CN112961914A - miR-3074-5p作为类风湿关节炎标志物的应用及其试剂盒 - Google Patents
miR-3074-5p作为类风湿关节炎标志物的应用及其试剂盒 Download PDFInfo
- Publication number
- CN112961914A CN112961914A CN202110171439.0A CN202110171439A CN112961914A CN 112961914 A CN112961914 A CN 112961914A CN 202110171439 A CN202110171439 A CN 202110171439A CN 112961914 A CN112961914 A CN 112961914A
- Authority
- CN
- China
- Prior art keywords
- mir
- kit
- rheumatoid arthritis
- tnfsf14
- expression level
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108091047945 miR-3074 stem-loop Proteins 0.000 title claims abstract description 92
- 108091050163 miR-3074-1 stem-loop Proteins 0.000 title claims abstract description 92
- 108091055582 miR-3074-2 stem-loop Proteins 0.000 title claims abstract description 92
- 206010039073 rheumatoid arthritis Diseases 0.000 title claims abstract description 61
- 239000003550 marker Substances 0.000 title description 4
- 108010065158 Tumor Necrosis Factor Ligand Superfamily Member 14 Proteins 0.000 claims abstract description 39
- 102000012883 Tumor Necrosis Factor Ligand Superfamily Member 14 Human genes 0.000 claims abstract description 34
- 239000002679 microRNA Substances 0.000 claims abstract description 31
- 108091070501 miRNA Proteins 0.000 claims abstract description 24
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 21
- 238000001514 detection method Methods 0.000 claims abstract description 20
- 108700011259 MicroRNAs Proteins 0.000 claims abstract description 15
- 238000002360 preparation method Methods 0.000 claims abstract description 9
- 239000003147 molecular marker Substances 0.000 claims abstract description 5
- 239000003112 inhibitor Substances 0.000 claims description 20
- 238000010839 reverse transcription Methods 0.000 claims description 11
- 238000009007 Diagnostic Kit Methods 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 102000004190 Enzymes Human genes 0.000 claims description 4
- 108090000790 Enzymes Proteins 0.000 claims description 4
- 239000011535 reaction buffer Substances 0.000 claims description 4
- 230000002441 reversible effect Effects 0.000 claims description 4
- 230000002401 inhibitory effect Effects 0.000 claims description 2
- 230000009467 reduction Effects 0.000 claims description 2
- 230000001225 therapeutic effect Effects 0.000 claims description 2
- 238000002405 diagnostic procedure Methods 0.000 claims 1
- 108020004999 messenger RNA Proteins 0.000 abstract description 16
- 238000003745 diagnosis Methods 0.000 abstract description 5
- 238000012360 testing method Methods 0.000 abstract description 3
- 230000005764 inhibitory process Effects 0.000 abstract description 2
- 230000003334 potential effect Effects 0.000 abstract 1
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 22
- 210000004027 cell Anatomy 0.000 description 17
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 13
- 238000003757 reverse transcription PCR Methods 0.000 description 12
- 239000005089 Luciferase Substances 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 9
