Application of METTL3 gene and detection method thereof

A gene and arthritis technology, applied in the application field of METTL3 gene in the preparation of auxiliary diagnostic preparations for rheumatoid arthritis, molecular targeted therapy preparations, and prognosis evaluation preparations. It can solve the problem of METTL3 gene and rheumatoid arthritis detection applications. Reporting and other issues

Active Publication Date: 2019-03-12
潍坊医学院第一附属医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the application of METTL3 gene in the detection and treatment of rheumatoid arthritis has not been reported

Method used

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  • Application of METTL3 gene and detection method thereof
  • Application of METTL3 gene and detection method thereof
  • Application of METTL3 gene and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] In order to explore the expression of key enzymes and regulatory proteins (METTL3, METTL4, FTO, ALKBH5, YTHDF1, YTHDF2) related to RNA m6A methylation modification in peripheral blood mononuclear cells of RA patients.

[0038] After the project has passed the review of the Ethics Committee, the applicant will systematically study the new diagnostic and therapeutic targets of human RA, extract PBMCs from the peripheral blood of RA patients, and detect METTL3, METTL4, FTO, ALKBH5, YTHDF1, YTHDF2 by real-time PCR gene expression.

[0039] In this embodiment, a total of 47 blood samples were collected from patients with active RA, and 30 blood samples from healthy people in the control group, a total of 77 samples (see Table 1 for specific information from 47 patients, and see Table 2 for sample characteristics)

[0040] Table 1 Sample Information 1

[0041]

[0042]

[0043]

[0044] Table 2 Sample Information 2

[0045]

[0046]

[0047] In this embodimen...

Embodiment 2

[0067] Example 2 METTL3 overexpression sequence and its application

[0068] This example designs and synthesizes the specific overexpression sequence Lv-METTL3 (its nucleotide sequence is shown in SEQ ID NO.3) for the full-length sequence of METTL3, and infects macrophages to overexpress the METTL3 gene in the cells , and further apply the overexpressed gene carrier to regulate the proliferation and inflammatory response of macrophages ( image 3 , 4 with 5).

[0069] The specific virus packaging and cell infection methods are as follows: (1) 293T cells (purchased from the Shanghai Chinese Academy of Sciences Cell Bank) were plated and cultured, and the density was adjusted to 1×10 5 / ml; (2) Prepare the plasmid calcium phosphate mixed transfection solution of the lentiviral packaging transfection system, and place it at room temperature for 20-25 minutes; The dye solution was evenly distributed, and cultured in an incubator; (3) After 6-8 hours, discard the cell supernata...

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Abstract

The invention relates to detection and application of a key gene (METTL3) in an m6A methylation modification process. A specific PCR primer is designed and synthetized according to an extracted totalRNA sequence; a real-time PCR technique is used for discovering that expression of METTL3 in a mononuclear cell of peripheral blood of rheumatoid arthritis patients is obviously improved and is obviously in a positive correlation relationship with CRP and ESR which are disease activity indexes of the rheumatoid arthritis, so that the gene METTL3 can be applied to preparation of preparations applied to auxiliary diagnosis and treatment or prognosis evaluation of the rheumatoid arthritis. Lv-METTL3 sequences with specific overexpression of the gene are simultaneously designed and synthetized; proliferation of macrophages and inflammatory response induced by LPS of the macrophages can be obviously inhibited by regulating and controlling an NF-kB signal path; the METTL3 can be used as a molecular targeted therapy tool of the rheumatoid arthritis.

Description

technical field [0001] The invention belongs to the field of molecular biology of rheumatism, and in particular relates to the expression, application and detection method of a key gene METTL3 in m6A methylation. Specifically, the invention specifically relates to the use of METTL3 gene in the preparation of auxiliary diagnostic preparations for rheumatoid arthritis , Molecular targeted therapy preparations or the application of prognosis evaluation preparations. Background technique [0002] Rheumatoid arthritis (Rheumatoid Arthritis, RA) is a common inflammatory and systemic autoimmune disease of unknown etiology. and vasculitis (Scott DL, et al. Rheumatoid arthritis. Lancet. 2010; 376:1094-108.). RA usually causes damage to organs other than joints, such as pulmonary fibrosis, pericarditis, and rheumatoid nodules on the skin (Turesson C. Extra-articular rheumatoid arthritis. Curr Opin Rheumatol. 2013; 25:360-366.) . RA is highly teratogenic and disabling, and seriously...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/686C12N15/11A61K31/7088A61P19/02A61P29/00
CPCA61K31/7088A61P19/02A61P29/00C12Q1/686C12Q1/6883C12Q2600/118C12Q2600/158C12Q2545/101C12Q2521/107
Inventor 王增艳王萍萍王增芳徐洋闫书山徐栋花王菁华
Owner 潍坊医学院第一附属医院
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