MiR-128-3p Regulates The Pathogenesis Of Rheumatoid Arthritis By Regulating TNFAIP3

Objective : To investigate the role of Mi R-128-3p,a highly expressed in the T cell of patients with rheumatoid arthritis(RA),in the pathogenesis of RA.Methods: 1?Firstly,20 patients diagnosed with RA according to the?2010 ACR/EULAR Rheumatoid Arthritis Classification Standard?were collected as subjects.At the same time,20 healthy volunteers were randomly selected as the control group.The median age is 43 years old.Two groups of 5ml peripheral venous blood were drawn as fresh anticoagulant samples.Blood was collected after 12 h at the last medicine ta?ing to minimize the effects of the drugs.2?T cells in peripheral blood mononuclear cell(PBMC)were isolated from the peripheral blood from people of RA and normal person were used,and The cells were cultured with PMA/ionin-containing medium for 18 hours,and the expression of CD69 and CD25 was detected.3?Real-time PCR was performed to detect the expression of the RA group and control group's Mi R-128-3p,while the protein expression of the RA group and control group's tumor necrosis factor-?-induced protein 3(TNFAIP3)was determined using Western blot.The levels of the RA group and control group's IL-6 and IL-17 were measured using enzyme-lin?ed immunosorbent assay(ELISA).4?Computer promoter analysis and prediction algorithm: computer startup using a public database.A sub-analysis was performed to predict the Mi R-128-3p target gene binding sequence and to synthesize the 3'-UTR of TNFAIP3,which contains the Mi R-128-3p binding site.In addition,several site mutations were randomly selected from the Mi R-128-3p binding site to constitute a TNFAIP3 mutant for comparative study.The dual luciferase assay confirmed that Mi R-128-3p binds to the 3'-UTR end of the coding gene for TNFAIP3.5?Pre-Mi R-128-3p or pre-Mi R-128-3p negative control synthesized with 100 n M using Lipofectamine 2000(Pre-Mi R Mi RNA precursor molecule and Pre-Mi R Mi RNA precursor negative)were transfected into HE?293 T cells.And added separately in the two groups.The Mi R-128-3p mimetic and the Mi R-128-3p inhibitor were each 100 nmol/l.The luciferase activity was measured again.6?The expression of CD69 and CD25 was detected using flow cytometry.p-I?B? was determined using Western blot.7?Established a RA mouse model to further determine the mechanism of action of Mi R-128-3p on RA in vivo,The RA mouse were divided into Mi R-128-3p inhibitor and control group(each group n = 6),the joint inflammation score was calculated.Results:(1)The expression of Mi R-128-3p was significantly increased,while TNFAIP3 was decreased in the T cells of RA patients.(2)the levels of IL-6 and IL-17 were also increased in the T cells of RA patients.(3)luciferase reporter assay was conducted,indicating TNFAIP3 containing 3'-UTR that Mi R-128-3p combined with.(4)Down-regulated Mi R-128-3p significantly increased the expression of TNFAIP3.(5)Down-regulated Mi R-128-3p significantly suppressed the expression of p-I?B? and decreased the ratio of CD69 and CD25.T cells co-transfected with si-TNFAIP3 abolished the effects of Mi R-128-3p ?noc?down.(6)The RA mouse model of the Mi R-128-3p inhibitor group had lower RA scores and milder arthritis symptoms than the control group.Conclusion: To further elucidate the nature of Mi R-128-3p ysregulation in RA,in this study,we compared the expression levels of Mi R-128-3p in T cells isolated from serum from RA patients and healthy individuals,and found that Mi R-128-3p is raised in RA.We further investigated the regulation of pro-inflammatory molecules by binding to the 3'-UTR end of the TNFAIP3-encoding gene,which inhibits the expression of TNFAIP3,an important negative regulator of the NF-?B signaling pathway,and thus NF-?B signal transduction pathway and T cell activity are up-regulated.The clinical symptoms of RA have increased.Mi R-128-3p was identified as a pro-inflammatory and joint disrupting factor in RA.