1) detecting at least one biomarker selected from the group consisting of miR-93-5p, miR-106b-5p, miR-301b-3p, and miR-762 in a body fluid collected from a subject Process,
Inspection methods, including:
　項２．　検出したバイオマーカーがmiR-93-5p、miR-106b-5p、及びmiR-301b-3pからなる群より選択される少なくとも1種のバイオマーカー（BM1）を含む場合、さらに、
（2a）前記工程（1）で検出された前記BM1の量又は濃度がカットオフ値以下である場合に、前記被検体が関節リウマチに罹患していると判定する工程、
を含む、項１に記載の検査方法。 Item 2. When the detected biomarkers include at least one biomarker (BM1) selected from the group consisting of miR-93-5p, miR-106b-5p, and miR-301b-3p,
(2a) a step of determining that the subject is suffering from rheumatoid arthritis when the amount or concentration of the BM1 detected in the step (1) is equal to or less than a cutoff value;
Item 1. The inspection method according to Item 1, comprising:
　項３．　検出したバイオマーカーがmiR-762（BM2）を含む場合、さらに、
（2b）前記工程（1）で検出された前記BM2の量又は濃度がカットオフ値以上である場合に、前記被検体が関節リウマチに罹患していると判定する工程、
を含む、項１に記載の検査方法。 Item 3. If the detected biomarkers include miR-762 (BM2),
(2b) a step of determining that the subject is suffering from rheumatoid arthritis when the amount or concentration of the BM2 detected in the step (1) is not less than a cutoff value;
Item 1. The inspection method according to Item 1, comprising:
　項４．　検出したバイオマーカーがmiR-93-5p、miR-106b-5p、及びmiR-301b-3pからなる群より選択される少なくとも1種のバイオマーカー（BM1）、並びにmiR-762（BM2）を含む場合、さらに、
（2c）前記工程（1）で検出された前記BM1の量又は濃度がカットオフ値以下であり、且つ前記工程（1）で検出された前記BM2の量又は濃度がカットオフ値以上である場合に、前記被検体が関節リウマチに罹患していると判定する工程、
を含む、項１に記載の検査方法。 Item 4. When the detected biomarkers include at least one biomarker (BM1) selected from the group consisting of miR-93-5p, miR-106b-5p, and miR-301b-3p, and miR-762 (BM2) ,further,
(2c) When the amount or concentration of the BM1 detected in the step (1) is equal to or less than a cutoff value, and the amount or concentration of the BM2 detected in the step (1) is equal to or more than a cutoff value. In the step of determining that the subject is suffering from rheumatoid arthritis,
Item 1. The inspection method according to Item 1, comprising:
　項５．　前記体液が全血、血漿、及び血清からなる群より選択される少なくとも1種である、項１～４のいずれかに記載の検査方法。 Item 5. Item 5. The test method according to any one of Items 1 to 4, wherein the body fluid is at least one selected from the group consisting of whole blood, plasma, and serum.
　項６．　前記被検体がヒトである、項１～５のいずれかに記載の検査方法。 Item 6.検 査 The method according to any one of Items 1 to 5, wherein the subject is a human.
　項７．　miR-93-5p、miR-106b-5p、miR-301b-3p、及びmiR-762からなる群より選択される少なくとも1種のバイオマーカーの検出剤を含む、関節リウマチの検査薬。 Item 7.検 査 A test agent for rheumatoid arthritis, comprising a detection agent for at least one biomarker selected from the group consisting of miR-93-5p, miR-106b-5p, miR-301b-3p, and miR-762.
　　項７Ａ．　関節リウマチの検査薬としての使用のための、miR-93-5p、miR-106b-5p、miR-301b-3p、及びmiR-762からなる群より選択される少なくとも1種のバイオマーカーの検出剤。 Item 7A. Agent for detecting at least one biomarker selected from the group consisting of miR-93-5p, miR-106b-5p, miR-301b-3p, and miR-762 for use as a test agent for rheumatoid arthritis .
　　項７Ｂ．関節リウマチを検査するための、miR-93-5p、miR-106b-5p、miR-301b-3p、及びmiR-762からなる群より選択される少なくとも1種のバイオマーカーの検出剤の使用。 Item 7B. Use of an agent for detecting at least one biomarker selected from the group consisting of miR-93-5p, miR-106b-5p, miR-301b-3p, and miR-762 for testing rheumatoid arthritis.
　　項７Ｃ．関節リウマチの検査薬の製造のための、miR-93-5p、miR-106b-5p、miR-301b-3p、及びmiR-762からなる群より選択される少なくとも1種のバイオマーカーの検出剤の使用。 Item 7C. For the manufacture of a test agent for rheumatoid arthritis, miR-93-5p, miR-106b-5p, miR-301b-3p, and a detection agent for at least one biomarker selected from the group consisting of miR-762 use.
　項８．　miR-93-5p、miR-106b-5p、miR-301b-3p、及びmiR-762からなる群より選択される少なくとも1種のバイオマーカーの検出剤を含む、関節リウマチの検査キット。 Item 8.検 査 A test kit for rheumatoid arthritis, comprising an agent for detecting at least one biomarker selected from the group consisting of miR-93-5p, miR-106b-5p, miR-301b-3p, and miR-762.
　項９．　被検物質で処理された動物から採取された体液における、miR-93-5p、miR-106b-5p、miR-301b-3p、及びmiR-762からなる群より選択される少なくとも1種のバイオマーカーの量又は濃度を指標とする、関節リウマチの予防又は治療剤の有効成分のスクリーニング方法。 Item 9. At least one biomarker selected from the group consisting of miR-93-5p, miR-106b-5p, miR-301b-3p, and miR-762 in a body fluid collected from an animal treated with the test substance A method for screening an active ingredient of a prophylactic or therapeutic agent for rheumatoid arthritis, using the amount or concentration of the drug as an index.
　項１０．　被検物質で処理された動物から採取された体液における、miR-93-5p、miR-106b-5p、miR-301b-3p、及びmiR-762からなる群より選択される少なくとも1種のバイオマーカーの量又は濃度を指標とする、関節リウマチの誘発性又は増悪性の評価方法。 Item 10. At least one biomarker selected from the group consisting of miR-93-5p, miR-106b-5p, miR-301b-3p, and miR-762 in a body fluid collected from an animal treated with the test substance For evaluating rheumatoid arthritis inducibility or malignancy using the amount or concentration of as an index.
　本発明によれば、関節リウマチのバイオマーカーを提供することができる。該バイオマーカーを利用することにより、関節リウマチの検査、関節リウマチの予防又は治療剤の有効成分のスクリーニング、関節リウマチの誘発性又は増悪性の評価等が可能になり得る。 According to the present invention, a biomarker for rheumatoid arthritis can be provided. By using the biomarker, rheumatoid arthritis can be examined, an active ingredient of a preventive or therapeutic agent for rheumatoid arthritis can be screened, and rheumatoid arthritis can be induced or aggravated.
