MiR-146b通过靶向TRAF6减轻狼疮性肾炎肾小球系膜细胞的损伤

MiR-146b alleviates glomerular mesangial cell damage in lupus nephritis by targeting TRAF6

目的:研究微RNA-146b(microRNA-146b,miR-146b)对狼疮性肾炎小鼠肾小球系膜细胞增殖、炎症因子分泌以及细胞周期的影响及其潜在机制。方法:采用双荧光素酶报告基因实验验证miR-146b和肿瘤坏死因子受体相关因子6(tumor necrosis factor receptor-associated factor 6,TRAF6)的靶向关系;实时定量聚合酶链反应(qRT-PCR)和蛋白质印迹法检测TRAF6蛋白的表达水平;将肾小球系膜细胞分为miR-control组(转染miR-146b模拟物阴性对照)、miR-146b mimic组(转染miR-146b模拟物)、miR-146b mimic+NC组(共转染miR-146b模拟物和空载质粒pcDNA-NC)和miR-146b mimic+TRAF6组(共转染miR-146b模拟物和TRAF6过表达质粒);采用细胞计数试剂盒8(CCK-8)和免疫荧光染色法检测细胞增殖;酶联免疫吸附试验(ELISA)检测细胞培养上清液中炎症因子的水平;流式细胞术分析细胞周期。结果:MiR-146b靶向结合TRAF6并负调控TRAF6的表达。与miR-control组比较,miR-146b mimic组细胞的增殖能力显著减弱,细胞培养上清液中炎症因子白细胞介素1β(interleukin-1β,IL-1β)、白细胞介素6(interleukin-6,IL-6)和肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)的水平降低,细胞发生G_(0)/G_(1)期阻滞;过表达TRAF6显著逆转了miR-146b对肾小球系膜细胞增殖和炎症因子分泌的抑制作用,减少G_(0)/G_(1)期细胞占比,促使细胞进入S期。结论:MiR-146b可能通过靶向TRAF6减轻狼疮性肾炎肾系膜细胞的炎性损伤。

Objective:To investigate the effects of microRNA-146b(miR-146b)on the proliferation,secretion of inflammatory factors,and cell cycle of mesangial cells in mice with lupus nephritis and its potential mechanism.Methods:The targeting relationship between miR-146b and tumor necrosis factor receptor-associated factor 6(TRAF6)was verified by dual luciferase reporter gene assay.Real-time quantitative polymerase chain reaction(qRT-PCR)and Western blotting were used to detect TRAF6 expression.The mesangial cells were divided into a miR-control group(negative control transfected with miR-146b mimic),a miR-146b mimic group(transfected with miR-146b mimic),a miR-146b mimic+NC group(co-transfected with miR-146b mimics and empty plasmid PCDNA-NC)and a miR-146b mimic+TRAF6 group(co-transfected with miR-146b mimics and TRAF6 overexpression plasmid).Cell counting kit 8(CCK-8)and immunofluorescent staining were used to detect cell proliferation.The levels of inflammatory cytokines in supernatant were detected by enzyme-linked immunosorbent assay(ELISA).Cell cycle was analyzed by flow cytometry.Results:MiR-146b targeted and negatively regulated the expression of TRAF6.Compared with miR-control group,the proliferation ability of cells in miR-146b mimic group was significantly decreased,the levels of inflammatory factors interleukin 1β(IL-1β),interleukin 6(IL-6)and tumor necrosis factorα(TNF-α)in cell supernatant were decreased,and the cells were arrested in G_(0)/G_(1) phase.Overexpression of TRAF6 significantly reversed the inhibitory effect of miR-146b on the proliferation and secretory of inflammatory factors in mesangial cells,reduced the proportion of G_(0)/G_(1) phase cells,and promoted the cells to enter into S phase.Conclusion:MiR-146b may alleviate the inflammatory damage of mesangial cells in lupus nephritis by targeting TRAF6.