miR-708-5p对类风湿关节炎滑膜成纤维细胞凋亡、炎症因子分泌和TLR4/NF-κB信号通路的影响

Effects of miR-708-5p on apoptosis,inflammatory factor secretion and TLR4/NF-κB signaling pathway in synovial fibroblasts of rheumatoid arthritis

目的:探讨miR-708-5p对类风湿关节炎滑膜成纤维细胞凋亡、炎性因子分泌的调控机制。方法:将体外培养的人类风湿关节炎滑膜成纤维细胞MH7A分为对照组(未处理)、miR-NC组(转染miR-NC)和miR-708-5p组(转染miR-708-5p模拟物)。采用qRT-PCR法检测转染48 h后各组细胞中miR-708-5p的表达,CCK-8法检测各组细胞存活率,流式细胞仪检测细胞的凋亡率,ELISA法检测细胞上清液中IL-6、IL-1β和TNF-α的含量,Western blot法检测细胞中Bcl-2、Bax、Cleaved Caspase-3、Toll样受体4(TLR4)、NF-κB p65和p-NF-κB p65蛋白的表达。结果:与对照组相比,miR-708-5p组细胞中miR-708-5p的表达水平、细胞凋亡率和Bax、Cleaved Caspase-3蛋白的表达水平均升高,而细胞存活率、细胞上清液中IL-6、IL-1β、TNF-α的含量以及TLR4、p-NF-κB p65蛋白的表达水平均降低(P<0.05)。结论:miR-708-5p可诱导类风湿关节炎滑膜成纤维细胞凋亡并减轻细胞炎症损伤,其作用机制可能与抑制TLR4/NF-κB信号通路的活化有关。

Aim:To investigate the regulatory mechanism of miR-708-5p on rheumatoid arthritis synovial fibroblast apoptosis and inflammatory factor′secretion.Methods:In vitro cultured human rheumatoid arthritis synovial fibroblasts MH7A were randomly divided into control group(untreated),miR-NC group(transfected miR-NC),miR-708-5p group(transfected miR-708-5p mimics).The expression of miR-708-5p was detected by qRT-PCR after transfection.The survival rate of cells was detected by CCK-8.The apoptosis rate of the cells were detected by flow cytometry.ELISA method was adopted to detect the contents of IL-6,IL-1β,and TNF-αin supernatant,and Western blot was adopted to detect the expressions of Bcl-2,Bax,Cleaved Caspase-3,and TLR4,NF-κB,p65 and p-NF-κB p65.Results:Compared with the control group,the expression level of miR-708-5p,the apoptotic rate and the protein expression levels of Bax and Cleaved Caspase-3 in miR-708-5p group were all significantly increased,while the cell survival rate and the levels of IL-6,IL-1βand TNF-αin supernatant and the protein expression levels of TLR4 and p-NF-κB p65 were significantly decreased(P<0.05).Conclusions:miR-708-5p could induce apoptosis of rheumatoid arthritis synovial fibroblasts and alleviate cellular inflammatory injury,and its mechanism may be related with inhibition of TLR4/NF-κB signaling pathway activation.