The role and mechanism of circular RNA circAZIN1 in regulating chondrocyte degeneration in osteoarthritis
Objective To investigate the role and mechanism of circular RNA(circRNA)has_circ_0003304(circAZIN1)in regulating chondrocyte degeneration in osteoarthritis(OA).Methods Gene chip was used to detect the expression level of circRNA during the chondrogenic differentiation of human adipose-de-rived stem cells(hADSC).The circAZIN1 was further screened through the chondrocyte inflammation model constructed with interleukin-β(IL-β)and tumor necrosis factor-α(TNF-α)from the top 10 circRNAs with the most differentiated expression detected by gene chip.Real-time fluorescence quantitative polymerase chain re-action PCR(qPCR)was used to detect the effect of circAZIN1 overexpression(transfected with pcDNA3.1-circAZIN1-EF1-ZsGreen plasmid)on the metabolism of chondrocyte extracellular matrix(ECM).RNA-Pull down test was conducted to detect the protein bound by circAZIN1,miRNA-circRNA Interactions predicted the microRNAs(miRNAs)and their sites that circAZIN1 may be sponge-adsorbed,further TargetMiner,miRDB and TargetScan databases were applied to predict miRNAs which circAZIN1 may sponge-adsorbed.circAZIN1,miRNA and downstream mRNA were detected to find whether there was mirror regulation phe-nomenon after overexpression of miRNA.Results CircAZIN1 had the most significant differential expression during the chondrogenic differentiation of hADSC(day 3 and day 21)and in the chondrocyty inflammation model constructed by IL-β,TNF-α(day 3 group vs.day 21 group,the conttrol group vs.the IL-β group,the control group vs.the TNF-α group).Overexpression of circAZIN1 could promote the ECM synthesis in chon-drocytes and inhibit its decomposition.RNA-Pull down test result showed that circAZIN1 obviously bound AGO2 protein,suggesting that circAZIN1 had a high possibility of sponge adsorbing miRNAs.Further data-base predicted that its downstream was hsa-miR-654-3p,and the downstream mRNA of hsa-miR-654-3p was CACNA1I.After the overexpression of hsa-miR-654-3p,qPCR test found that circAZIN1,hsa-miR-654-3p and CACNA1I had a mirror image regulation phenomenon.Conclusion circAZIN1 inhibits the silencing effect of CACNA1I through sponge adsorption of hsa-miR-654-3p and thus plays a role in inhibiting chondrocyte de-generation,which provides a reference for the study of the regulatory mechanism of circRNA in the develop-ment of OA.
万方数据
维普