試験例2のスクリーニング結果を示す図である。プロットは各々のmiRNAを示す。横軸は、関節リウマチの患者群における発現量を対照群における発現量で除した値の対数値を示し、縦軸は発現量に差がないという帰無仮説を否定する際の偽発見率を、底を10とした対数変換を行い負の符号をつけたものである。FIG. 9 is a view showing a screening result of Test Example 2. The plot shows each miRNA. The horizontal axis shows the logarithmic value of the value obtained by dividing the expression level in the rheumatoid arthritis patient group by the expression level in the control group, and the vertical axis shows the false discovery rate when negating the null hypothesis that there is no difference in the expression level. , A logarithmic conversion with a base of 10 and a negative sign. 試験例3における、hsa-miR-93-5pで診断する場合の、早期関節リウマチ患者（30名）に対する受信者動作特性（ROC）曲線を示す。縦軸は感度を示し、横軸は1－特異度を示す。図中にROC曲線に基づいて算出された曲線下面積（Area Under the Curve, AUC）も示す。FIG. 8 shows a receiver operating characteristic (ROC) curve for early rheumatoid arthritis patients (30 patients) in the case of diagnosis with hsa-miR-93-5p in Test Example 3. FIG. The vertical axis indicates sensitivity, and the horizontal axis indicates 1-specificity. The figure also shows the area under the curve (Area Under the Curve, AUC) calculated based on the ROC curve. 試験例3における、hsa-miR-106b-5pで診断する場合の、早期関節リウマチ患者（30名）に対する受信者動作特性（ROC）曲線を示す。縦軸は感度を示し、横軸は1－特異度を示す。図中にROC曲線に基づいて算出された曲線下面積（Area Under the Curve, AUC）も示す。FIG. 8 shows a receiver operating characteristic (ROC) curve for an early rheumatoid arthritis patient (30 patients) in the case of diagnosis with hsa-miR-106b-5p in Test Example 3. FIG. The vertical axis indicates sensitivity, and the horizontal axis indicates 1-specificity. The figure also shows the area under the curve (Area Under the Curve, AUC) calculated based on the ROC curve. 試験例3における、hsa-miR-301b-3pで診断する場合の、早期関節リウマチ患者（30名）に対する受信者動作特性（ROC）曲線を示す。縦軸は感度を示し、横軸は1－特異度を示す。図中にROC曲線に基づいて算出された曲線下面積（Area Under the Curve, AUC）も示す。FIG. 9 shows a receiver operating characteristic (ROC) curve for early rheumatoid arthritis patients (30 patients) in the case of diagnosis with hsa-miR-301b-3p in Test Example 3. FIG. The vertical axis indicates sensitivity, and the horizontal axis indicates 1-specificity. The figure also shows the area under the curve (Area Under the Curve, AUC) calculated based on the ROC curve. 試験例3における、hsa-miR-762で診断する場合の、早期関節リウマチ患者（30名）に対する受信者動作特性（ROC）曲線を示す。縦軸は感度を示し、横軸は1－特異度を示す。図中にROC曲線に基づいて算出された曲線下面積（Area Under the Curve, AUC）も示す。FIG. 8 shows a receiver operating characteristic (ROC) curve for early rheumatoid arthritis patients (30 patients) in the case of diagnosis with hsa-miR-762 in Test Example 3. FIG. The vertical axis indicates sensitivity, and the horizontal axis indicates 1-specificity. The figure also shows the area under the curve (Area Under the Curve, AUC) calculated based on the ROC curve. 試験例3における、全てのバイオマーカー（hsa-miR-93-5p、hsa-miR-106b-5p、hsa-miR-301b-3p、hsa-miR-762）を組合わせて診断する場合の、早期関節リウマチ患者（30名）に対する受信者動作特性（ROC）曲線を示す。縦軸は感度を示し、横軸は1－特異度を示す。図中にROC曲線に基づいて算出された曲線下面積（Area Under the Curve, AUC）も示す。In the case of diagnosing a combination of all biomarkers (hsa-miR-93-5p, hsa-miR-106b-5p, hsa-miR-301b-3p, hsa-miR-762) in Test Example 3, Fig. 4 shows receiver operating characteristic (ROC) curves for rheumatoid arthritis patients (30 patients). The vertical axis indicates sensitivity, and the horizontal axis indicates 1-specificity. The figure also shows the area under the curve (Area Under the Curve, AUC) calculated based on the ROC curve. 試験例3における、hsa-miR-93-5pで診断する場合の、抗CCP抗体陰性関節リウマチ患者（14名）に対する受信者動作特性（ROC）曲線を示す。縦軸は感度を示し、横軸は1－特異度を示す。図中にROC曲線に基づいて算出された曲線下面積（Area Under the Curve, AUC）も示す。FIG. 9 shows a receiver operating characteristic (ROC) curve for anti-CCP antibody-negative rheumatoid arthritis patients (14 patients) in the case of diagnosis with hsa-miR-93-5p in Test Example 3. FIG. The vertical axis indicates sensitivity, and the horizontal axis indicates 1-specificity. The figure also shows the area under the curve (Area Under the Curve, AUC) calculated based on the ROC curve. 試験例3における、hsa-miR-106b-5pで診断する場合の、抗CCP抗体陰性関節リウマチ患者（14名）に対する受信者動作特性（ROC）曲線を示す。縦軸は感度を示し、横軸は1－特異度を示す。図中にROC曲線に基づいて算出された曲線下面積（Area Under the Curve, AUC）も示す。FIG. 8 shows a receiver operating characteristic (ROC) curve for an anti-CCP antibody-negative rheumatoid arthritis patient (14 patients) in the case of diagnosis with hsa-miR-106b-5p in Test Example 3. FIG. The vertical axis indicates sensitivity, and the horizontal axis indicates 1-specificity. The figure also shows the area under the curve (Area Under the Curve, AUC) calculated based on the ROC curve. 試験例3における、hsa-miR-301b-3pで診断する場合の、抗CCP抗体陰性関節リウマチ患者（14名）に対する受信者動作特性（ROC）曲線を示す。縦軸は感度を示し、横軸は1－特異度を示す。図中にROC曲線に基づいて算出された曲線下面積（Area Under the Curve, AUC）も示す。FIG. 9 shows a receiver operating characteristic (ROC) curve for an anti-CCP antibody-negative rheumatoid arthritis patient (14 patients) in the case of diagnosis with hsa-miR-301b-3p in Test Example 3. FIG. The vertical axis indicates sensitivity, and the horizontal axis indicates 1-specificity. The figure also shows the area under the curve (Area Under the Curve, AUC) calculated based on the ROC curve. 試験例3における、hsa-miR-762で診断する場合の、抗CCP抗体陰性関節リウマチ患者（14名）に対する受信者動作特性（ROC）曲線を示す。縦軸は感度を示し、横軸は1－特異度を示す。図中にROC曲線に基づいて算出された曲線下面積（Area Under the Curve, AUC）も示す。FIG. 9 shows a receiver operating characteristic (ROC) curve for an anti-CCP antibody-negative rheumatoid arthritis patient (14 patients) in the case of diagnosis with hsa-miR-762 in Test Example 3. FIG. The vertical axis indicates sensitivity, and the horizontal axis indicates 1-specificity. The figure also shows the area under the curve (Area （Under the Curve, AUC) calculated based on the ROC curve. 試験例3における、全てのバイオマーカー（hsa-miR-93-5p、hsa-miR-106b-5p、hsa-miR-301b-3p、hsa-miR-762）を組合わせて診断する場合の、抗CCP抗体陰性関節リウマチ患者（14名）に対する受信者動作特性（ROC）曲線を示す。縦軸は感度を示し、横軸は1－特異度を示す。図中にROC曲線に基づいて算出された曲線下面積（Area Under the Curve, AUC）も示す。In Test Example 3, anti-diagnosis when all biomarkers (hsa-miR-93-5p, hsa-miR-106b-5p, hsa-miR-301b-3p, hsa-miR-762) are diagnosed in combination Fig. 4 shows a receiver operating characteristic (ROC) curve for CCP antibody-negative rheumatoid arthritis patients (14 patients). The vertical axis indicates sensitivity, and the horizontal axis indicates 1-specificity. The figure also shows the area under the curve (Area Under the Curve, AUC) calculated based on the ROC curve.
　本明細書中において、「含有」及び「含む」なる表現については、「含有」、「含む」、「実質的にからなる」及び「のみからなる」という概念を含む。 に お い て In this specification, the expressions “contain” and “contain” include the concepts of “contain”, “contain”, “consisting essentially of” and “consisting only of”.
　1．関節リウマチの検査方法
　本発明は、その一態様において、関節リウマチを検査する方法であって、（1）被検体から採取された体液における、miR-93-5p、miR-106b-5p、miR-301b-3p、及びmiR-762からなる群より選択される少なくとも1種のバイオマーカーを検出する工程、を含む、検査方法（本明細書において、「本発明の関節リウマチ検査方法」と示すこともある。）に関する。以下、これについて説明する。 1． Method for testing rheumatoid arthritis In one embodiment, the present invention relates to a method for testing rheumatoid arthritis, comprising: (1) miR-93-5p, miR-106b-5p, miR- in a body fluid collected from a subject; A step of detecting at least one biomarker selected from the group consisting of 301b-3p and miR-762, including a step of detecting the biomarker (herein, also referred to as "the rheumatoid arthritis test method of the present invention". There is.) Hereinafter, this will be described.
　1-1．工程（1）
　検査対象である関節リウマチの種類は、特に制限されない。関節リウマチの各種分類基準における全てのクラス、グレード、ステージの関節リウマチが検査対象となる。関節リウマチには、抗CCP抗体陰性の関節リウマチも包含される。 1-1. Process (1)
The type of rheumatoid arthritis to be tested is not particularly limited. Rheumatoid arthritis of all classes, grades and stages in various classification criteria for rheumatoid arthritis is to be tested. Rheumatoid arthritis also includes anti-CCP antibody negative rheumatoid arthritis.
　被検体は、本発明の検査方法の対象生物であり、その生物種は特に制限されない。被検体の生物種としては、例えばヒト、サル、マウス、ラット、イヌ、ネコ、ウサギなどの種々の哺乳類動物が挙げられ、好ましくはヒトが挙げられる。 (4) The subject is the target organism of the test method of the present invention, and the species is not particularly limited. Examples of the species of the subject include various mammals such as humans, monkeys, mice, rats, dogs, cats and rabbits, and humans are preferred.
　被検体の状態は、特に制限されない。被検体としては、例えば関節リウマチに罹患しているかどうか不明な検体、関節リウマチに罹患していると既に別の方法により判定されている検体、関節リウマチに罹患していないと既に別の方法により判定されている検体、関節リウマチの治療中の検体等が挙げられる。 状態 The state of the subject is not particularly limited. As a subject, for example, a sample that is unknown whether it is suffering from rheumatoid arthritis, a sample that has already been determined to be suffering from rheumatoid arthritis, a method that is already suffering from rheumatoid arthritis by another method Examples include a sample that has been determined and a sample that is undergoing treatment for rheumatoid arthritis.
　体液は、特に制限されない。体液としては、例えば全血、血清、血漿、髄液、唾液、関節液、尿、組織液（気管支肺胞洗浄液を含む）、汗、涙、喀痰、鼻汁などが挙げられ、好ましくは全血、血清、血漿、髄液が挙げられ、より好ましくは全血、血清、血漿が挙げられる。体液は、好ましくは末梢血単核球を含む体液である。体液は、1種単独で採用してもよいし、2種以上を組み合わせて採用してもよい。 The body fluid is not particularly limited. Examples of the body fluid include whole blood, serum, plasma, cerebrospinal fluid, saliva, synovial fluid, urine, tissue fluid (including bronchoalveolar lavage fluid), sweat, tears, sputum, nasal discharge, and the like, and preferably whole blood, serum , Plasma and cerebrospinal fluid, more preferably whole blood, serum and plasma. The body fluid is preferably a body fluid containing peripheral blood mononuclear cells. The body fluid may be used alone or in combination of two or more.
