Mechanism Of MiRNA-146a Regulating TLR4 Signaling Pathway In Osteoarthritis Chondrocytes

Objective:Osteoarthritis(OA)is a degenerative disease,which is caused by aging,obesity,strain,trauma,joint congenital abnormality,joint deformity and many other factors,such as articular cartilage degeneration and injury,joint edge and subchondral reactive hyperplasia.At present,the basic research on osteoarthritis was relatively backward,its pathogenesis was not clear,and there were obvious obstacles to the precise and efficient treatment.Therefore,it was urgent to conduct in-depth in vitro research and clinical basic research on osteoarthritis.This study was mainly to explore the role and mechanism of miRNA-146 A mediated TLR4 signaling pathway in osteoarthritis.Methods:In this study,an animal model of OA was successfully established,and primary chondrocytes were isolated to establish an in vitro chondrocyte OA model.Bioinformatics analysis was used to predicte the gene binding sites between miR-146 a and TLR4.The binding of miR-146 a to TLR4 gene was detected by dual luciferase reporter genes.CCK8 assay detected cell activity.The m RNA expressions of miR-146 a,TLR4,Aggrecan,MMP-3,MMP-13,Collagen I/II/X were detected by real-time quantitative PCR.Flow cytometry analysed cell apoptosis.The protein expressions of TLR4,Aggrecan,MMP-3,MMP-13 and Collagen I/II in chondrocytes were detected by Western Blotting.Spearman correlation coefficient was used to analyze the correlation between miR-146 a and TLR4 gene expression.Results:The animal OA model constructed in this study showed that compared with control group,miR-146 a expression was significantly decreased in model group,while TLR4 expression was significantly increased in model group.Primary chondrocytes were extracted and an in vitro chondrocyte OA model was constructed.It was found that compared with control group,the expression of chondrocyte Collagen I/II in the model group was decreased,cell proliferation was inhibited,miR-146 a expression was decreased,and TLR4 expression was increased.Spearman analysis showed a significant negative correlation between miR-146 a and TLR4 gene expression.Bioinformatics analysis showed that miR-146 a could target TLR4 gene site,and dual luciferase assay showed that miR-146 a could target TLR4 gene.Overexpression of miR-146 a inhibited the expression of TLR4 as well as MMP-3 and MMP-13.Inhibition of miR-146 a activated the TLR4 signaling pathway,significantly increased the expressions of MMP-3 and MMP-13,and significantly decreased the expressions of Aggrecan and Collagen I/II.Interference with TLR4 blocked the activation of the miR-146a/TLR4 signaling pathway.Conclusion:The expression of miR-146 a was decreased in osteoarthritis,and the expression of TLR4 was increased in osteoarthritis.miR-146 a can protect osteoarthritis by targeting to inhibit the activation of TLR4 signaling pathway.