circARF3 improves TNF-α-induced inflammatory damage of ATDC5 chondrocytes by down-regulating miR-195 expression
The purpose of this study was to explore the effects of circular RNA(circRNA)ADP ribosylation factor 3(ARF3)on the inflammatory damage of ATDC5 chondrocytes induced by TNF-α through miR-195 regulating.To this end,ATDC5 chondrocytes were divided into control(Cont)group,TNF-α group,pCMV-BC group,pCMV-ARF3-NC group,pCMV-ARF3 group,inhibitor-NC group,miR-195a inhibitor group,miR-195b inhibitor group,pCMV-ARF3+mimics-NC group,pCMV-ARF3+miR-195a mimics group and pCMV-ARF3+miR-195b mimics group.qRT-PCR was performed to measure the expression of ARF3 mRNA and miR-195a/b in each ATDC5 chondrocyte group.FACS was performed to measure the cell apoptosis rate and ELISA was used to measure the levels of intercellular adhesion molecule 1(ICAM-1),IL-1β,IL-6,and cyclooxygenase 2(COX-2).RNA pull-down and dual-luciferase report gene experiment were set out to analyze the binding ability of circARF3 to miR-195a and miR-195b.Western blotting was run to measure the protein expressions of collagen Ⅱ(COL2),proteoglycan Aggrecan,matrix metalloproteinase 13(MMP13),MMP1,cleaved caspase 3(cl-caspase-3),and cl-caspase-9.The results showed that compared to the Cont group,the expression of ARF3mRNA,and the levels of COL2 and Aggrecan proteins in ATDC5 cells of the TNF-α group were significantly decreased(all with P＜0.05).In contrast,the expressions of miR-195a and miR-195b,the protein levels of cl-caspase-3,cl-caspase-9,MMP13,and MMP1,cell apoptosis rate,and the levels of COX-2,IL-6,IL-1β,and ICAM-1 in the supernatant were all significantly increased(all with P＜0.05).Over-expressing ARF3 or knock-down of miR-195a and miR-195b inhibited cell apoptosis,inflammatory factors release,and extracellular matrix(ECM)degradation.The result showed that ARF3 bounded and down-regulated the expressions of miR-195a and miR-195b.Over-expression of miR-195a or miR-195b partially reversed the inhibitory effects of high-expressed ARF3 on ATDC5 apoptosis,inflammatory responses and ECM degradation.Therefore,circARF3 may be used as competing endogenous RNA(ceRNA)to absorb miR-195a and miR-195b.Over-expression of ARF3 can inhibit the apoptosis,inflammation,and ECM degradation of ATDC5 cells induced by TNF-α through down-regulating the expressions of miR-195a and miR-195b.
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