miR-29a对类风湿关节炎成纤维样滑膜细胞增殖和凋亡的影响

目的探讨miR-29a对类风湿关节炎(RA)成纤维样滑膜细胞(FLS)活力和凋亡的影响。方法分离培养健康人FLS(n-FLS)和RA患者FLS(RA-FLS);Western blot检测n-FLS和RA-FLS中标志物CHI3L1蛋白表达;将miR-29a mimics、si-NFAT5及miR-29a+NFAT5转染RA-FLSs,转染48 h后,RT-qPCR检测miR-29a和NFAT5 mRNA表达;MTT法及流式细胞计量术分别检测各组细胞活力和凋亡率;Western blot检测p38、p-p38、cleaved-caspase3蛋白表达;通过双荧光报告基因检测系统检测过表达或抑制miR-29a后NFAT5表达,验证miR-29a和NFAT5的靶向关系。结果与n-FLS比较,RA-FLS中CHI3L1蛋白表达明显升高(P<0.05);过表达miR-29a或抑制NFAT5后,RA-FLS细胞活力明显降低,凋亡率明显升高,p-p38表达明显降低,cleaved-caspase3表达明显升高(P<0.05)。miR-29a与野生型NFAT5 3′UTR共转染组RA-FLS的荧光素酶活性明显降低,而anti-miR-29a与野生型NFAT5 3′UTR共转染组荧光素酶活性明显升高(P<0.05);过表达miR-29a可明显下调RA-FLS中NFAT5表达,抑制miR-29a表达可明显上调NFAT5表达(P<0.05);过表达NFAT5可减弱过表达miR-29a对RA-FLS活力和凋亡的影响(P<0.05)。结论 miR-29a可通过靶向NFAT5下调p38MAPK信号通路抑制RA-FLS活力及诱导细胞凋亡。

Effect of miR-29a on proliferation and apoptosis of fibroblast-like synoviocyte in rheumatoid arthritis

Objective To investigate the effect of miR-29 a on the activity and apoptosis of fibroblast-like synoviocyte(FLS) in rheumatoid arthritis(RA). Methods The normal human FLS(n-FLS) and RA patients FLS(RA-FLS) were isolated and cultured,and the expression level of CHI3 L1 protein was detected by Western blot. miR-29 a mimics, si-NFAT5 and miR-29 a+NFAT5 were transfected into RA-FLS for 48 h. Then,the expression levels of miR-29 a and NFAT5 mRNA were detected by RT-qPCR, the cell viability and apoptosis were detected by MTT assay and flow cytometry,respectively and the expression of p38, p-p38, cleaved-caspase3 proteins were detected by Western blot. Targeting relationship between miR-29 a and NFAT5 was validated by dual fluorescent reporter gene detection system. Results Compared with n-FLS, the expression level of CHI3 L1 protein in RA-FLS was increased significantly(P<0.05). After over-expression of miR-29 a or inhibition of NFAT5, the cell viability decreased, the apoptosis rate increased, the expression of p-p38 decreased, and the expression of cleaved-caspase3 increased significantly(P<0.05). Luciferase activity of RA-FLS was significantly decreased in miR-29 a and wild-type NFAT5 3′UTR co-transfection group, but increased in anti-miR-29 a and wild-type NFAT5 3′UTR co-transfection group(P<0.05). Over-expression of miR-29 a significantly decreased the expression of NFAT5, and inhibition of miR-29 a significantly increased the expression of NFAT5(P<0.05).Over-expression of NFAT5 can weaken the effect of over-expression of miR-29 a on RA-FLS activity and apoptosis(P<0.05). Conclusions miR-29 a can inhibit the activity of RA-FLS and induce its apoptosis by targeting NFAT5 to down-regulate p38 MAPK signaling pathway.