　体液は、当業者に公知の方法で被検体から採取することができる。例えば、全血は、注射器などを用いた採血によって採取することができる。血清は、全血から血球及び特定の血液凝固因子を除去した部分であり、例えば、全血を凝固させた後の上澄みとして得ることができる。血漿は、全血から血球を除去した部分であり、例えば、全血を凝固させない条件下で遠心分離に供した際の上澄みとして得ることができる。 Body fluid can be collected from a subject by a method known to those skilled in the art. For example, whole blood can be collected by blood collection using a syringe or the like. Serum is a portion of whole blood from which blood cells and specific blood clotting factors have been removed, and can be obtained, for example, as a supernatant after coagulating whole blood. Plasma is a portion of whole blood from which blood cells have been removed, and can be obtained, for example, as a supernatant when subjected to centrifugation under conditions that do not allow whole blood to coagulate.
　工程（1）の検出対象は、miR-93-5p、miR-106b-5p、miR-301b-3p、及びmiR-762からなる群より選択される少なくとも1種のバイオマーカー（本明細書において、これらをまとめて「対象バイオマーカー」と示すこともある。）である。 The detection target in step (1) is at least one biomarker selected from the group consisting of miR-93-5p, miR-106b-5p, miR-301b-3p, and miR-762 (in the present specification, These are sometimes collectively referred to as “target biomarkers.”).
　対象バイオマーカーは、関節リウマチにおいて発現量が変化しているバイオマーカーであり、これを指標とすることにより関節リウマチを鑑別可能である。 The target biomarker is a biomarker whose expression level is changed in rheumatoid arthritis, and using this as an index, rheumatoid arthritis can be distinguished.
　対象バイオマーカー中、miR-93-5p、miR-106b-5p、及びmiR-301b-3pからなる群より選択される少なくとも1種のバイオマーカー（BM1）は、関節リウマチ検体における量が健常検体における量よりも低い対象バイオマーカーである。 Among the target biomarkers, at least one biomarker (BM1) selected from the group consisting of miR-93-5p, miR-106b-5p, and miR-301b-3p has an amount in a rheumatoid arthritis sample in a healthy sample. The target biomarker is lower than the amount.
　対象バイオマーカー中、miR-762（BM2）は、関節リウマチ検体における量が健常検体における量よりも高い対象バイオマーカーである。 Mi Among target biomarkers, miR-762 (BM2) is a target biomarker whose amount in rheumatoid arthritis samples is higher than that in healthy samples.
　対象バイオマーカーは、公知のデータベース（例えば、miRBase：http://www.mirbase.org/）でその塩基配列等を特定することができる。例えば、ヒトの場合、その塩基配列は以下の通りである。
>hsa-miR-93-5p MIMAT0000093
CAAAGUGCUGUUCGUGCAGGUAG（配列番号1）
>hsa-miR-106b-5p MIMAT0000680
UAAAGUGCUGACAGUGCAGAU（配列番号2）
>hsa-miR-301b-3p MIMAT0004958
CAGUGCAAUGAUAUUGUCAAAGC（配列番号3）
>hsa-miR-762 MIMAT0010313
GGGGCUGGGGCCGGGGCCGAGC（配列番号4）。 The base sequence of the target biomarker can be specified in a known database (for example, miRBase: http://www.mirbase.org/). For example, in the case of human, its base sequence is as follows.
> hsa-miR-93-5p MIMAT0000093
CAAAGUGCUGUUCGUGCAGGUAG (SEQ ID NO: 1)
> hsa-miR-106b-5p MIMAT0000680
UAAAGUGCUGACAGUGCAGAU (SEQ ID NO: 2)
> hsa-miR-301b-3p MIMAT0004958
CAGUGCAAUGAUAUUGUCAAAGC (SEQ ID NO: 3)
> hsa-miR-762 MIMAT0010313
GGGGCUGGGGCCGGGGCCGAGC (SEQ ID NO: 4).
　対象バイオマーカーは、成熟miRNAであることが望ましいが、前駆体（例えばpri-miRNA、pre-miRNA等）であってもよい。 The target biomarker is preferably a mature miRNA, but may be a precursor (eg, pri-miRNA, pre-miRNA, etc.).
　工程（1）における対象バイオマーカーの数は、1種のみでもよいが、2種以上、3種以上、又は4種の組み合わせであってもよい。より多くの対象バイオマーカーを組み合わせることにより、関節リウマチの検査等を、より正確に行うことが可能になる。 数 The number of target biomarkers in step (1) may be only one, but may be two or more, three or more, or a combination of four. By combining more target biomarkers, it becomes possible to more accurately perform an examination of rheumatoid arthritis and the like.
　また、工程（1）における対象バイオマーカーは、miR-106b-5p、miR-301b-3p、及びmiR-762からなる群より選択される少なくとも1種を含むことが好ましい。 In addition, the target biomarker in the step (1) preferably contains at least one selected from the group consisting of miR-106b-5p, miR-301b-3p, and miR-762.
　検出は、通常は、対象バイオマーカーの量又は濃度を測定することによって行われる。「濃度」とは、絶対濃度に限らず、相対濃度や、単位体積辺りの重量や、絶対濃度を知るために測定した生データなどでもよい。 Detection is usually performed by measuring the amount or concentration of the target biomarker. The “concentration” is not limited to the absolute concentration, but may be a relative concentration, a weight per unit volume, or raw data measured to know the absolute concentration.
　対象バイオマーカーを検出する方法としては、対象バイオマーカーの一部又は全部を特異的に検出できる方法であれば特に制限されない。検出方法としては、具体的には、例えば、RNA-seq解析法、RT-PCR法、核酸チップ解析法、ノーザンブロット法等を挙げることができる。 方法 The method for detecting the target biomarker is not particularly limited as long as it can specifically detect a part or all of the target biomarker. Specific examples of the detection method include, for example, RNA-seq analysis, RT-PCR, nucleic acid chip analysis, and Northern blot.
　RNA-seq解析法を利用する場合は、具体的には、被検体由来のRNAから常法に従ってcDNAを調製して、次世代シークエンサー等を用いてシークエンス解析を行い、得られたデータに基づいてマッピング、遺伝子発現解析、発現量解析等を行い、発現量情報を得る方法が挙げられる。 When using the RNA-seq analysis method, specifically, cDNA is prepared from RNA from the subject in accordance with a conventional method, sequence analysis is performed using a next-generation sequencer, etc., and based on the obtained data, A method of obtaining expression level information by performing mapping, gene expression analysis, expression level analysis, and the like.
　RT-PCR法を利用する場合は、具体的には、被検体由来のRNAから常法に従ってcDNAを調製して、これを鋳型として標的領域が増幅できるように、一対のプライマー（上記cDNA（－鎖）に結合する正鎖、＋鎖に結合する逆鎖）をこれとハイブリダイズさせて、常法に従ってPCR法を行い、得られた増幅二本鎖DNAを検出する方法を例示することができる。なお、増幅された二本鎖DNAの検出は、上記PCRを予めRIや蛍光物質で標識しておいたプライマーを用いて行うことによって産生される標識二本鎖DNAを検出する方法、産生された二本鎖DNAを常法に従ってナイロンメンブレン等にトランスファーさせて、標識プローブを使用してこれとハイブリダイズさせて検出する方法などを用いることができる。 When the RT-PCR method is used, specifically, a cDNA is prepared from RNA derived from a subject according to a conventional method, and a pair of primers (the cDNA (- A method for detecting the amplified double-stranded DNA obtained by hybridizing the positive strand binding to the strand) and the reverse strand binding to the + strand) and performing PCR according to a conventional method can be exemplified. . The detection of the amplified double-stranded DNA was performed by detecting the labeled double-stranded DNA produced by performing the above PCR using primers that had been labeled with RI or a fluorescent substance in advance. A method in which double-stranded DNA is transferred to a nylon membrane or the like according to a conventional method, hybridized with a labeled probe and detected, or the like can be used.
　核酸チップ解析を利用する場合は、核酸プローブ（1本鎖又は2本鎖）を貼り付けた核酸チップを用意し、これに被検体由来のRNAまたは該RNAから常法によって調製された核酸とハイブリダイズさせて、形成された二本鎖を検出する方法を挙げることができる。 When using nucleic acid chip analysis, prepare a nucleic acid chip to which a nucleic acid probe (single-stranded or double-stranded) is attached, and hybridize it with RNA derived from a subject or a nucleic acid prepared from the RNA by a conventional method. There can be mentioned a method of detecting a double strand formed by soybean.
