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A kind of microrna biomarker and its application for the diagnosis of rheumatoid arthritis

A biomarker and rheumatoid technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of small heritability and genetic factors to be discovered, and achieve excellent sensitivity and specificity Effect

Active Publication Date: 2021-04-30
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, the molecular mechanism of RA pathogenesis has not yet been studied maturely. The genetic susceptibility loci that have been found together explain the heritability of RA is very small (15%), and there are still a large number of genetic factors to be discovered.

Method used

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  • A kind of microrna biomarker and its application for the diagnosis of rheumatoid arthritis
  • A kind of microrna biomarker and its application for the diagnosis of rheumatoid arthritis
  • A kind of microrna biomarker and its application for the diagnosis of rheumatoid arthritis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Chip Screening of Differentially Expressed microRNA (miRNA) in Peripheral Blood Mononuclear Cells

[0043] 1. After obtaining the informed consent of the patients, the peripheral blood samples of 25 patients diagnosed with rheumatoid arthritis and the peripheral blood samples of 18 healthy people were collected from the First People's Hospital Affiliated to Soochow University and treated with sodium citrate as normal controls. Coagulation tube.

[0044] 2. Obtain peripheral blood mononuclear cells from patients with rheumatoid arthritis and normal persons by density gradient centrifugation. The specific steps are as follows:

[0045] (1) Centrifuge the peripheral blood sample to make it layered, at 2000 rpm for 2 minutes.

[0046] (2) Add 15ml of separation liquid into the leucosep separation tube, and centrifuge at 5100g for 1 minute.

[0047] (3) Concentrate the supernatant of the stratified peripheral blood sample into a 15ml centrifuge tube, and centrif...

Embodiment 2

[0058] Example 2: qRT-PCR experiment of miRNA in peripheral blood mononuclear cells

[0059] 1. According to the miRNA microarray results, 9 miRNAs were selected from 18 differentially expressed miRNAs for qRT-PCR verification according to the following conditions. The specific conditions are: (1) the miRNA has not been reported before and has been verified by RT-PCR in the RA population; (2) the miRNA has a high FC value; (3) the miRNA has a high expression in the chip Level. Selected hsa-miR-99b-5p, hsa-miR-26b-5p, hsa-miR-7641, hsa-miR-142-5p, hsa-miR-29c-3p, hsa-miR-3607-5p, hsa - See Table 1 for the sequences of miR-3651, hsa-miR-4448, and hsa-miR-1184. The qRT-PCR detection of miRNA was performed on the peripheral blood mononuclear cells of patients with rheumatoid arthritis and normal controls. Strict quality control was implemented throughout the study, and each sample was continuously tested at least three times.

[0060] Table 1 Primer sequence list

[0061]

...

Embodiment 3

[0067] Example 3: Effect of specific miRNA (miR-99b-5p) on cell growth and inflammatory cytokines in Jurkat immune cells

[0068]The foregoing examples show that hsa-miR-99b-5p, hsa-miR-26b-5p, and hsa-miR-7641 have a higher diagnostic value for the detection of peripheral blood mononuclear cells in rheumatoid arthritis (high sensitivity and specificity ), this example carries out the association research between miR-99b-5p and rheumatoid arthritis.

[0069] 1. Construction of miR-99b-5p overexpression cell line in Jurkat cells using pCDH-CMV-MCS-EF1-copGFP vector

[0070] 1.1 Construction of miR-99b-5p lentiviral vector

[0071] Primers were designed according to the complete sequence of hsa-miR-99b-5p and its upstream and downstream sequences of 1000 bp, and the PCR product of the target sequence was prepared. The obtained PCR products were verified by electrophoresis. After the verification is correct, the PCR product is purified (Qiaquick PCR Purification Kit), digested...

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Abstract

The invention discloses a microRNA biomarker for diagnosing rheumatoid arthritis and an application thereof, belonging to the technical field of biological detection. These 3 microRNA biomarkers provided by the present invention and their corresponding primer sets and probes can be used to prepare diagnostic kits, and they have excellent performance when applied to the diagnosis of rheumatoid arthritis in peripheral blood mononuclear cell samples. sensitivity and specificity. The combined AUC value of the three microRNAs of the present invention can reach 0.745, and the sensitivity and specificity are 73.5% and 76.7% respectively. These three microRNAs can be used as biomarkers for the diagnosis of human rheumatoid arthritis peripheral blood mononuclear cell samples, which is conducive to promoting the development of early diagnosis, predictive treatment, and monitoring recurrence of rheumatoid arthritis in my country.

Description

technical field [0001] The invention relates to a microRNA biomarker for diagnosing rheumatoid arthritis and an application thereof, belonging to the technical field of biological detection. Background technique [0002] Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by erosive arthritis. About 1% of the world's population currently suffers from this disease, among which the prevalence rate in developed countries is 0.5-1%, and the annual new cases are 5-50 / 100,000. The prevalence rate in mainland China is 0.2-0.36%. The total is more than 5 million. The clinical manifestations are symmetrical and persistent joint swelling and pain, often accompanied by bone loss, bone erosion, loss of articular cartilage, synovial inflammation and joint capsule swelling. If the patient does not control the disease, it will gradually lead to muscular and skeletal insufficiency, decreased physical function, and induced commensal diseases. The late disabilit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6883C12N15/113
CPCC12Q1/6883C12Q2600/158C12Q2600/178
Inventor 雷署丰朱晓炜武龙飞邓飞艳莫兴波
Owner SUZHOU UNIV
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