干扰RNCR2表达通过负调控miR-130b保护高糖诱导的肾小球系膜细胞损伤

Interfering RNCR2 expression protects against glomerular mesangial cell damage induced by high glucose via negative regulation of miR-130b

目的探讨RNCR2对高糖诱导的肾小球系膜细胞损伤的影响及作用机制。方法体外培养肾小球系膜细胞SV40-MES-13,分为对照组、高糖组、高糖+si-NC组、高糖+si-RNCR2组、高糖+miR-NC组、高糖+miR-130b组、高糖+si-RNCR2+anti-miR-NC组和高糖+si-RNCR2+anti-miR-130b组,RT-qPCR检测RNCR2、miR-130b、MCP-1和IL-6的mRNA表达水平,酶联免疫吸附法检测MDA和SOD水平,流式细胞术检测细胞凋亡,Western印迹法检测Caspase3蛋白表达水平。双荧光素酶报告基因实验验证RNCR2与miR-130b调控关系。结果与对照组比较,高糖组SV40-MES-13细胞中RNCR2水平(3.66±0.11比0.97±0.09)、MDA含量[(14.52±0.60)nmol/ml比(5.71±0.44)nmol/ml]、细胞凋亡率、Caspase3蛋白及MCP-1和IL-6的mRNA水平均升高(P<0.05),SOD水平[(14.84±0.43)U/ml比(39.59±1.19)U/ml]降低(P<0.05)。与高糖+si-NC组比较,高糖+si-RNCR2组SV40-MES-13细胞中MDA含量、细胞凋亡率、Caspase3蛋白及MCP-1和IL-6的mRNA水平均降低(P<0.05),SOD水平升高(P<0.05)。RNCR2在SV40-MES-13细胞中负调控miR-130b表达。与高糖+miR-NC组比较,高糖+miR-130b组SV40-MES-13细胞中MDA含量、细胞凋亡率、Caspase3蛋白及MCP-1和IL-6的mRNA水平均降低(P<0.05),SOD水平升高(P<0.05)。与高糖+si-RNCR2+anti-miR-NC组比较,高糖+si-RNCR2+anti-miR-130b组SV40-MES-13细胞中MDA含量、细胞凋亡率、Caspase3蛋白及MCP-1和IL-6的mRNA水平均升高(P<0.05),SOD水平降低(P<0.05)。结论干扰RNCR2表达可能通过负调控miR-130b降低高糖诱导的肾小球系膜细胞SV40-MES-13氧化应激、炎症反应和凋亡,保护细胞损伤。

Objective To investigate the effect and mechanism of RNCR2 on glomerular mesangial cell injury induced by high glucose.Methods Glomerular mesangial cells SV40-MES-13 were cultured in vitro and divided into control group,high glucose group,high glucose+si-NC group,high glucose+si-RNCR2 group,high glucose+miR-NC group,high glucose+miR-130b group,high glucose+si-RNCR2+anti-miR-NC group and high glucose+si-RNCR2+anti-miR-130b group.RT-qPCR was used to detect the expression levels of RNCR2,miR-130b,MCP-1 and IL-6 mRNA.Enzyme-linked immunosorbent assay was used to detect the MDA and SOD levels.Flow cytometry was used to detect cell apoptosis.Western blotting was used to detect Caspase3 protein expression level.Dual luciferase reporter gene assay was used to verify the regulatory relationship between RNCR2 and miR-130b.Results Compared with the control group,the high glucose groups of SV40-MES-13 cells showed higher RNCR2 expression(3.66±0.11 vs.0.97±0.09),MDA content[(14.52±0.60)nmol/ml vs(5.71±0.44)nmol/ml],apoptosis rate,levels of Caspase3 protein,MCP-1 and IL-6 mRNA(P<0.05),and lower SOD level[(14.84±0.43)U/ml vs(39.59±1.19)U/ml](P<0.05).Compared with the high glucose+si-NC group,SV40-MES-13 cells in the high glucose+si-RNCR2 group showed lowered MDA content,apoptosis rate,levels of Caspase3 protein,MCP-1 and IL-6 mRNA(P<0.05),but higher SOD level(P<0.05).RNCR2 was found to negatively regulate the miR-130b expression in SV40-MES-13 cells.Compared with the high glucose+miR-NC group,SV40-MES-13 cells in the high glucose+miR-130b group showed lower MDA content,apoptosis rate,levels of Caspase3 protein and MCP-1 and IL-6 mRNA(P<0.05),but higher SOD level(P<0.05).Compared with the high glucose+si-RNCR2+anti-miR-NC group,SV40-MES-13 cells in the high glucose+si-RNCR2+anti-miR-130b group showed higher MDA content,apoptosis rate,levels of Caspase3 protein,MCP-1 and IL-6 mRNA(P<0.05),but lower SOD level(P<0.05).Conclusion Interfering RNCR2 expression may reduce oxidative stress,inflammation response and apoptosis of glomerular mesangial cells SV40-MES-13 induced by high glucose via negative regulation of miR-130b,and thereby protect against cell damage.