PRMT1通过调控ZEB1对高糖诱导的肾小管上皮细胞凋亡和上皮—间充质转化的影响

The effects of PRMT1 on renal tubular epithelial cell apoptosis and epithelial-mesenchymal transition induced by high glucose by regulating ZEB1

目的:探究PRMT1对高糖诱导的肾小管上皮细胞损伤的作用及其可能的作用机制。方法:体外培养人肾小管上皮细胞系HK-2,分别以含葡萄糖5.5 mmol/L、30 mmol/L的DMEM培养基培养,分为空白对照组、高糖组;对HK-2细胞进行转染处理,分为空白对照组、si-NC组、si-PRMT1组、si-NC+oe-NC组、si-PRMT1+oe-NC组、si-NC+oe-ZEB1组、si-PRMT1+oe-ZEB1组;CCK-8检测细胞增殖;流式细胞术检测细胞凋亡;qRT-PCR检测细胞PRMT1和ZEB1 mRNA表达;Western blotting检测细胞PRMT1、ZEB1、E-cadherin、Fibronectin和α-SMA蛋白表达;CHIP-PCR和CHIP-qPCR实验检测PRMT1对ZEB1启动子的调控作用。结果:与空白对照组相比,高糖组细胞增殖能力和E-cadherin蛋白表达明显降低(P<0.05),细胞凋亡率、Fibronectin和α-SMA蛋白表达明显升高(P<0.05),PRMT1和ZEB1表达明显升高(P<0.05)。PRMT1通过与ZEB1启动子区域结合调控ZEB1的表达。与空白对照组相比,si-NC组细胞各指标差异均无统计学意义(P>0.05);与si-NC+oe-NC组相比,si-PRMT1+oe-NC组细胞中PRMT1、ZEB1、Fibronectin和α-SMA蛋白表达、细胞凋亡率明显降低(P<0.05),增殖能力和E-cadherin蛋白表达明显升高(P<0.05),si-NC+oe-ZEB1组细胞中PRMT1蛋白表达差异无统计学意义(P>0.05),ZEB1、Fibronectin和α-SMA蛋白表达和细胞凋亡率明显升高(P<0.05),增殖能力和E-cadherin蛋白表达明显降低(P<0.05);与si-PRMT1+oe-NC组相比,si-PRMT1+oe-ZEB1组细胞中PRMT1蛋白表达差异无统计学意义(P>0.05),ZEB1、Fibronectin和α-SMA蛋白表达和细胞凋亡率明显升高(P<0.05),增殖能力和E-cadherin蛋白表达明显降低(P<0.05);与si-NC+oe-ZEB1组相比,si-PRMT1+oeZEB1组细胞中PRMT1、ZEB1、Fibronectin和α-SMA蛋白表达、细胞凋亡率明显降低(P<0.05),增殖能力和E-cadherin蛋白表达明显升高(P<0.05)。结论:PRMT1通过上调ZEB1表达促进高糖诱导的肾小管上皮细胞凋亡和上皮-间充质转化。

Objective:To explore the effect of PRMT1 on renal tubular epithelial cell injury induced by high glu-cose and its possible mechanism.Methods:The human renal tubular epithelial cell line HK-2 was cultured in DMEM medium containing 5.5 mmol/L glucose(blank control group)and 30 mmol/L glucose(high glucose group),respectively.Transfected cells were divided into si-NC group,si-PRMT1 group,si-NC+OE-NC group,si-PRMT1+OE-NC group,si-NC+OE-NC group,si-NC+OE-ZEB1 group,and si-PRMT1+OE-ZEB1 group.CCK-8 assay was used to detect the cell proliferation.Flow cytometry was used to detect cell apoptosis.qRT-PCR was used to detect the expression of PRMT1 and ZEB1 mRNA.Western blotting was used to detect the expression of PRMT1,ZEB1,E-cadherin,Fibronectin,andα-SMA protein.CHIP-PCR and CHIP-qPCR experiments were used to detect the regulatory effect of PRMT1 on the ZEB1 promoter.Results:Compared with the blank control group,the cell proliferation ability and the expression of E-cadherin protein in the high glucose group were signif-icantly reduced,while the apoptosis rate and the ex-pression of Fibronectin,α-SMA,PRMT1 and ZEB1 were significantly increased(P<0.05).PRMT1 regulated the expression of ZEB1 by binding to the ZEB1 pro-moter region.There was no significant difference in all indexes between the blank control group and si-NC group(P>0.05).Compared with si-NC+oe-NC group,the expression of PRMT1,ZEB1,Fibronectin andα-SMA pro-tein and cell apoptosis rate insi-PRMT1+oe-NC group were significantly reduced,while the cell proliferation abil-ity and the expression of E-cadherin protein were significantly increased(P<0.05).There was no significant dif-ference in the expression of PRMT1 protein in the si-NC+oe-ZEB1 group(P>0.05),the expression of ZEB1,Fi-bronectin andα-SMA protein and the rate of apoptosis were significant increased(P<0.05),proliferation ability and E-cadherin protein expression were significantly reduced(P<0.05).The expression level of PRMT1 protein in si-PRMT1+oe-NCgroup and si-PRMT1+oe-ZEB1 group was similar(P>0.05).The expression of ZEB1,Fi-bronectin andα-SMA protein and the apoptosis rate of cells in si-PRMT1+oe-ZEB1 group were significantly in-creased,while the proliferation ability and the expression of E-cadherin protein were significantly reduced(P<0.05).Compared with si-NC+oe-ZEB1 group,the expression of PRMT1,ZEB1,Fibronectin andα-SMA protein and the apoptosis rate of cells in si-PRMT1+oe-ZEB1 group were significantly reduced,while the proliferation ability and the expression of E-cadherin protein were significantly increased(P<0.05).Conclusion:PRMT1 pro-motes renal tubular epithelial cell apoptosis and epithelial-mesenchymal transition induced by high glucose through up-regulating the expression of ZEB1.