miR-515-5p targeting Toll-like receptor 4 regulates myeloid differentiation primary response gene 88/nuclear factor-kappa B pathway to inhibit apoptosis and inflamma-tory response of osteoarthritis chondrocytes
Objective To explore the molecular mechanism of miR-515-5p in inhibiting chondrocyte apoptosis and alleviating inflammatory response in osteoarthritis(OA).Methods Human cartilage cell line C28/I2 was cultured in vitro and treated with 10 ng/mL interleukin 1β(IL-1 β)for 24 hours to construct an in vitro OA model.C28/I2 cells were transfected with miR mimics,mimics negative control(NC),over expression(oe)-NC,and oe-Toll-like receptor 4(TLR4),respectively,and then treated with 10 ng/mL IL-1 β for 24 hours to establish OA model.Cell proliferation capacity was detected by cell counting kit 8 and 5-Ethynyl-2'-deoxyuridine,cell apoptosis and cell cycle were detected by flow cytometry,and B-cell lymphoma 2 protion(Bcl-2),Bcl-2-associated X protein(Bax),cleaved-Caspase-3,TLR4,myeloid differentiation primary response gene 88(MyD88),p65 and phosphorylated p65(p-p65)protein expression levels were detected by Western blot.Real-time fluorescence quantitative PCR was used to detect mRNA expression levels of miR-515-5p and TLR4,and ELISA was used to detect pro-inflammatory factor prostaglandin E2(PGE2),tumor necrosis factorα(TNF-α),and IL-6 levels in cell supernatant.The potential binding sites between miR-515-5p and TLR4 were predicted by BiBiServ2 database,and the targeting relationship between miR-515-5p and TLR4 was verified by dual luciferase reporting assay.Results After the treatment of C28/I2 cells with IL-1β,the expressions of miR-515-5p and Bcl-2 protein and the proliferation ability of C28/I2 cells significantly reduced.The expression levels of Bax and cleaved-Caspase-3 protein,the levels of pro-inflammatory factors(PGE2,TNF-α,IL-6)in the supernatant of C28/I2 cells,and the apoptosis of C28/I2 cells significantly increased.In addition,the proportion of the cells at S phase and G2 phase decreased significantly,and the proportion of cells at G1 phase increased significantly,suggesting that the cell cycle was blocked after IL-1β treatment.After transfection with miR mimics,the expression level of miR-515-5p in the cells significantly up-regulated,partially reversing the apoptosis of OA chondrocytes induced by IL-1β,and alleviating the cycle arrest and inflammatory response of OA chondrocytes.After treating C28/I2 cells with IL-1β,the mRNA and protein levels of TLR4 significantly increased.Overexpression of miR-515-5p targeted inhibition of TLR4 expression and blocked activation of MyD88/nuclear factor κB(NF-κB)pathway.Overexpression of TLR4 could partially reverse the effect of miR mimics on IL-1β-induced apoptosis and inflammation of OA chondrocytes.Conclusion miR-515-5p negatively regulates the expression of TLR4,inhibits the activation of MyD88/NF-KB pathway and apoptosis of OA chondrocytes,and effectively alleviates the inflammatory response of the cells.
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