miR-146b在高糖环境下对肾小球系膜细胞的作用研究

目的探讨miR-146b在高糖环境下对肾小球系膜细胞的作用。方法构建高糖培养环境,实时荧光定量PCR(qRT-PCR)检测miR-146b在大鼠肾小管上皮细胞(NRK-52E)、足细胞(podocyte)及肾小球系膜细胞(HBZY-1)中的表达水平;western blot分析过表达/敲减miR-146b前后,HBZY-1细胞中YAP及磷酸化YAP(p-YAP)蛋白的表达水平;细胞免疫荧光法检测YAP蛋白的表达情况。结果体外经高糖刺激后,HBZY-1细胞中miR-146b的表达水平(2.197±0.166)明显升高(t=7.215,P0.01)。western blot结果显示,高糖组YAP蛋白(0.753±0.008)的表达水平升高(t=32.17,P0.001),p-YAP蛋白(0.372±0.005)的表达水平降低(t=39.56,P0.001);免疫荧光结果显示,高糖组YAP蛋白荧光强度(53.620±4.229)明显升高(t=5.410,P0.01)。过表达miR-146b后,western blot结果显示,YAP蛋白(0.883±0.005)表达上调(t=10.71,P0.001),而p-YAP蛋白(0.606±0.006)表达下调(t=44.16,P0.001);免疫荧光结果显示,YAP荧光强度(25.70±1.589)增强(t=10.45,P0.001)。敲减miR-146b后,western blot结果显示,YAP蛋白(0.706±0.002)表达下调(t=22.15,P0.001),p-YAP蛋白(0.774±0.003)表达上调(t=25.61,P0.001);免疫荧光结果显示,YAP荧光强度(8.900±1.022)减弱(t=11.56,P0.001)。结论 miR-146b在高糖刺激的HBZY-1细胞中表达上调,可能通过调控YAP信号参与肾细胞损伤。

Effect of miR-146b on glomerular mesangial cells in high glucose environment

Abstract:?Objective?To investigate the effect of miR-146b on glomerular mesangial cells in high glucose environment.?Methods?The high glucose culture environment was constructed, and the expression levels of miR-146b in rat renal tubular epithelial cells (NRK-52E), podocytes and glomerular mesangial cells (HBZY-1) were detected by qRT-PCR. The expression levels of YAP and phosphorylated YAP (p-YAP) proteins in HBZY-1 cells were detected by western blot before and after overexpression or knockdown of miR-146b. The expression of YAP protein was also detected by cellular immunofluorescence.?Results?The expression levels of miR-146b (2.197±0.166) in HBZY-1 cells increased significantly after high glucose stimulation?in?vitro?(t=7.215,P<0.01). western blot results showed that the expression levels of YAP protein (0.753±0.008) in the high glucose group increased (t=32.17,P<0.001), while that of p-YAP protein (0.372±0.005) decreased (t=39.56,P<0.001). The immunofluorescence results showed that the fluorescence intensity of YAP protein (53.620±4.229) in the high glucose group increased significantly (t=5.410,P<0.01). After miR-146b was over-expressed, the expression level of YAP protein (0.883±0.005) was up-regulated (t=10.71,P<0.001), while that of p-YAP protein (0.606±0.006) down-regulated (t= 44.16,P<0.001). Meanwhile, immunofluorescence results showed that the fluorescence intensity of YAP (25.70±1.589) was enhanced (t=10.45,P<0.001). After miR-146b was knocked down, the expression level of YAP protein (0.706±0.002) was down-regulated (t=22.15,P<0.001), while that of p-YAP protein (0.774±0.003) up-regulated (t=25.61,P<0.001). Meanwhile, immunofluorescence results showed that the fluorescence intensity of YAP (8.900±1.022) was reduced (t=11.56,P<0.001).?Conclusion?miR-146b is up-regulated in HBZY-1 cells stimulated by high glucose, which may be involved in renal cell injury by regulating YAP signal.