　ノーザンブロット法を利用する場合は、具体的には、プローブを放射性同位元素（32P、33Pなど：RI）や蛍光物質などで標識し、それを、常法に従ってナイロンメンブレン等にトランスファーした上記発現系由来のmRNAとハイブリダイズさせた後、形成された診断薬と被検者の試料由来のmRNAとの二重鎖を、プローブの標識物（RI若しくは蛍光物質などの標識物質）に由来するシグナルを放射線検出器又は蛍光検出器などで検出、測定する方法を例示することができる。 When the Northern blot method is used, specifically, the above-described expression system in which the probe is labeled with a radioisotope (eg, 32P, 33P: RI) or a fluorescent substance and then transferred to a nylon membrane or the like according to a conventional method. After hybridization with the derived mRNA, the duplex of the formed diagnostic agent and the mRNA derived from the subject's sample is converted into a signal derived from the probe label (a labeling substance such as RI or a fluorescent substance). A method of detecting and measuring with a radiation detector or a fluorescence detector can be exemplified.
　工程（1）を含む本発明の検査方法によれば、関節リウマチの検出指標である対象バイオマーカーの量及び／又は濃度を提供することができ、これにより関節リウマチの検出などを補助することができる。 According to the test method of the present invention including the step (1), it is possible to provide the amount and / or concentration of the target biomarker, which is a detection index for rheumatoid arthritis, and thereby assist in detecting rheumatoid arthritis and the like. it can.
　工程（1）を含む本発明の検査方法による検査結果は、治療効果判定、関節リウマチの病態解明、関節リウマチの予後予測、患者層別化、治療方法の選択（個別化医療、治療反応性）等に利用し得る。 The test results of the test method of the present invention including the step (1) are used to determine the therapeutic effect, elucidate the pathology of rheumatoid arthritis, predict the prognosis of rheumatoid arthritis, stratify patients, select a treatment method (individualized medicine, treatment responsiveness) And so on.
　1-2．工程（2）
　本発明の検査方法は、一態様として、検出したバイオマーカーがmiR-93-5p、miR-106b-5p、及びmiR-301b-3pからなる群より選択される少なくとも1種のバイオマーカー（BM1）を含む場合、さらに、
（2a）前記工程（1）で検出された前記BM1の量又は濃度がカットオフ値以下である場合に、前記被検体が関節リウマチに罹患していると判定する工程、
を含むことが好ましい。該工程2aを含む本発明の検査方法によれば、関節リウマチを判定することが可能となる。 1-2. Process (2)
In one aspect of the test method of the present invention, the detected biomarker is at least one biomarker (BM1) selected from the group consisting of miR-93-5p, miR-106b-5p, and miR-301b-3p. If you include
(2a) a step of determining that the subject is suffering from rheumatoid arthritis when the amount or concentration of the BM1 detected in the step (1) is equal to or less than a cutoff value;
It is preferable to include According to the test method of the present invention including the step 2a, rheumatoid arthritis can be determined.
　本発明の検査方法は、一態様として、検出したバイオマーカーがmiR-762（BM2）を含む場合、さらに、
（2b）前記工程（1）で検出された前記BM2の量又は濃度がカットオフ値以上である場合に、前記被検体が関節リウマチに罹患していると判定する工程、
を含むことが好ましい。該工程2bを含む本発明の検査方法によれば、関節リウマチを判定することが可能となる。 In one embodiment, the test method of the present invention further comprises, when the detected biomarker includes miR-762 (BM2),
(2b) a step of determining that the subject is suffering from rheumatoid arthritis when the amount or concentration of the BM2 detected in the step (1) is not less than a cutoff value;
It is preferable to include According to the test method of the present invention including the step 2b, it is possible to determine rheumatoid arthritis.
　本発明の検査方法は、一態様として、検出したバイオマーカーがmiR-93-5p、miR-106b-5p、及びmiR-301b-3pからなる群より選択される少なくとも1種のバイオマーカー（BM1）、並びにmiR-762（BM2）を含む場合、さらに、
（2c）前記工程（1）で検出された前記BM1の量又は濃度がカットオフ値以下であり、且つ前記工程（1）で検出された前記BM2の量又は濃度がカットオフ値以上である場合に、前記被検体が関節リウマチに罹患していると判定する工程、
を含むことが好ましい。該工程2cを含む本発明の検査方法によれば、関節リウマチを判定することが可能となる。 In one aspect of the test method of the present invention, the detected biomarker is at least one biomarker (BM1) selected from the group consisting of miR-93-5p, miR-106b-5p, and miR-301b-3p. , And miR-762 (BM2),
(2c) When the amount or concentration of the BM1 detected in the step (1) is equal to or less than a cutoff value, and the amount or concentration of the BM2 detected in the step (1) is equal to or more than a cutoff value. In the step of determining that the subject is suffering from rheumatoid arthritis,
It is preferable to include According to the test method of the present invention including the step 2c, rheumatoid arthritis can be determined.
　カットオフ値は、感度、特異度、陽性的中率、陰性的中率などの観点から当業者が適宜設定することができ、例えば、関節リウマチに罹患していない被検体から採取された体液における対象バイオマーカーの量及び／又は濃度に基づいて、その都度定められた値、或いは予め定められた値とすることができる。カットオフ値は、例えば、関節リウマチに罹患していない被検体から採取された体液における対象バイオマーカーの量及び／又は濃度（被検体が複数の場合は、平均値、中央値など）の、例えば0.5～1.5倍の値とすることができる。 Cut-off value, sensitivity, specificity, positive predictive value, can be appropriately set by those skilled in the art from the viewpoint of negative predictive value, for example, in a body fluid collected from a subject not suffering from rheumatoid arthritis Based on the amount and / or concentration of the target biomarker, a predetermined value or a predetermined value can be used. The cut-off value is, for example, the amount and / or concentration of the target biomarker in a body fluid collected from a subject not suffering from rheumatoid arthritis (for a plurality of subjects, an average value, a median, etc.), for example, The value can be 0.5 to 1.5 times.
　工程（2）の好ましい一態様においては、被検体が関節リウマチの治療中の検体である場合、カットオフ値を、例えば同一検体についての過去の試料における対象バイオマーカーの量及び／又は濃度に基づいた値とすることにより、治療効果を判定することができる。 In a preferred embodiment of the step (2), when the subject is a subject undergoing treatment for rheumatoid arthritis, the cutoff value is determined based on, for example, the amount and / or concentration of the target biomarker in a past sample of the same sample. By setting these values, the therapeutic effect can be determined.
　2．関節リウマチのより高い精度での診断
　工程（2）を含む本発明の検査方法により、被検体が関節リウマチに罹患していると判定された場合、本発明の検査方法に、さらに関節リウマチの医師による診断を適用する工程を組み合わせることによって、より高い精度で関節リウマチを診断することができる。また、本発明の検査方法はより正確に関節リウマチを検出できるので、本発明の検査方法に上記工程を組み合わせることによって、より効率的且つより正確に「関節リウマチに罹患している」と診断できる。 2． When the subject is determined to be suffering from rheumatoid arthritis by the test method of the present invention including the diagnostic step (2) with higher accuracy for rheumatoid arthritis, the test method of the present invention is further added to the rheumatoid arthritis doctor. By combining the steps of applying the diagnosis according to the above, rheumatoid arthritis can be diagnosed with higher accuracy. In addition, since the test method of the present invention can more accurately detect rheumatoid arthritis, by combining the above-described steps with the test method of the present invention, it is possible to more efficiently and more accurately diagnose "affected by rheumatoid arthritis". .
　3．関節リウマチの治療
　工程（2）を含む本発明の検査方法により被検体が関節リウマチに罹患していると判定された場合は本発明の検査方法に対してさらに、或いは上記「2．関節リウマチのより高い精度での診断」に記載の様に関節リウマチに罹患していると診断された場合は本発明の検査方法と医師による診断を適用する工程との組合せに対してさらに、（3）関節リウマチに罹患していると判定又は診断された被検体に対して、該疾患の治療を行う工程を行うことによって、被検体の該疾患を治療することが可能となる。また、本発明の検査方法はより正確に関節リウマチを検出できるので、本発明の検査方法に対して、或いは本発明の検査方法と医師による診断を適用する工程との組合せに対して工程3を組み合わせることによって、関節リウマチに罹患している被検体をより効率的に、より確実に治療できる。 3． When the subject is determined to be suffering from rheumatoid arthritis by the test method of the present invention including the treatment step (2) for rheumatoid arthritis, the test method of the present invention is further applied, or the above-mentioned “2. If it is diagnosed as having rheumatoid arthritis as described in “Diagnosis with Higher Accuracy”, the combination of the test method of the present invention and the step of applying a diagnosis by a physician, By performing a step of treating the disease for a subject determined or diagnosed as having rheumatism, the disease of the subject can be treated. In addition, since the test method of the present invention can more accurately detect rheumatoid arthritis, Step 3 is performed for the test method of the present invention or for a combination of the test method of the present invention and a step of applying a diagnosis by a doctor. By the combination, a subject suffering from rheumatoid arthritis can be treated more efficiently and more reliably.
　関節リウマチの治療方法は、特に制限されないが、代表的には投薬治療が挙げられる。投薬治療に用いる医薬としては、特に制限されないが、例えば抗リウマチ薬、生物学的製剤（バイオ医薬品）、非ステロイド性抗炎症薬、ステロイド（副腎皮質ステロイド）等が挙げられる。医薬は、1種、2種、又は3種以上を組み合わせて用いることができる。 The method of treating rheumatoid arthritis is not particularly limited, but typically includes medication. The drug used for the drug treatment is not particularly limited, and examples thereof include antirheumatic drugs, biological preparations (biopharmaceuticals), non-steroidal anti-inflammatory drugs, and steroids (corticosteroids). One, two, or three or more pharmaceuticals can be used in combination.
