miR-377调控THEM4对高糖诱导的人近端肾小管上皮细胞EMT的作用

Effects of miR-377 on high glucose-induced EMT in human proximal tubular epithelial cells by regulating THEM4

目的:探讨微小RNA-377(miR-377)对硫酯酶超家族成员4(THEM4)的靶向作用及对高糖诱导的人近端肾小管上皮细胞上皮间充质转化(EMT)的影响。方法:将人近端肾小管上皮细胞HK-2分为正常葡萄糖组(NG组:5 mmol/L葡萄糖)、高糖组(HG组:30 mmol/L葡萄糖)、HG+miR-NC组(HG+转染miR-NC)、HG+miR-377组(HG+转染miR-377)、HG+antimiR-NC组(HG+转染anti-miR-NC)、HG+anti-miR-377组(HG+转染anti-miR-377)、HG+miR-377+pcDNA3.1组(HG+共转染miR-377+pcDNA3.1)、HG+miR-377+pcDNA3.1-THEM4组(HG+共转染miR-377+pcDNA3.1-THEM4)、HG+miR-377+si-NC组(HG+共转染miR-377+si-NC)和HG+miR-377+si-THEM4组(HG+共转染miR-377+si-THEM4)。qPCR检测miR-377表达,Western blot检测Vimentin、E-cadherin、THEM4蛋白表达,生物信息学预测和双荧光素酶实验分析miR-377与THEM4的靶向关系。结果:与NG组相比,HG诱导的HK-2细胞中miR-377表达量、Vimentin蛋白表达量明显增加,E-cadherin蛋白水平显著降低(P<0.05)。与HG+miR-NC组相比,HG+miR-377组HK-2细胞的miR-377表达量、Vimentin蛋白表达量显著升高,E-cadherin蛋白水平显著降低(P<0.05)。与HG+anti-miR-NC组相比,HG+anti-miR-377组HK-2细胞中miR-377表达量、Vimentin蛋白水平显著降低(P<0.05),E-cadherin蛋白表达量显著升高(P<0.05)。miR-377靶向调控THEM4表达。与HG+miR-377+pcDNA3.1组相比,HG+miR-377+pcDNA3.1-THEM4组HK-2细胞中THEM4、E-cadherin蛋白水平明显升高,Vimentin蛋白表达量显著降低(P<0.05)。与HG+miR-377+si-NC组相比,HG+miR-377+si-THEM4组HK-2细胞的THEM4、E-cadherin蛋白表达量明显降低,Vimentin蛋白水平明显升高(P<0.05)。结论:miR-377通过靶向THEM4调控高糖诱导的人近端肾小管上皮细胞EMT。

Objective:To investigate the effect of microRNA-377(miR-377)on thioesterase superfamily member 4(THEM4 and its effect on high glucose-induced epithelial-mesenchymal transition(EMT)in human proximal tubular epithelial cells.Methods:Human proximal tubular epithelial cells HK-2 were divided into normal glucose group(NG group:5 mmol/L glucose),high glucose group(HG group:30 mmol/L glucose),HG+miR-NC group(HG+transfection of miR-NC),HG+miR-377 group(HG+transfection of miR-377),HG+anti-miR-NC group(HG+transfection of anti-miR-NC),HG+anti-miR-377 group(HG+transfected anti-miR-377),HG+miR-377+pcDNA3.1 group(HG+co-transfected miR-377+pcDNA3.1),HG+miR-377+pcDNA3.1-THEM4 group(HG+co-transfected miR-377+pcDNA3.1-THEM4),HG+miR-377+si-NC group(HG+co-transfected miR-377+si-NC),HG+miR-377+si-THEM4 group(HG+co-transfected miR-377+si-THEM4). qPCR was used to detect expression of miR-377,Western blot was applied to determine expressions of Vimentin,E-cadherin and THEM4 proteins. Bioinformatics prediction and dual luciferase assay analyzed targeting relationship of miR-377 and THEM4.Results:Compared with NG group,expression levels of miR-377 and Vimentin protein in HG-induced HK-2 cells were significantly increased,while level of E-cadherin protein was greatly decreased(P<0.05). Compared with HG+miR-NC group,HG+miR-377 group could markedly improve expression levels of miR-377 and Vimentin protein in HKG cells,while obviously reduce level of E-cadherin protein(P<0.05). Compared with HG+anti-miR-NC group,expression levels of miR-377 and Vimentin protein in HK-2 cells of HG+anti-miR-377 group were statistically lower(P<0.05),while expression of E-cadherin protein was remarkably increased(P<0.05). miR-377 targeted regulated expression of THEM4. Compared with HG+miR-377+pcDNA3.1 group,HG+miR-377+pcDNA3.1-THEM4 group could evidently raise levels of THEM4 and E-cadherin in HK-2 cells,and apparently decrease expression of Vimentin protein(P<0.05). Compared with HG+miR-377+si-NC group,expression levels of THEM4 and E-cadherin protein in HKG cells of HG+miR-377+si-THEM4 group were dramatically decreased,and Vimentin protein level was notably increased(P<0.05).Conclusion:miR-377 regulates high glucose-induced EMT in human proximal tubular epithelial cells by targeting THEM4.