- 238000001890 transfection Methods 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 6
- 239000013642 negative control Substances 0.000 description 6
- 108060001084 Luciferase Proteins 0.000 description 5
- 238000012761 co-transfection Methods 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- 108090000331 Firefly luciferases Proteins 0.000 description 4
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 230000000295 complement effect Effects 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 4
- 230000002596 correlated effect Effects 0.000 description 4
- 230000009977 dual effect Effects 0.000 description 4
- 239000012091 fetal bovine serum Substances 0.000 description 4
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 4
- 238000002844 melting Methods 0.000 description 4
- 230000008018 melting Effects 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 4
- 102100032752 C-reactive protein Human genes 0.000 description 3
- 238000011529 RT qPCR Methods 0.000 description 3
- 210000001744 T-lymphocyte Anatomy 0.000 description 3
- 102100040247 Tumor necrosis factor Human genes 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 210000001503 joint Anatomy 0.000 description 3
- 230000008506 pathogenesis Effects 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 208000037976 chronic inflammation Diseases 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 239000000104 diagnostic biomarker Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 230000009266 disease activity Effects 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 238000013399 early diagnosis Methods 0.000 description 2
- 239000012636 effector Substances 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 238000003670 luciferase enzyme activity assay Methods 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 239000012096 transfection reagent Substances 0.000 description 2
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- 102100022717 Atypical chemokine receptor 1 Human genes 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 108010074051 C-Reactive Protein Proteins 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 101000678879 Homo sapiens Atypical chemokine receptor 1 Proteins 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 108090001005 Interleukin-6 Proteins 0.000 description 1
- 206010023232 Joint swelling Diseases 0.000 description 1
- 238000001276 Kolmogorov–Smirnov test Methods 0.000 description 1
- 102000012220 Member 14 Tumor Necrosis Factor Receptors Human genes 0.000 description 1
- 108010061593 Member 14 Tumor Necrosis Factor Receptors Proteins 0.000 description 1
- 101100185408 Mus musculus Mug2 gene Proteins 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 108010052090 Renilla Luciferases Proteins 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 241000700584 Simplexvirus Species 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 108091026822 U6 spliceosomal RNA Proteins 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000003356 anti-rheumatic effect Effects 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 239000003435 antirheumatic agent Substances 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000005784 autoimmunity Effects 0.000 description 1