　4．関節リウマチの検査薬
　本発明は、その一態様において、miR-93-5p、miR-106b-5p、miR-301b-3p、及びmiR-762からなる群より選択される少なくとも1種のバイオマーカーの検出剤（本明細書において、「本発明の検査薬」と示すこともある。）を含む、関節リウマチの検査薬（本明細書において、「本発明の検査薬」と示すこともある。）に関する。以下、これについて説明する。 Four. Test agent for rheumatoid arthritis In one aspect, the present invention provides at least one biomarker selected from the group consisting of miR-93-5p, miR-106b-5p, miR-301b-3p, and miR-762. A test agent for rheumatoid arthritis containing a detection agent (herein, sometimes referred to as “test agent of the present invention”) (herein, sometimes referred to as “test agent of the present invention”). About. Hereinafter, this will be described.
　miR-93-5p、miR-106b-5p、miR-301b-3p、miR-762、関節リウマチ等については、上記「1．関節リウマチの検査方法」における定義と同様である。 MiR-93-5p, miR-106b-5p, miR-301b-3p, miR-762, rheumatoid arthritis and the like are the same as defined in the above “1. Method for testing rheumatoid arthritis”.
　本発明の検出剤は、対象バイオマーカーを特異的に検出できるものである限り特に制限されない。該検出剤としては、例えば対象バイオマーカーに対するプライマー、プローブ等が挙げられる。 検 出 The detection agent of the present invention is not particularly limited as long as it can specifically detect the target biomarker. Examples of the detection agent include primers and probes for the target biomarker.
　本発明の検出剤は、その機能が著しく損なわれない限りにおいて、修飾が施されていてもよい。修飾としては、例えば、標識物、例えば蛍光色素、酵素、タンパク質、放射性同位体、化学発光物質、ビオチン等の付加が挙げられる。 修飾 The detection agent of the present invention may be modified as long as its function is not significantly impaired. Examples of the modification include addition of a label, for example, a fluorescent dye, an enzyme, a protein, a radioisotope, a chemiluminescent substance, biotin, and the like.
　本発明において用いられる蛍光色素としては、一般にヌクレオチドを標識して、核酸の検出や定量に用いられるものが好適に使用でき、例えば、HEX（4,7,2’,4’,5’,7’-hexachloro-6-carboxylfluorescein、緑色蛍光色素）、フルオレセイン（fluorescein）、NED（商品名、アプライドバイオシステムズ社製、黄色蛍光色素）、あるいは、6-FAM（商品名、アプライドバイオシステムズ社製、黄緑色蛍光色素）、ローダミン（rhodamin）またはその誘導体〔例えば、テトラメチルローダミン（TMR）〕を挙げることができるが、これらに限定されない。蛍光色素でヌクレオチドを標識する方法は、公知の標識法のうち適当なものを使用することができる〔Nature Biotechnology, 14, 303-308 (1996)参照〕。また、市販の蛍光標識キットを使用することもできる（例えば、アマシャム・ファルマシア社製、オリゴヌクレオチドECL 3’-オリゴラベリングシステム等）。 As the fluorescent dye used in the present invention, generally used are those which label nucleotides and are used for the detection and quantification of nucleic acids. For example, HEX (4,7,2 ', 4', 5 ', 7 '-hexachloro-6-carboxylfluorescein, green fluorescent dye), fluorescein (fluorescein), NED (trade name, manufactured by Applied Biosystems, yellow fluorescent dye), or 6-FAM (trade name, manufactured by Applied Biosystems, yellow) Green fluorescent dye), rhodamine or a derivative thereof (eg, tetramethylrhodamine (TMR)), but is not limited thereto. As a method for labeling nucleotides with a fluorescent dye, an appropriate one of known labeling methods can be used [see Nature Biotechnology, {14, {303-308} (1996)]. Alternatively, a commercially available fluorescent labeling kit can be used (for example, oligonucleotide ECL '3'-oligolabeling system, manufactured by Amersham Pharmacia).
　本発明の検出剤は、任意の固相に固定化して用いることもできる。このため本発明の検査薬は、検出剤を固定化した基板（例えばプローブを固定化したマイクロアレイチップ等。）の形態として提供することができる。 検 出 The detection agent of the present invention can also be used by immobilizing it on any solid phase. Therefore, the test agent of the present invention can be provided in the form of a substrate on which a detection agent is immobilized (for example, a microarray chip on which a probe is immobilized).
　固定化に使用される固相は、ポリヌクレオチド等を固定化できるものであれば特に制限されることなく、例えばガラス板、ナイロンメンブレン、マイクロビーズ、シリコンチップ、キャピラリーまたはその他の基板等を挙げることができる。固相への検出剤の固定は、特に制限されない。固定方法は、例えばマイクロアレイであれば、市販のスポッター（Amersham社製など）を利用するなど、固定化プローブの種類に応じて当該技術分野で周知である〔例えば、photolithographic技術(Affymetrix社)、インクジェット技術(Rosetta Inpharmatics社)によるオリゴヌクレオチドのin situ合成等〕。 The solid phase used for immobilization is not particularly limited as long as it can immobilize a polynucleotide or the like, and examples thereof include a glass plate, a nylon membrane, microbeads, a silicon chip, a capillary, and other substrates. Can be. Immobilization of the detection agent on the solid phase is not particularly limited. The immobilization method is well known in the art depending on the type of the immobilized probe, for example, using a commercially available spotter (for example, Amersham) if it is a microarray [for example, photolithographic technology (Affymetrix), In-situ synthesis of oligonucleotides by inkjet technology (Rosetta Inpharmatics).
　プライマーやプローブ等は、対象バイオマーカーやそれに由来する核酸等を選択的に（特異的に）認識するものであれば、特に限定されない。ここで、「選択的に（特異的に）認識する」とは、例えばノーザンブロット法においては、対象バイオマーカーが特異的に検出できること、またRT-PCR法においては対象バイオマーカー若しくはそれに由来する核酸（cDNA等）が特異的に増幅されることを意味するが、それに限定されることなく、当業者が上記検出物または増幅物が対象バイオマーカーに由来するものであると判断できるものであればよい。 The primer and probe are not particularly limited as long as they selectively (specifically) recognize the target biomarker and the nucleic acid derived therefrom. Here, “selectively (specifically) recognizes” means, for example, that a target biomarker can be specifically detected in Northern blotting, and a target biomarker or a nucleic acid derived therefrom in RT-PCR. (CDNA, etc.) is specifically amplified, but is not limited thereto, provided that a person skilled in the art can determine that the above-mentioned detected substance or amplified substance is derived from the target biomarker. Good.
　プライマーやプローブの具体例としては、下記（a）に記載するポリヌクレオチド並びに下記（b）に記載するポリヌクレオチド：
　（a）対象バイオマーカーの塩基配列において連続する少なくとも15塩基を有するポリヌクレオチド及び／若しくは当該ポリヌクレオチドに相補的なポリヌクレオチド、並びに
　（b）対象バイオマーカーの塩基配列若しくはそれに相補的な塩基配列にストリンジェントな条件下でハイブリダイズする、少なくとも15塩基を有するポリヌクレオチド
からなる群より選択される少なくとも１種が挙げられる。 Specific examples of primers and probes include the polynucleotide described in (a) below and the polynucleotide described in (b) below:
(A) a polynucleotide having at least 15 consecutive nucleotides in the base sequence of the target biomarker and / or a polynucleotide complementary to the polynucleotide; and (b) a base sequence of the target biomarker or a base sequence complementary thereto. At least one selected from the group consisting of polynucleotides having at least 15 bases that hybridize under stringent conditions is included.
　相補的なポリヌクレオチド又は相補的な塩基配列（相補鎖、逆鎖）とは、対象バイオマーカーの塩基配列からなるポリヌクレオチドの全長配列、または該塩基配列において少なくとも連続した15塩基長の塩基配列を有するその部分配列（ここでは便宜上、これらを「正鎖」ともいう）に対して、A:TおよびG:Cといった塩基対関係に基づいて、塩基的に相補的な関係にあるポリヌクレオチド又は塩基配列を意味するものである。ただし、かかる相補鎖は、対象とする正鎖の塩基配列と完全に相補配列を形成する場合に限らず、対象とする正鎖とストリンジェントな条件でハイブリダイズすることができる程度の相補関係を有するものであってもよい。なお、ここでストリンジェントな条件は、Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego CA) に教示されるように、複合体或いはプローブを結合する核酸の融解温度(Tm)に基づいて決定することができる。例えばハイブリダイズ後の洗浄条件として、通常「1×SSC、0.1%SDS、37℃」程度の条件を挙げることができる。相補鎖はかかる条件で洗浄しても対象とする正鎖とハイブリダイズ状態を維持するものであることが好ましい。特に制限されないが、より厳しいハイブリダイズ条件として「0.5×SSC、0.1%SDS、42℃」程度、さらに厳しいハイブリダイズ条件として「0.1×SSC、0.1%SDS、65℃」程度の洗浄条件を挙げることができる。具体的には、このような相補鎖として、対象の正鎖の塩基配列と完全に相補的な関係にある塩基配列からなる鎖、並びに該鎖と少なくとも90％、好ましくは95％、より好ましくは98％以上、さらに好ましくは99％以上の同一性を有する塩基配列からなる鎖を例示することができる。 A complementary polynucleotide or a complementary nucleotide sequence (complementary strand, reverse strand) refers to a full-length sequence of a polynucleotide comprising a nucleotide sequence of a target biomarker, or a nucleotide sequence of at least 15 nucleotides continuous in the nucleotide sequence. A polynucleotide or base having a base complementary relationship to its partial sequence (for convenience, these are also referred to as “positive chains”) based on base pairing such as A: T and G: C. Means an array. However, such a complementary strand is not limited to a case where it forms a completely complementary sequence with the base sequence of the target positive strand, but has a complementary relationship that allows hybridization with the target positive strand under stringent conditions. You may have. Note that the stringent conditions bind the complex or probe as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques in Enzymology, Vol. 152, Academic Press, San Diego CA). It can be determined based on the melting temperature (Tm) of the nucleic acid. For example, as washing conditions after hybridization, there can be usually mentioned conditions of about “1 × SSC, 0.1% SDS, 37 ° C.”. The complementary strand is preferably one that maintains a hybridized state with the target positive strand even when washed under such conditions. Although not particularly limited, the more stringent hybridization conditions are about 0.5 × SSC, 0.1% SDS, 42 ° C., and the more stringent hybridization conditions are about 0.1 × SSC, 0.1% SDS, 65 ° C. Can be. Specifically, as such a complementary strand, a strand consisting of a nucleotide sequence completely complementary to the nucleotide sequence of the target positive strand, and at least 90%, preferably 95%, more preferably A chain consisting of a base sequence having 98% or more, more preferably 99% or more identity can be exemplified.
