环状RNA circAZIN1在骨关节炎中调控软骨细胞退变的作用机制

The role and mechanism of circular RNA circAZIN1 in regulating chondrocyte degeneration in osteoarthritis

目的研究环状RNA(circRNA)hsa_circ_0003304(circAZIN1)在骨关节炎(OA)中调控软骨细胞退变的作用及机制。方法基因芯片检测人脂肪源性干细胞(hADSC)成软骨分化过程中circRNA的表达水平变化。从基因芯片检测差异化表达最明显的前10位circRNA中,用IL-β及TNF-α构建的软骨细胞炎症模型进一步筛选出circAZIN1,过表达circAZIN1(转染pcDNA3.1-circAZIN1-EF1-ZsGreen质粒)后实时荧光定量PCR(qPCR)检测其对软骨细胞外基质(ECM)代谢的影响。RNA-蛋白体外结合(RNA pull down)试验检测circAZIN1结合的蛋白,miRNA-circRNA Interactions预测circAZIN1可能海绵吸附的微RNA(miRNA)及其位点,进一步利用TargetMiner、miRDB及TargetScan数据库预测circAZIN1可能海绵吸附的miRNA及下游mRNA,过表达miRNA后检测circAZIN1、miRNA及下游的mRNA是否存在镜像调控现象。结果circAZIN1在hADSC成软骨分化过程中(第3天和第21天)、IL-β及TNF-α构建的软骨细胞炎症模型中差异化表达最明显(第3天组vs.第21天组、对照组vs.IL-β组、对照组vs.TNF-α组);过表达circAZIN1可促进软骨细胞ECM的合成、抑制其分解;RNA-Pull down试验检测到circAZIN1明显结合AGO2蛋白,提示circAZIN1海绵吸附miRNAs的可能性大,进一步数据库预测circAZIN1的下游miRNA为hsa-miR-654-3p,而hsa-miR-654-3p的下游mRNA为CACNA1I,最后过表达hsa-miR-654-3p后qPCR检测证实circAZIN1、hsa-miR-654-3p及CACNA1I间存在镜像调控现象。结论circAZIN1通过海绵吸附hsa-miR-654-3p继而抑制CACNA1I的沉默效应从而发挥抑制软骨细胞退变的作用,这为circRNA在OA发生、发展中的调控机制研究提供了参考。

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-derived 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 reaction 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 phenomenon 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-βgroup,the control group vs.the TNF-αgroup).Overexpression of circAZIN1 could promote the ECM synthesis in chondrocytes 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 database 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 degeneration,which provides a reference for the study of the regulatory mechanism of circRNA in the development of OA.