- 230000008436 biogenesis Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 230000006020 chronic inflammation Effects 0.000 description 1
- 208000037893 chronic inflammatory disorder Diseases 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000010219 correlation analysis Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 230000035622 drinking Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000006481 glucose medium Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000011132 hemopoiesis Effects 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 210000003125 jurkat cell Anatomy 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 108091027963 non-coding RNA Proteins 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 235000017924 poor diet Nutrition 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 230000011218 segmentation Effects 0.000 description 1
- 230000000391 smoking effect Effects 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 201000004595 synovitis Diseases 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- 230000009752 translational inhibition Effects 0.000 description 1
- 241001529453 unidentified herpesvirus Species 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Analytical Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Physical Education & Sports Medicine (AREA)
- Biophysics (AREA)
- Rheumatology (AREA)
- Physics & Mathematics (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Pathology (AREA)
- Microbiology (AREA)
- Epidemiology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明涉及miR‑3074‑5p的新用途，miR‑3074‑5p可作为microRNA分子标志物在制备类风湿关节炎的诊断试剂或试剂盒中进行应用。在miR‑3074‑5p抑制表达组中TNFSF14的mRNA表达水平明显高于对照组，通过计算受试者工作特征曲线(ROC)和斯皮尔曼相关性检验评估miRNA在RA中的诊断价值，确认其在RA诊断中的潜在作用。miR‑3074‑5p可以被认为是一个新的类风湿关节炎的检测或治疗靶点，为RA的诊断和治疗提供新应用方向。miR‑3074‑5p作为类风湿关节炎的检测或治疗靶点时，其AUC＝1，被认为是及其可信的检测指标。
Description
技术领域
本发明属于生物分子技术领域，涉及miR-3074-5p作为类风湿关节炎标志物的应用及其试剂盒。
背景技术
类风湿关节炎(Rheumatoid arthritis,RA)是一种常见的以滑膜炎、滑膜增生、骨软骨破坏为特征的慢性炎症疾病，最终可导致软骨或骨损伤，甚至是残疾。RA在女性中的发病率高于男性，遗传因素、环境因素和生活方式是风湿性关节炎的高危因素，如吸烟、饮酒、饮食不良等，但其发病机制尚不清楚。RA的早期诊断对于积极治疗关节或器官的不可逆损伤以及通过广泛使用常规药物和抗风湿性关节炎药物(DMARDs)控制慢性炎症至关重要。然而，部分类风湿因子和环瓜氨酸抗体均为阴性的RA患者可能会被误诊，延误可能导致临床预后恶化。因此，探讨RA的病因病机对RA的早期预防、早期诊断和有效干预具有重要价值。
小分子核糖核酸(microRNA，miRNA)是一种18-25nt长的内源性非编码RNA，广泛分布在多个真核生物，通过调节mRNA的降解或翻译抑制来调节一个或多个基因的表达。miRNA的合成和功能的分子机制已被广泛研究。随着分子生物学的发展，miRNA可以影响免疫系统的各个方面，从造血到效应功能，但其在RA中的作用机制尚未明确。
TNFSF14(tumor necrosis factor superfamily 14),又名LIGHT[lymphotoxin-like,exhibits inducible expression,and competes with HSV glycoprotein D(gD)for HVEM,a receptor expressed by T lymphocytes],或HVEM-L(Herpesvirus entrymediator-Ligand),是Mauri等于1998年在对单纯疱疹病毒活化T细胞的研究中发现的TNF超家族的第14个成员。LIGHT是一个多功能、多效应分子，在体内的异常表达参与了机体自身免疫，与一些自身免疫性疾病有关。
发明内容
有鉴于此，本发明的目的在于提供一种miR-3074-5p作为microRNA分子标志物在制备类风湿关节炎的诊断试剂或试剂盒中的应用，以及检测miR-3074-5p表达水平的引物的相关应用，还提供一种检测类风湿关节炎的诊断试剂盒。
为达到上述目的，本发明提供如下技术方案：
1.miR-3074-5p作为microRNA分子标志物在制备类风湿关节炎的诊断试剂或试剂盒中的应用。