　プライマーやプローブ等は、例えば対象バイオマーカーの塩基配列をもとに、例えば各種設計プログラムを利用して設計することができる。具体的には前記対象バイオマーカーの塩基配列を設計プログラムにかけて得られる、プライマーまたはプローブの候補配列、若しくは少なくとも該配列を一部に含む配列を、プライマーまたはプローブとして使用することができる。 Primers and probes can be designed based on the base sequence of the target biomarker, for example, using various design programs. Specifically, a primer or probe candidate sequence obtained by subjecting the base sequence of the target biomarker to a design program, or a sequence partially containing at least the sequence can be used as the primer or probe.
　プライマーやプローブ等の塩基長は、上述のように連続する少なくとも15塩基の長さを有するものであれば特に制限されず、用途に応じて適宜設定することができる。塩基長としては、例えばプライマーとして用いる場合であれば、例えば15塩基～35塩基が例示でき、例えばプローブとして用いる場合であれば、例えば15塩基～35塩基が例示できる。 塩 基 The base length of the primer or probe is not particularly limited as long as it has a length of at least 15 consecutive bases as described above, and can be appropriately set according to the application. The base length is, for example, 15 to 35 bases when used as a primer, and for example, 15 to 35 bases when used as a probe.
　本発明の検査薬には、本発明の検出剤以外の、他の検出剤（例えば、他のmiRNA等の核酸を検出するためのプローブや、抗体等）が含まれていてもよい。この場合の本発明の検査薬は、関節リウマチの他に、他の疾患や状態も検査することができる検査薬であってもよい。この場合、本発明の検出剤は、関節リウマチ検査用の検出剤として含まれている。この観点から、本発明の検査薬は、その一態様において、本発明の検出剤からなる関節リウマチ検査用検出剤を含む、関節リウマチの検査薬である。 検 査 The test agent of the present invention may contain other detection agents (eg, a probe for detecting a nucleic acid such as another miRNA, an antibody, etc.) other than the detection agent of the present invention. In this case, the test agent of the present invention may be a test agent capable of testing other diseases and conditions in addition to rheumatoid arthritis. In this case, the detection agent of the present invention is included as a detection agent for a rheumatoid arthritis test. From this viewpoint, the test agent of the present invention is, in one aspect thereof, a test agent for rheumatoid arthritis, which contains a detection agent for rheumatoid arthritis test comprising the detection agent of the present invention.
　本発明の検査薬は、組成物の形態であってもよい。該組成物には、必要に応じて他の成分が含まれていてもよい。他の成分としては、例えば基剤、担体、溶剤、分散剤、乳化剤、緩衝剤、安定剤、賦形剤、結合剤、崩壊剤、滑沢剤、増粘剤、保湿剤、着色料、香料、キレート剤等が挙げられる。 検 査 The test agent of the present invention may be in the form of a composition. The composition may contain other components as necessary. Other components include, for example, bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, fragrances And chelating agents.
　本発明の検査薬は、キットの形態であってもよい。該キットには、上記検出剤或いはこれを含む上記組成物のほかに、被検体の体液における対象バイオマーカーの検出に使用し得るものを含んでいてもよい。このようなものの具体例としては、各種試薬（例えば緩衝液等）、器具（例えば体液の精製、分離用器具）等が挙げられる。 検 査 The test agent of the present invention may be in the form of a kit. The kit may contain, besides the above-mentioned detection agent or the above-mentioned composition containing the same, a kit that can be used for detection of a target biomarker in a body fluid of a subject. Specific examples of such a substance include various reagents (for example, a buffer solution or the like) and instruments (for example, instruments for purifying and separating body fluids).
　5．関節リウマチの予防又は治療剤の有効成分のスクリーニング方法
　本発明は、その一態様において、被検物質で処理された動物から採取された体液における、miR-93-5p、miR-106b-5p、miR-301b-3p、及びmiR-762からなる群より選択される少なくとも1種のバイオマーカーの量又は濃度を指標とする、関節リウマチの予防又は治療剤の有効成分のスクリーニング方法（本明細書において、「本発明の有効成分スクリーニング方法」と示すこともある。）に関する。以下、これについて説明する。 Five. The present invention relates to, in one embodiment, miR-93-5p, miR-106b-5p, miR in a body fluid collected from an animal treated with a test substance. -301b-3p, and an amount or concentration of at least one biomarker selected from the group consisting of miR-762 as an index, a method for screening an active ingredient of a preventive or therapeutic agent for rheumatoid arthritis (herein, It may also be referred to as “the active ingredient screening method of the present invention.” Hereinafter, this will be described.
　体液、miR-93-5p、miR-106b-5p、miR-301b-3p、miR-762、関節リウマチ、対象バイオマーカーの量又は濃度の測定等については、上記「1．関節リウマチの検査方法」における定義と同様である。 For body fluid, miR-93-5p, miR-106b-5p, miR-301b-3p, miR-762, rheumatoid arthritis, measurement of the amount or concentration of a target biomarker, etc., see “1. Is the same as the definition in
　動物の生物種は特に制限されない。動物の生物種としては、例えばヒト、サル、マウス、ラット、イヌ、ネコ、ウサギなどの種々の哺乳類動物が挙げられる。 Animal species are not particularly limited. Examples of animal species include various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits.
　被検物質としては、天然に存在する化合物又は人工に作られた化合物を問わず広く使用することができる。また、精製された化合物に限らず、多種の化合物を混合した組成物や、動植物の抽出液も使用することができる。化合物には、低分子化合物に限らず、タンパク質、核酸、多糖類等の高分子化合物も包含される。 As the test substance, any of naturally occurring compounds or artificially produced compounds can be widely used. In addition, not only a purified compound but also a composition in which various kinds of compounds are mixed, and an extract of animals and plants can be used. The compound is not limited to a low molecular compound, but also includes a high molecular compound such as a protein, a nucleic acid, and a polysaccharide.
　本発明の有効成分スクリーニング方法は、より具体的には、指標とするバイオマーカーが、miR-93-5p、miR-106b-5p、及びmiR-301b-3pからなる群より選択される少なくとも1種のバイオマーカー（BM1）である場合、上記指標の値が、被検物質で処理されていない動物から採取された体液における対応バイオマーカーの量又は濃度（対照値）よりも高い場合に、前記被検物質を関節リウマチの予防又は治療剤の有効成分（或いは、関節リウマチの予防又は治療剤の有効成分の候補物質）として選択する工程を含む。 More specifically, the active ingredient screening method of the present invention, the biomarker as an indicator, at least one selected from the group consisting of miR-93-5p, miR-106b-5p, and miR-301b-3p If the value of the above index is higher than the amount or concentration (control value) of the corresponding biomarker in a body fluid collected from an animal not treated with the test substance, Selecting a test substance as an active ingredient of a prophylactic or therapeutic agent for rheumatoid arthritis (or a candidate substance as an active ingredient of a prophylactic or therapeutic agent for rheumatoid arthritis).
　本発明の有効成分スクリーニング方法は、別の具体例として、指標とするバイオマーカーが、miR-762（BM2）である場合、上記指標の値が、被検物質で処理されていない動物から採取された体液における対応バイオマーカーの量又は濃度（対照値）よりも低い場合に、前記被検物質を関節リウマチの予防又は治療剤の有効成分（或いは、関節リウマチの予防又は治療剤の有効成分の候補物質）として選択する工程を含む。 As another specific example, when the biomarker serving as an index is miR-762 (BM2), the value of the index is collected from an animal that has not been treated with the test substance. When the amount of the corresponding biomarker in the body fluid is lower than the amount or concentration (control value) of the corresponding biomarker, the test substance is used as an active ingredient of a prophylactic or therapeutic agent for rheumatoid arthritis (or a candidate for an active ingredient of a preventive or therapeutic agent for rheumatoid arthritis). As a substance).
