WO2020054474A1 - Méthode d'examen de la polyarthrite rhumatoïde - Google Patents

Méthode d'examen de la polyarthrite rhumatoïde 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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English (en)
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/ja
Publication of WO2020054474A1 publication Critical patent/WO2020054474A1/fr
Priority to JP2024031786A priority patent/JP2024051168A/ja

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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.

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Abstract

La présente invention aborde le problème de la fourniture d'un nouveau biomarqueur pour la polyarthrite rhumatoïde et une méthode d'utilisation de celui-ci. Ce problème est résolu par une méthode d'examen de la polyarthrite rhumatoïde, ladite méthode comprenant (1) une étape de détection d'au moins un type de biomarqueur choisi dans le groupe comprenant miR-93-5p, miR-106b-5p, miR-301b-3p et miR-762 dans un liquide biologique prélevé chez un sujet.
PCT/JP2019/034364 2018-09-13 2019-09-02 Méthode d'examen de la polyarthrite rhumatoïde WO2020054474A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014034685A1 (fr) * 2012-08-27 2014-03-06 国立大学法人京都大学 Diagnostic de la polyarthrite rhumatoïde au moyen de microarn
CN107243012A (zh) * 2017-05-25 2017-10-13 江苏大学 一种exosomes装载的miR‑93‑5p在治疗类风湿性关节炎中的应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014034685A1 (fr) * 2012-08-27 2014-03-06 国立大学法人京都大学 Diagnostic de la polyarthrite rhumatoïde au moyen de microarn
CN107243012A (zh) * 2017-05-25 2017-10-13 江苏大学 一种exosomes装载的miR‑93‑5p在治疗类风湿性关节炎中的应用

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SAKAUE S. ET AL.: "Integration of genetics and miRNA-target gene network identified disease biology implicated in tissue specificity", NUCLEIC ACIDS RESEARCH, vol. 46, no. 22, 8 November 2018 (2018-11-08), pages 11898 - 11909, XP055693604 *
TAG Y. ET AL.: "Downregulation of miR-106b attenuates inflammatory responses and joint damage in collagen-induced arthritis", RHEUMATOLOGY, vol. 56, no. 10, October 2017 (2017-10-01), pages 1804 - 1813, XP055693607 *

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