2.检测miR-3074-5p表达水平的引物在制备类风湿关节炎的诊断试剂或试剂盒中的应用。
进一步，所述miR-3074-5p表达水平为外周血单个核细胞中的miR-3074-5p表达水平。
进一步，检测miR-3074-5p表达水平的引物的序列为SEQ ID NO:6。
3.一种检测类风湿关节炎的诊断试剂盒，所述诊断试剂盒包括检测miR-3074-5p表达水平的引物。
进一步，检测miR-3074-5p表达水平的引物的序列为SEQ ID NO:6。
进一步，试剂盒还包括反转录试剂部分和荧光定量检测试剂部分。
进一步，反转录试剂部分由2×miRNA RT Reaction Buffer、miRNA RT EnzymeMix、RNase-Free ddH2O组成。
进一步，荧光定量检测试剂部分由2×miRcute Plus miRNA PreMix、10μMReverse Primer、ddH2O组成。
进一步，2×miRcute Plus miRNA PreMix中包含SYBR和ROX染料。
进一步，反转录体系总体积20μl，总RNA为2～5μl，2×miRNA RT Reaction Buffer10μl、miRNA RT Enzyme Mix 2μl，最后RNase-Free ddH2O补至20μl。
进一步，荧光定量检测体系总体积20μl或50μl，20μl体系组成为2×miRcute PlusmiRNA PreMix 10μl、10μM Reverse Primer 0.4μl、Forward Primer 0.4～0.8μl、cDNA1-2μl、余量为ddH2O。
进一步，2×miRcute Plus miRNA PreMix中包含SYBR和ROX染料。
4、miR-3074-5p抑制剂在制备抑制TNFSF14表达量降低的试剂中的应用。
5、miR-3074-5p抑制剂在制备类风湿关节炎的治疗试剂或试剂盒中的应用。
本发明的有益效果在于：本发明通过检测RA外周血单个核细胞(Peripheralblood mononuclear cells,PBMC)中不同基因的mRNA表达，发现TNFSF14 mRNA水平在RA的PBMCs存在显著表达差异，进一步利用生信预测可能与其相互作用的microRNA，再通过大量双荧光素酶报告系统和功能性实验等方法进行验证；最后用RT-PCR检测到miR-3074-5p水平在RA的PBMCs存在表达差异，并在miR-3074-5p不同表达状态下检测TNFSF14的表达水平，结果显示TNFSF14的基因mRNA表达量在miR-3074-5p过表达的细胞系中表达量明显下调，而在miR-3074-5p抑制表达组中TNFSF14的mRNA表达水平明显高于对照组。由此可以得出TNFSF14表达受到miR-3074-5p的调控作用，结合双荧光素酶报告系统可以认为TNFSF14是miR-3074-5p下游的靶基因。再进一步通过ROC曲线评估miR-3074-5p在RA中的临床诊断价值，确认其在RA诊断中的潜在作用。miR-3074-5p与RA PBMCs中的anti-CCP水平呈正相关，TNFSF14表达水平与RA PBMCs中RA-IgG、RF和DAS28呈负相关。这些结果都表明miR-3074-5p通过靶向TNFSF14参与RA的发生、发展过程，可以被认为是一个新的类风湿关节炎的检测或治疗靶点，为RA的诊断和治疗提供新应用方向。miR-3074-5p作为类风湿关节炎的检测或治疗靶点时，其AUC＝1，被认为是及其可信的检测指标。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚，本发明提供如下附图进行说明：
图1为PBMC中TNFSF14的mRNA水平相对表达量差异比较；
图2为作用于TNFSF14的microRNAs韦恩图；
图3为TNFSF14的microRNAs相互作用的网络图；
图4为miR-3074-5p与TNFSF14基因存在的多个互补结合位点示意图；
图5为各RT-PCR结果，其中，a为用miR-3074-5p mimics转染后，miR-3074-5p的表达水平；b，miR-3074-5p模拟物转染后，TNFSF14的表达水平；c，转染miR-3074-5p抑制剂后，miR-3074-5p的表达水平。d，转染miR-3074-5p抑制剂后，TNFSF14的表达水平。
图6为双荧光素酶活性检测结果；
图7为miR-3074-5p的熔解曲线；
图8为TNFSF14的熔解曲线；
图9为RA和HC的定量RT-PCR结果及比较；
图10为RA和HC组的PBMC中miR-3074-5p的ROC分析；
图11为RA患者中miR-3074-5p、TNFSF14表达与部分临床参数的关系。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
以下实施例中的定量试验，均设置三次重复实验，数据为三次重复实验的平均值或平均值±标准差。
主要试剂：
1、RNA提取试剂盒为日本TaKaRa公司、miRNA逆转录试剂盒和荧光定量PCR试剂盒为天根公司。
2、野生型(WT)和突变型(MUT)双荧光素酶载体由广州锐博构建。
3、miR-3074-5p模拟物(mimics)、阴性对照模拟物(NC mimics)、miR-3074-5p抑制剂(inhibitor)和阴性对照抑制剂(NC inhibitor)由上海吉玛公司合成。
4、双荧光素酶检测试剂盒购于北京普洛麦格公司。
5、胎牛血清(FBS)、细胞培养基(RPMI-1640、DMEM)和转染试剂(Lipofectamine3000)均来源为美国Thermo Fisher公司。
主要仪器：
Nanodrop-2000核酸定量仪来源为美国Thermo Fisher公司，高速冷冻离心机来源为德国Eppendorf公司，qRT-PCR仪来源为美国Bio-Rad公司。
实施例1
研究对象：2019年3月至2020年10月在陆军军医大学西南医院招募22例RA患者。RA疾病活动度由疾病活动度评分28(DAS28)来定义。此外，本研究纳入22名健康人(Healthycontrol,HC)作为对照(表1)。研究由西南医院伦理委员会批准并监督，所有参与者均给出了书面知情同意书。人T淋巴细胞白血病细胞，Jurkat(Clone E6-1)和人胚肾细胞293T细胞(HEK-293T)购自中国科学院上海细胞库。
表1研究人群的临床特征
其中RF:rheumatoid factor(类风湿因子)；anti-CCP:anti-cyclocitrullineantibody(抗环瓜氨酸抗体)；TJC:number of tenderness joints(压痛关节数)；SJC:Number of joint swelling(关节肿胀数)；ESR:Erythrocyte sedimentation rate(红细胞沉降率)；CRP:C-reactive protein(C反应蛋白)；DAS28:Condition score of patientswith rheumatoid arthritis(类风湿关节炎患者的病情评分)。所有数据均表示为平均值±SD。N/A(Not applicable)：不适用。