　対応バイオマーカーとは、指標としている対象バイオマーカーと同じmiRNAを意味する。 The corresponding biomarker means the same miRNA as the target biomarker used as an index.
　「高い」とは、例えば指標の値が、対照値の2倍、5倍、10倍、20倍、50倍、100倍であることを意味する。 “High” means that, for example, the index value is twice, five times, ten times, twenty times, fifty times, and one hundred times the control value.
　「低い」とは、例えば指標の値が、対照値の1/2、1/5、1/10、1/20、1/50、1/100であることを意味する。 “Low” means, for example, that the index value is 1/2, 1/5, 1/10, 1/20, 1/50, 1/100 of the control value.
　6．関節リウマチの誘発性又は増悪性の評価方法
　本発明は、その一態様において、被検物質で処理された動物から採取された体液における、miR-93-5p、miR-106b-5p、miR-301b-3p、及びmiR-762からなる群より選択される少なくとも1種のバイオマーカーの量又は濃度を指標とする、関節リウマチの誘発性又は増悪性の評価方法（本明細書において、「本発明の毒性評価方法」と示すこともある。）に関する。以下、これについて説明する。 6． Method for assessing rheumatoid arthritis inducibility or malignancy The present invention, in one embodiment, in a body fluid collected from an animal treated with a test substance, miR-93-5p, miR-106b-5p, miR-301b -3p, and a method for evaluating the induction or malignancy of rheumatoid arthritis, using as an index the amount or concentration of at least one biomarker selected from the group consisting of miR-762 (in the present specification, Toxicity evaluation method "). Hereinafter, this will be described.
　体液、miR-93-5p、miR-106b-5p、miR-301b-3p、miR-762、関節リウマチ、対象バイオマーカーの量又は濃度の測定、動物の生物種、被検物質等については、上記「1．関節リウマチの検査方法」及び「6．関節リウマチの予防又は治療剤の有効成分のスクリーニング方法」における定義と同様である。 Body fluid, miR-93-5p, miR-106b-5p, miR-301b-3p, miR-762, rheumatoid arthritis, measurement of the amount or concentration of the target biomarker, animal species, test substance, etc. This is the same as the definition in “1. Method for testing rheumatoid arthritis” and “6. Method for screening active ingredient of preventive or therapeutic agent for rheumatoid arthritis”.
　本発明の毒性評価方法は、より具体的には、指標とするバイオマーカーが、miR-93-5p、miR-106b-5p、及びmiR-301b-3pからなる群より選択される少なくとも1種のバイオマーカー（BM1）である場合、上記指標の値が、被検物質で処理されていない動物から採取された体液における対応バイオマーカーの量又は濃度（対照値）よりも低い場合に、前記被検物質を関節リウマチの誘発性又は増悪性があると判定する工程を含む。 The toxicity evaluation method of the present invention, more specifically, the biomarker as an index, miR-93-5p, miR-106b-5p, and at least one selected from the group consisting of miR-301b-3p In the case of a biomarker (BM1), when the value of the above index is lower than the amount or concentration (control value) of the corresponding biomarker in a body fluid collected from an animal not treated with the test substance, Determining the substance to be rheumatoid arthritis-induced or malignant.
　本発明の毒性評価方法は、別の具体例として、指標とするバイオマーカーが、miR-762（BM2）である場合、上記指標の値が、被検物質で処理されていない動物から採取された体液における対応バイオマーカーの量又は濃度（対照値）よりも高い場合に、前記被検物質を関節リウマチの誘発性又は増悪性があると判定する工程を含む。 As another specific example, in the toxicity evaluation method of the present invention, when the biomarker serving as an index is miR-762 (BM2), the value of the index is obtained from an animal not treated with the test substance. If the amount or concentration of the corresponding biomarker in the body fluid is higher than the control value, the test substance is determined to be rheumatoid arthritis-inducing or malignant.
　対応バイオマーカーとは、指標としている対象バイオマーカーと同じmiRNAを意味する。 The corresponding biomarker means the same miRNA as the target biomarker used as an index.
　「高い」とは、例えば指標の値が、対照値の2倍、5倍、10倍、20倍、50倍、100倍であることを意味する。 “High” means that, for example, the index value is twice, five times, ten times, twenty times, fifty times, and one hundred times the control value.
　「低い」とは、例えば指標の値が、対照値の1/2、1/5、1/10、1/20、1/50、1/100であることを意味する。 “Low” means, for example, that the index value is 1/2, 1/5, 1/10, 1/20, 1/50, 1/100 of the control value.
　以下に、実施例に基づいて本発明を詳細に説明するが、本発明はこれらの実施例によって限定されるものではない。 Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.
　試験例1．関節リウマチバイオマーカーのスクリーニング1
　MIGWAS解析パイプラインにおいては、データベースに登録されたマイクロRNAを対象に、複数のアルゴリズムを用いて予測した標的遺伝子のリストを作成する。対象マイクロRNAをコードする遺伝子と標的遺伝子の双方において、該当疾患のゲノムワイド関連解析における要約統計量から算出される遺伝子レベルのP値が閾値未満である、すなわち疾患感受性が認められているかを判定する。さらに、データベースに登録された細胞組織別に測定されたマイクロRNA発現プロファイル情報を取得し、各組織別にマイクロRNAの発現情報を正規化し、組織特異的に発現量の高いマイクロRNAを同定する。それらのマイクロRNAを対象に、各組織別のMIGWAS解析パイプラインを実施することで、実際の生体内の特定の組織において機能しているマイクロRNAを優先的にスクリーニングすることが可能となる。 Test example 1. Screening for rheumatoid arthritis biomarkers 1
In the MIGWAS analysis pipeline, a list of target genes predicted using a plurality of algorithms is created for microRNAs registered in the database. For both the gene encoding the target microRNA and the target gene, determine whether the gene-level P value calculated from the summary statistics in the genome-wide association analysis of the relevant disease is below the threshold, that is, whether disease susceptibility is recognized I do. Furthermore, microRNA expression profile information measured for each cell tissue registered in the database is obtained, the microRNA expression information is normalized for each tissue, and a microRNA having a high expression level is identified in a tissue-specific manner. By performing a MIGWAS analysis pipeline for each tissue with respect to those microRNAs, it is possible to preferentially screen microRNAs that function in a specific tissue in an actual living body.
　関節リウマチの大規模ゲノムワイド関連解析結果（患者群19243名および対照群61565名）に対して、MlGWAS解析パイプラインを適用し、バイオマーカー候補として48個のマイクロRNAを同定した。 (4) The MlGWAS analysis pipeline was applied to the results of a large-scale genome-wide association analysis of rheumatoid arthritis (19243 patients and 61565 controls), and 48 microRNAs were identified as biomarker candidates.
　試験例2．関節リウマチバイオマーカーのスクリーニング2
　独立した関節リウマチ患者群30名および対照群33名から採取された末梢血単核球より抽出したマイクロRNA分画における発現情報の検討を行った。次世代シークエンサーHiSeq2500を用いて詳細な発現情報を取得したところ、関節リウマチにおける発現量が有意に健常人と異なるマイクロRNA、すなわちバイオマーカー候補として94個のマイクロRNAを同定した。 Test example 2. Screening for rheumatoid arthritis biomarkers 2
The expression information in microRNA fractions extracted from peripheral blood mononuclear cells collected from 30 independent rheumatoid arthritis patients and 33 control groups was examined. When detailed expression information was obtained using the next-generation sequencer HiSeq2500, microRNAs whose expression levels in rheumatoid arthritis were significantly different from those of healthy individuals, that is, 94 microRNAs were identified as biomarker candidates.
