circARF3下调miR-195减轻TNF-α诱导的ATDC5软骨细胞炎症损伤  

circARF3 improves TNF-α-induced inflammatory damage of ATDC5 chondrocytes by down-regulating miR-195 expression

作　　者：吴焘[1] 张国秋[1] 李超[1] 张兰涛 WU Tao;ZHANG Guo-qiu;LI Chao;ZHANG Lan-tao(Department of Joint Surgery,Qinghai University Affiliated Hospital,Xining 810001,China)

机构地区：[1]青海大学附属医院关节外科,西宁810001

出　　处：《现代免疫学》2024年第3期206-213,220,共9页Current Immunology

基　　金：青海大学中青年科研基金项目(2018-QYY-8)。

摘　　要：为探讨环状RNA(circular RNA,circRNA)ADP核糖基化因子3(ADP ribosylation factor 3,ARF3)调节miR-195对TNF-α诱导的ATDC5软骨细胞炎症损伤的影响,将ATDC5细胞分为对照组(control,Cont)、TNF-α组、pCMV-BC组、pCMV-ARF3-NC组、pCMV-ARF3组、inhibitor-NC组、miR-195a inhibitor组、miR-195b inhibitor组、pCMV-ARF3+mimics-NC组、pCMV-ARF3+miR-195a mimics组和pCMV-ARF3+miR-195b mimics组。qRT-PCR检测各组ATDC5细胞中ARF3的mRNA及miR-195a/b相对表达量;FACS检测ATDC5细胞的凋亡水平;ELISA检测细胞培养上清液中细胞间黏附分子1(intercellular adhesion molecule 1,ICAM-1)、IL-1β、IL-6和环氧合酶2(cyclooxygenase 2,COX-2)水平;RNA pull-down和双荧光素酶报告基因实验验证circARF3与miR-195a、miR-195b的结合情况;Western blotting检测Ⅱ型胶原(collagenⅡ,COL2)、蛋白聚糖Aggrecan、基质金属蛋白酶13(matrix metalloproteinase 13,MMP13)、MMP1、剪切caspase 3(cleaved caspase 3,cl-caspase-3)和cl-caspase-9蛋白表达量。结果显示,与Cont组相比,TNF-α组ATDC5细胞ARF3 mRNA、COL2和Aggrecan蛋白表达量显著降低(均P<0.05),而miR-195a、miR-195b相对表达量、cl-caspase-3、cl-caspase-9、MMP13和MMP1蛋白表达量、ATDC5细胞凋亡率及上清液中COX-2、IL-6、IL-1β和ICAM-1水平显著升高(均P<0.05)。过表达ARF3或低表达miR-195a、miR-195b可抑制ATDC5细胞凋亡、炎性因子释放及细胞外基质(extracellular matrix,ECM)降解。circARF3可结合并靶向下调miR-195a和miR-195b表达。过表达miR-195a或miR-195b可部分逆转高表达ARF3对ATDC5细胞凋亡、炎症应答和ECM降解的抑制效应。由此,circARF3或作为竞争性内源RNA(competing endogenous RNA,ceRNA)吸附miR-195a和miR-195b,过表达ARF3可通过下调miR-195a和miR-195b表达抑制TNF-α诱导的ATDC5细胞凋亡、炎症应答和ECM降解。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-αthrough miR-195 regulating.To this end,ATDC5 chondrocytes were divided into control(Cont)group,TNF-αgroup,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 collagenⅡ(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-αgroup 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

关 键 词：环状核糖核酸腺苷二磷酸核糖基化因子3 微小核糖核酸195 肿瘤坏死因子α ATDC5细胞 凋亡 炎症 

分 类 号：R446.62[医药卫生—诊断学]