实施例2
本实施例中使用的mRNA和GAPDH的引物由英潍捷基有限公司(上海，中国)设计和合成。用于检测基因mRNA表达水平的引物如下表2所示：
表2
| 名称 | 引物序列 | 序列号 |
| TNFSF14 | 5'-ATACAAGAGCGAAGGTCTCACG-3'(F) | SEQ ID No.1 |
| TNFSF14 | 5'-CTGAGTCTCCCATAACAGCGG-3'(R) | SEQ ID No.2 |
| GAPDH | 5'-GGAGTCCACTGGCGTCTTC-3'(F) | SEQ ID No.3 |
| GAPDH | 5'-GCTGATGATCTTGAGGCTGTTG-3'(R) | SEQ ID No.4 |
用GAPDH水平标准化circRNA的相对表达水平。
图8为TNFSF14的熔解曲线(箭头所指的曲线)，22例RA患者PBMC中TNFSF14的mRNA水平相对表达量为(0.59±0.36)，与22例HC相比(1.41±0.39)，差异有统计学意义(P<0.0001)，具有显著差异，结果如图1所示，结果提示RA患者PBMC中TNFSF14 mRNA的相对表达量显著低于健康人组。
实施例3
进一步探究TNFSF14参与RA发病可能的机制，我们通过miRDB(http://www.mirdb.org/),miRwalk(http://mirwalk.umm.uni-heidelberg.de/)和TargetScan(http://www.targetscan)预测可能作用于TNFSF14的microRNAs。韦恩图分析发现71个共同交叉的microRNAs(图2)，其中蓝色代表miRDB，黄色代表miRwalk，绿色代表TargetScan。并通过cytoscape作出它们相互作用的网络图(图3)。并通过Targetscan软件分析各microRNAs与TNFSF14基因的多个互补结合位点，图4为miR-3074-5p与TNFSF14基因存在的多个互补结合位点示意图。进一步通过实验研究证明各microRNAs与TNFSF14基因的潜在关系，以发现和证明microRNAs可作为RA诊断分子标志物的潜能。经过重重实验筛选和证实，TNFSF14是miR-3074-5p的潜在靶基因，以下只记载与本发明技术方案相关的内容，其余实验在此不提。
实施例4
为进一步证实TNFSF14是miR-3074-5p的潜在靶基因，我们在细胞系Jurkat中转染miR-3074-5p minics和miR-3074-5p inhibitor，NC minics和NC inhibitor作为阴性对照。miRNA inhibitor为化学合成的经过甲氧基修饰设计的成熟miRNA互补单链。专门针对细胞中特异的靶miRNA的抑制剂，可高效地抑制生物体内源型miRNA的活性。
1、细胞培养与转染：Jurkat细胞用RPMI-1640培养基(10％FBS、1％青霉素链霉素)，37℃5％CO2恒温孵箱培养，第二天换液。将细胞分为转染组(miR-3074-5p mimics、miR-3074-5p inhibitor)，阴性对照组(NC mimics、NC inhibitor)和只加转染试剂的空白对照组。转染用12孔板，每孔5×105个jurkat细胞，使用Opti-MEMTM培养基分别稀释带转染的质粒和Lipofectamine 3000，5min后将两者按1:1比例轻轻充分混匀并在室温下静置15min，每个培养孔加入质粒-脂质体复合物，前后轻轻摇动细胞培养板使复合物与培养板中培养液混匀，最后将细胞放置于细胞培养箱中培养，48h之后收集细胞用于后续RT-PCR实验。
图5为各RT-PCR结果，其中，a为用miR-3074-5p mimics转染后，通过RT-PCR检测miR-3074-5p的表达水平；b，miR-3074-5p模拟物转染后，通过RT-PCR检测TNFSF14的表达水平；c，转染miR-3074-5p抑制剂后，通过RT-PCR检测miR-3074-5p的表达水平。d，转染miR-3074-5p抑制剂后，通过RT-PCR检测TNFSF14的表达水平。各RT-PCR结果显示，miR-3074-5pminics转染jurkat之后，miR-3074-5p mRNA表达增加，TNFSF14 mRNA表达减少，与阴性对照组比较，差异有统计学意义(P<0.05)(图5a，5b)。miR-3074-5p inhibitor转染Jurkat细胞之后，miR-3074-5p mRNA表达减少，TNFSF14 mRNA表达增加，与阴性对照组比较，差异有统计学意义(P<0.05)(图5c，5d)。功能实验综合显示miR-3074-5p可有效调控TNFSF14表达。
2、双荧光素酶报告系统检测荧光素酶活性：将TNFSF14-3'UTR构建到双荧光素酶质粒中，分miR-3074-5p mimics+TNFSF14-3'UTR-WT、NC mimics+TNFSF14-3'UTR-WT、miR-3074-5p mimics+TNFSF14-3'UTR-MUT和NC mimics+TNFSF14-3'UTR-MUT 4组共转染于293T细胞中。
将293T细胞置于DMEM高糖培养基+10％FBS、1％青霉素链霉素，37℃5％CO2恒温孵箱里培养，待293T细胞密度达到50％-70％时，按照以下分组转染细胞：miR-3074-5pmimics+TNFSF14-3'UTR-WT共转染组、NC mimics+TNFSF14-3'UTR-WT共转染组、miR-3074-5p mimics+TNFSF14-3'UTR-MUT共转染组和NC mimics+TNFSF14-3'UTR-MUT共转染组。48h后收集各转染组的293T细胞，弃去细胞培养液后加入200μl被动细胞裂解液，摇床上晃动20min充分裂解细胞后4000rpm/min离心5min，按照双荧光素酶报告基因检测试剂盒说明书吸取上清液测定萤火虫荧光素酶和海肾荧光素酶活性。萤火虫荧光素酶作为内参，计算两者相对荧光强度的比值，以比值作为相对荧光素酶活性的结果。每个转染组独立重复3次实验，对3次测定的实验数据进行统计分析。
图6为双荧光素酶活性检测结果，其中a为带有miR-3074-5p的TNFSF14 3-UTR上结合位点的突变示意图；b为TNFSF14 3-UTR和miR-3074-5p共转染对293T细胞荧光的影响。检测结果表明，miR-3074-5p mimics+TNFSF14 3'UTR-WT共转染组低于对照组，差异有统计学意义(P<0.05)；突变TNFSF14-3'UTR与miR-3074-5p的结合位点之后，miR-3074-5p mimics+TNFSF14-3'UTR-MUT共转染组与对照组比较，差异无统计学意义(P>0.05)(图6b)。实验结果表明miR-3074-5p通过靶向TNFSF14-3'UTR负向调控TNFSF14的表达。
实施例5
为了验证miR-3074-5p在RA中的表达水平是否与预测的一致，我们在22例RA患者和22例健康对照组中进行了定量RT-PCR。