　試験例3．スクリーニング結果の解析
　試験例1で得られた候補miRNAと試験例2で得られた候補miRNAとで重複している4つのmiRNAを、信頼性が高いバイオマーカーとして抽出した（hsa-miR-93-5p、hsa-miR-106b-5p、hsa-miR-301b-3p、hsa-miR-762、FDR補正後Q値＜0.05）。各miRNAの関節リウマチ患者と健常人での発現量を評価したボルケーノプロットを図１に示す。3個のマイクロRNA（hsa-miR-93-5p、hsa-miR-106b-5p、hsa-miR-301b-3p）においては患者群で発現レベルが有意に低く、1個のマイクロRNA（hsa-miR-762）においては患者群で発現レベルが有意に高かった。本発明に含まれるマイクロRNA（hsa-miR-93-5p、hsa-miR-106b-5p、hsa-miR-301b-3p、hsa-miR-762）は、関節リウマチの患者群および対照群との間での異なる発現レベルを有し、その比（オッズ比）は各々、0.696倍、0.570倍、0.516倍、2.23倍である。この詳細データを表１に示す。 Test example 3. Analysis of Screening Results Four miRNAs overlapping the candidate miRNA obtained in Test Example 1 and the candidate miRNA obtained in Test Example 2 were extracted as highly reliable biomarkers (hsa-miR-93- 5p, hsa-miR-106b-5p, hsa-miR-301b-3p, hsa-miR-762, Q value after FDR correction <0.05). FIG. 1 shows a Volcano plot in which the expression levels of each miRNA in rheumatoid arthritis patients and healthy subjects were evaluated. In three microRNAs (hsa-miR-93-5p, hsa-miR-106b-5p and hsa-miR-301b-3p), the expression level was significantly lower in the patient group, and one microRNA (hsa-miR-93-5p, hsa-miR-106b-5p) In miR-762), the expression level was significantly higher in the patient group. The microRNAs (hsa-miR-93-5p, hsa-miR-106b-5p, hsa-miR-301b-3p, hsa-miR-762) included in the present invention were compared with those of a rheumatoid arthritis patient group and a control group. And the ratios (odds ratio) are 0.696-fold, 0.570-fold, 0.516-fold, and 2.23-fold, respectively. The detailed data is shown in Table 1.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　これら4個のマイクロRNAを組み合わせることで、10.86倍という高い比率をもって患者群と対照群を区別することが可能となり、関節リウマチの診断に寄与すると考えられる。さらに、同定したバイオマーカーそれぞれで診断する場合及び全てを組合わせて診断する場合の、早期関節リウマチ患者（30名）に対する受信者動作特性（ROC）曲線を作成した結果を図２～６に示す。特に、全てを組合わせて診断する場合は、感度70%、特異度70%と高精度であることが確認された。 組 み 合 わ せ By combining these four microRNAs, it becomes possible to distinguish between the patient group and the control group at a ratio as high as 10.86 times, which is thought to contribute to the diagnosis of rheumatoid arthritis. Furthermore, the results of creating a receiver operating characteristic (ROC) curve for early rheumatoid arthritis patients (30 patients) in the case of diagnosing with each of the identified biomarkers and in the case of diagnosing all of them in combination are shown in FIGS. . In particular, when all were combined and diagnosed, it was confirmed that the sensitivity was 70% and the specificity was 70% and the accuracy was high.
　さらに、上記解析を抗CCP抗体陰性関節リウマチ患者についても行った。本発明に含まれるマイクロRNA（hsa-miR-93-5p、hsa-miR-106b-5p、hsa-miR-301b-3p、hsa-miR-762）は、抗CCP抗体陰性関節リウマチの患者群および対照群との間での異なる発現レベルを有し、その比（オッズ比）は各々、0.733倍、0.641倍、0.567倍、2.65倍である。この詳細データを表２に示す。 The above analysis was also performed on anti-CCP antibody negative rheumatoid arthritis patients. The microRNA (hsa-miR-93-5p, hsa-miR-106b-5p, hsa-miR-301b-3p, hsa-miR-762) included in the present invention is a group of patients with anti-CCP antibody negative rheumatoid arthritis and It has different expression levels with the control group, the ratios (odds ratio) are 0.733, 0.641, 0.567 and 2.65, respectively. Table 2 shows the detailed data.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　これら4つのマイクロRNAを組み合わせることで、9.95倍という高い比率をもって抗CCP抗体陰性関節リウマチ群と対照群を区別することが可能である。さらに、同定したバイオマーカーそれぞれで診断する場合及び全てを組合わせて診断する場合の、抗CCP抗体陰性関節リウマチ患者（14名）に対する受信者動作特性（ROC）曲線を作成した結果を図７～１１に示す。特に、全てを組合わせて診断する場合は、感度50%、特異度94%と高精度であることが確認された。このことから、早期リウマチ患者における抗CCP抗体での偽陰性（見逃し例）を診断することができる。 (4) By combining these four microRNAs, it is possible to distinguish the anti-CCP antibody-negative rheumatoid arthritis group from the control group at a high ratio of 9.95 times. Furthermore, the results of creating a receiver operating characteristic (ROC) curve for anti-CCP antibody-negative rheumatoid arthritis patients (14 patients) in the case of diagnosing with each of the identified biomarkers and in the case of diagnosing in combination of all are shown in FIG. 11 is shown. In particular, when all were combined for diagnosis, it was confirmed that the sensitivity was 50% and the specificity was 94%, and the accuracy was high. From this, it is possible to diagnose false negatives (missing cases) with anti-CCP antibodies in early rheumatic patients.

Claims (10)

  1. 関節リウマチを検査する方法であって、
    （1）被検体から採取された体液における、miR-93-5p、miR-106b-5p、miR-301b-3p、及びmiR-762からなる群より選択される少なくとも1種のバイオマーカーを検出する工程、
    を含む、検査方法。     A method for testing rheumatoid arthritis,
    (1) detecting at least one biomarker selected from the group consisting of miR-93-5p, miR-106b-5p, miR-301b-3p, and miR-762 in a body fluid collected from a subject Process,
    Inspection methods, including:
  2. 検出したバイオマーカーがmiR-93-5p、miR-106b-5p、及びmiR-301b-3pからなる群より選択される少なくとも1種のバイオマーカー（BM1）を含む場合、さらに、
    （2a）前記工程（1）で検出された前記BM1の量又は濃度がカットオフ値以下である場合に、前記被検体が関節リウマチに罹患していると判定する工程、
    を含む、請求項１に記載の検査方法。     When the detected biomarkers include at least one biomarker (BM1) selected from the group consisting of miR-93-5p, miR-106b-5p, and miR-301b-3p,
    (2a) a step of determining that the subject is suffering from rheumatoid arthritis when the amount or concentration of the BM1 detected in the step (1) is equal to or less than a cutoff value;
    The inspection method according to claim 1, comprising:
  3. 検出したバイオマーカーがmiR-762（BM2）を含む場合、さらに、
    （2b）前記工程（1）で検出された前記BM2の量又は濃度がカットオフ値以上である場合に、前記被検体が関節リウマチに罹患していると判定する工程、
    を含む、請求項１に記載の検査方法。     If the detected biomarkers include miR-762 (BM2),
    (2b) a step of determining that the subject is suffering from rheumatoid arthritis when the amount or concentration of the BM2 detected in the step (1) is not less than a cutoff value;
    The inspection method according to claim 1, comprising:
  4. 検出したバイオマーカーがmiR-93-5p、miR-106b-5p、及びmiR-301b-3pからなる群より選択される少なくとも1種のバイオマーカー（BM1）、並びにmiR-762（BM2）を含む場合、さらに、
    （2c）前記工程（1）で検出された前記BM1の量又は濃度がカットオフ値以下であり、且つ前記工程（1）で検出された前記BM2の量又は濃度がカットオフ値以上である場合に、前記被検体が関節リウマチに罹患していると判定する工程、
    を含む、請求項１に記載の検査方法。     When the detected biomarkers include at least one biomarker (BM1) selected from the group consisting of miR-93-5p, miR-106b-5p, and miR-301b-3p, and miR-762 (BM2) ,further,
    (2c) When the amount or concentration of the BM1 detected in the step (1) is equal to or less than a cutoff value, and the amount or concentration of the BM2 detected in the step (1) is equal to or more than a cutoff value. In the step of determining that the subject is suffering from rheumatoid arthritis,
    The inspection method according to claim 1, comprising:
  5. 前記体液が全血、血漿、及び血清からなる群より選択される少なくとも1種である、請求項１～４のいずれかに記載の検査方法。 The test method according to claim 1, wherein the body fluid is at least one selected from the group consisting of whole blood, plasma, and serum.
  6. 前記被検体がヒトである、請求項１～５のいずれかに記載の検査方法。 The test method according to any one of claims 1 to 5, wherein the subject is a human.
  7. miR-93-5p、miR-106b-5p、miR-301b-3p、及びmiR-762からなる群より選択される少なくとも1種のバイオマーカーの検出剤を含む、関節リウマチの検査薬。 A test agent for rheumatoid arthritis, comprising a detection agent for at least one biomarker selected from the group consisting of miR-93-5p, miR-106b-5p, miR-301b-3p, and miR-762.
  8. miR-93-5p、miR-106b-5p、miR-301b-3p、及びmiR-762からなる群より選択される少なくとも1種のバイオマーカーの検出剤を含む、関節リウマチの検査キット。 A test kit for rheumatoid arthritis, comprising an agent for detecting at least one biomarker selected from the group consisting of miR-93-5p, miR-106b-5p, miR-301b-3p, and miR-762.
  9. 被検物質で処理された動物から採取された体液における、miR-93-5p、miR-106b-5p、miR-301b-3p、及びmiR-762からなる群より選択される少なくとも1種のバイオマーカーの量又は濃度を指標とする、関節リウマチの予防又は治療剤の有効成分のスクリーニング方法。 At least one biomarker selected from the group consisting of miR-93-5p, miR-106b-5p, miR-301b-3p, and miR-762 in a body fluid collected from an animal treated with the test substance A method for screening an active ingredient of a prophylactic or therapeutic agent for rheumatoid arthritis, using the amount or concentration of as an index.
  10. 被検物質で処理された動物から採取された体液における、miR-93-5p、miR-106b-5p、miR-301b-3p、及びmiR-762からなる群より選択される少なくとも1種のバイオマーカーの量又は濃度を指標とする、関節リウマチの誘発性又は増悪性の評価方法。 At least one biomarker selected from the group consisting of miR-93-5p, miR-106b-5p, miR-301b-3p, and miR-762 in a body fluid collected from an animal treated with the test substance A method for evaluating rheumatoid arthritis inducibility or malignancy using the amount or concentration of as an index.
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CN107243012A (en) * 2017-05-25 2017-10-13 江苏大学 A kind of applications of the 5p of miR 93 that exosomes is loaded in treatment rheumatoid arthritis

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