外周血单个核细胞的制备及总RNA的提取，(TaKaRa，Code No.9108Q)：实验前从每个受试者中取约5ml全血，储存在乙二胺四乙酸(EDTA))抗凝管中。采用室温密度梯度离心法(2000rpm，20min)从供者新鲜血液提取PBMCs。随后，根据说明书用Trizol提取总RNA，nanodrop 2000和1％琼脂糖凝胶用于测定RNA浓度和纯度，将总的RNA保存在-80℃以备进一步实验。
将总RNA反转录为cDNA，选用miRcute增强型miRNA cDNA第一链合成试剂盒(KR211)，反转录体系总体积20μl，具体体系成分见下表3：
表3：反转录体系
| 试剂组分 | 体积 | 终浓度 |
| 总RNA | 2-5μl | 可达2μg |
| 2×miRNA RT Reaction Buffer | 10μl | 1× |
| miRNA RT Enzyme Mix | 2μl | - |
| RNase-Free ddH<sub>2</sub>O | 补至20μl | - |
逆转录miRNA为cDNA：42℃60min加poly A尾和逆转录反应，95℃灭活逆转录酶3min，-20℃保存。
实时定量聚合酶链反应：使用miRcute增强型miRNA荧光定量检测试剂盒(FP411)，定量RT-PCR体系总体积20μl或50μl，具体体系成分见下表4：
表4：
本实施例中使用的microRNA和U6的正向引物由天根生物技术合成(北京，中国)，U6snRNA作为microRNA规范化的内源性对照。通用反向引物在天根逆转录试剂盒中提供。U6Forward Primer为5'-CGCAAGGATGACACGCAAATTC-3'(SEQ ID No.5)，miR-3074-5p的Forward Primer为5’-TTCCTGCTGAACTGAGCCA-3’(SEQ ID No.6)。
将上述cDNA(稀释10倍)为模板，用SEQ ID No.6所示的miR-3074-5p PCR引物进行qRT-PCR反应。qRT-PCR反应条件：95℃预变性30s，95℃变性15s，60℃退火30s，共40个循环。反应结束后根据扩增曲线分析扩增结果，得到了每个样本的Ct值，按照2-ΔΔCt法计算目的基因和内参U6的相对表达量相对表达量。每个样本独立重复三次以上。
图7为miR-3074-5p的熔解曲线，图9为定量PCR检测miR-3074-5p在RA和HC组PBMC中的表达结果及比较；图10为RA和HC组的PBMC中miR-3074-5p的ROC分析。结果表明，22例RA患者PBMC中miR-3074-5p的相对表达量为(1.64±0.34)，与22例HC相比(0.77±0.25)，差异有统计学意义(P<0.001)，RA患者PBMC中miR-3074-5p的相对表达量高于健康人组。进一步，我们使用ROC曲线及曲线下面积(area under the curve，AUC)分析来验证miR-3074-5p作为RA诊断分子标志物的潜能。其中AUC反映了检测效能，AUC越接近1.0，检测方法真实性越高；0.5＜AUC＜0.7检测效能较低；0.7＜AUC＜0.85检测效能一般；0.85＜AUC＜1，检测效能很好；AUC＝1，检测效能完美。ACU的CI值95％。
由曲线下面积(area under The curve,AUC)表明，miR-3074-5p在PBMC中可以区分RA患者与HC(图10)，miR-3074-5p可以作为RA PBMC中的诊断生物标记物。随后，我们分析了RA患者PBMC中miR-3074-5p、TNFSF14的表达与年龄、病程、TNF-α、IL-6、RA-IgG、RA-IgM、RA-IgA、SJC、TJC、ESR、CRP和DAS28等指标的关系。结果显示，miR-3074-5p水平与CCP呈正相关(p<0.001)(图11中a)。TNFSF14水平与RA中RF、RA-IgG、DAS28水平呈负相关，(p<0.001)，(图11中b为TNFSF14水平与RA中RF水平关系)。因此，miR-3074-5p不仅可以作为RA PBMC中的诊断生物标记物进行应用，还至少可以部分反映疾病的严重程度。
以上实验统计学方法：采用双尾Student's t检验和Mann-Whitney's t检验分别评估组间表达的正态分布参数和偏态分布参数。相关分析采用单样本Kolmogorov-Smirnov检验，组间正态分布时采用Pearson相关检验；非正态分布时采用Spearman相关检验分析。采用SPSS22.0统计软件分析，P<0.05为差异有统计学意义。
最后说明的是，以上优选实施例仅用以说明本发明的技术方案而非限制，尽管通过上述优选实施例已经对本发明进行了详细的描述，但本领域技术人员应当理解，可以在形式上和细节上对其作出各种各样的改变，而不偏离本发明权利要求书所限定的范围。
序列表
<110> 安徽省立医院（中国科学技术大学附属第一医院）
中国人民解放军陆军军医大学
<120> miR-3074-5p作为类风湿关节炎标志物的应用及其试剂盒
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 1
atacaagagc gaaggtctca cg 22
<210> 2
<211> 21
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 2
ctgagtctcc cataacagcg g 21
<210> 3
<211> 19
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 3
ggagtccact ggcgtcttc 19
<210> 4
<211> 22
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 4
gctgatgatc ttgaggctgt tg 22
<210> 5
<211> 22
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 5
cgcaaggatg acacgcaaat tc 22
<210> 6
<211> 19
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 6
ttcctgctga actgagcca 19
Claims (10)
1.miR-3074-5p作为microRNA分子标志物在制备类风湿关节炎的诊断试剂或试剂盒中的应用。
2.检测miR-3074-5p表达水平的引物在制备类风湿关节炎的诊断试剂或试剂盒中的应用。
3.根据权利要求2所述的应用，其特征在于，检测miR-3074-5p表达水平的引物的序列为SEQ ID NO:6。
4.一种检测类风湿关节炎的诊断试剂盒，其特征在于，所述诊断试剂盒包括检测miR-3074-5p表达水平的引物。
5.根据权利要求5所述的诊断试剂盒，其特征在于，检测miR-3074-5p表达水平的引物的序列为SEQ ID NO:6。
6.根据权利要求4或5所述的诊断试剂盒，其特征在于，试剂盒还包括反转录试剂部分和荧光定量检测试剂部分。
7.根据权利要求6所述的诊断试剂盒，其特征在于，反转录试剂部分由2×miRNA RTReaction Buffer、miRNA RT Enzyme Mix、RNase-Free ddH2O组成。
8.根据权利要求6所述的诊断试剂盒，其特征在于，荧光定量检测试剂部分由2×miRcute Plus miRNA PreMix、10μM Reverse Primer、ddH2O组成。
9.miR-3074-5p抑制剂在制备抑制TNFSF14表达量降低的试剂中的应用。
10.miR-3074-5p抑制剂在制备类风湿关节炎的治疗试剂或试剂盒中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110171439.0A CN112961914B (zh) | 2021-02-07 | 2021-02-07 | miR-3074-5p作为类风湿关节炎标志物的应用及其试剂盒 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110171439.0A CN112961914B (zh) | 2021-02-07 | 2021-02-07 | miR-3074-5p作为类风湿关节炎标志物的应用及其试剂盒 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112961914A true CN112961914A (zh) | 2021-06-15 |
| CN112961914B CN112961914B (zh) | 2022-08-30 |
Family
ID=76275416
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110171439.0A Active CN112961914B (zh) | 2021-02-07 | 2021-02-07 | miR-3074-5p作为类风湿关节炎标志物的应用及其试剂盒 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112961914B (zh) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101643730A (zh) * | 2009-07-27 | 2010-02-10 | 北京大学人民医院 | 微小RNA-146a在类风湿关节炎治疗中的用途 |
| CN103805696A (zh) * | 2013-12-12 | 2014-05-21 | 焦志军 | 一种诊断类风湿关节炎的microRNA分子标志物及其检测试剂盒 |
| US20140147454A1 (en) * | 2012-11-26 | 2014-05-29 | Moderna Therapeutics, Inc. | Terminally modified rna |
| CN106492223A (zh) * | 2016-09-29 | 2017-03-15 | 哈尔滨医科大学 | microRNA‑34a‑5p抑制剂在制备治疗类风湿关节炎药物中的应用 |
| US20180305689A1 (en) * | 2015-04-22 | 2018-10-25 | Mina Therapeutics Limited | Sarna compositions and methods of use |
-
2021
- 2021-02-07 CN CN202110171439.0A patent/CN112961914B/zh active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101643730A (zh) * | 2009-07-27 | 2010-02-10 | 北京大学人民医院 | 微小RNA-146a在类风湿关节炎治疗中的用途 |
| US20140147454A1 (en) * | 2012-11-26 | 2014-05-29 | Moderna Therapeutics, Inc. | Terminally modified rna |
| CN103805696A (zh) * | 2013-12-12 | 2014-05-21 | 焦志军 | 一种诊断类风湿关节炎的microRNA分子标志物及其检测试剂盒 |
| US20180305689A1 (en) * | 2015-04-22 | 2018-10-25 | Mina Therapeutics Limited | Sarna compositions and methods of use |
| CN106492223A (zh) * | 2016-09-29 | 2017-03-15 | 哈尔滨医科大学 | microRNA‑34a‑5p抑制剂在制备治疗类风湿关节炎药物中的应用 |
Non-Patent Citations (3)
| Title |
|---|
| YAN GU ET AL.: ""Aberrant Placental Villus Expression of miR-486-3p" and miR-3074-5p in Recurrent Miscarriage Patients and Uterine Expression of These MicroRNAs during Early Pregnancy in Mice", 《GYNECOL OBSTET INVEST》 * |
| ZHUYAN JIANG ET AL.: ""circPTPN22 as a novel biomarker and ceRNA in peripheral blood mononuclear cells of rheumatoid arthritis"", 《MOLECULAR MEDICINE REPORTS》 * |
| 蒋柱燕等: ""miR-3074-5p通过靶向TNFSF14参与类风湿关节炎的发生发展"", 《免疫学杂志》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112961914B (zh) | 2022-08-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Smigielska-Czepiel et al. | Comprehensive analysis of miRNA expression in T-cell subsets of rheumatoid arthritis patients reveals defined signatures of naive and memory Tregs | |
| Nakasa et al. | Expression of microRNA‐146 in rheumatoid arthritis synovial tissue | |
| Rossato et al. | Association of MicroRNA‐618 expression with altered frequency and activation of plasmacytoid dendritic cells in patients with systemic sclerosis | |
| Ho et al. | MicroRNA profiling in pediatric pilocytic astrocytoma reveals biologically relevant targets, including PBX3, NFIB, and METAP2 | |
| Hirota et al. | Genome-wide gene expression profiles of clear cell renal cell carcinoma: identification of molecular targets for treatment of renal cell carcinoma | |
| Kalpachidou et al. | Non-coding RNAs in neuropathic pain | |
| Townsend | Molecular and cellular heterogeneity in the Rheumatoid Arthritis synovium: clinical correlates of synovitis | |
| Zou et al. | Involvement of long noncoding RNAs in the pathogenesis of autoimmune diseases | |
| Ibrahim et al. | Inflammatory gene networks in term human decidual cells define a potential signature for cytokine-mediated parturition | |
| Gimondi et al. | Circulating miRNA panel for prediction of acute graft-versus-host disease in lymphoma patients undergoing matched unrelated hematopoietic stem cell transplantation | |
| WO2014014518A1 (en) | Methods for treating, preventing and predicting risk of developing breast cancer | |
| Schena et al. | microRNAs in glomerular diseases from pathophysiology to potential treatment target | |
| EP3933050A1 (en) | Application of circular rna in preparing drug for treating systemic lupus erythematosus | |
| WO2015143062A1 (en) | Dux4-induced gene expression in facioscapulohumeral muscular dystrophy (fshd) | |
| Jabandziev et al. | The emerging role of noncoding RNAs in pediatric inflammatory bowel disease | |
| Tofigh et al. | Rheumatoid arthritis and non-coding RNAs; how to trigger inflammation | |
| Qiu et al. | Comprehensive transcriptome analysis reveals competing endogenous RNA networks during avian leukosis virus, subgroup j-induced tumorigenesis in chickens | |
| WO2013009705A2 (en) | Biomarkers, methods, and compositions for inhibiting a multi-cancer mesenchymal transition mechanism | |
| CN112961914B (zh) | miR-3074-5p作为类风湿关节炎标志物的应用及其试剂盒 | |
| CN113980968B (zh) | 一种新的ra标志长链非编码rna及其应用 | |
| Romero et al. | Primary mediastinal large B-cell lymphoma: Transcriptional regulation by miR-92a through FOXP1 targeting | |
| CN112795640B (zh) | 三种microRNA作为RA标志物的应用及其试剂盒 | |
| CN102899405A (zh) | 检测肺癌肿瘤细胞rna变化的试剂盒 | |
| CN111534587A (zh) | 分子标志物5-tRF-His、乳腺癌检测试剂盒及其应用 | |
| Wang et al. | MicroRNA‐7 deficiency ameliorates d‐galactose‐induced aging in mice by regulating senescence of Kupffer cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20240110 Address after: 230000 No.9 Lujiang Road, Luyang District, Hefei City, Anhui Province Patentee after: Anhui Provincial Hospital (the First Affiliated Hospital of China University of science and Technology) Address before: 230000 No.9 Lujiang Road, Luyang District, Hefei City, Anhui Province Patentee before: Anhui Provincial Hospital (the First Affiliated Hospital of China University of science and Technology) Patentee before: THIRD MILITARY MEDICAL University |
|
| TR01 | Transfer of patent right |