shRNA干扰β-catenin对TGF-β1诱导的肾小管上皮细胞转分化的作用

Short hairpin RNA of β-catenin attenuates TGF-β1-induced epithelial mesenchymal transition in human proximal tubular epithelial cells

目的构建特异性针对β-catenin基因的shRNA干扰载体,转染TGF-β1诱导的人近端肾小管上皮细胞株HKCs,研究其对肾小管上皮细胞转分化的影响。方法将构建的shRNA-β-catenin干扰载体转染TGF-β1诱导的HKC细胞,RT-PCR、Western blot检测β-catenin的mRNA及蛋白表达水平,筛选抑制效果最佳载体;RT-PCR、细胞免疫荧光、Western blot检测肾小管上皮细胞转分化相关蛋白E-cadherin、α-SMA的mRNA及蛋白表达水平。结果经酶切和测序鉴定,成功构建的shRNA-β-catenin表达载体转染HKC细胞,β-catenin在mRNA及蛋白表达水平均低于转染空白载体组(P<0.05),并筛选shRNA-β-catenin-1进入实验组;转染shRNA-β-catenin-1组细胞β-catenin在mRNA及蛋白表达水平均低于TGF-β1诱导组和空载体组(P<0.05),且β-catenin转核显著减弱(P<0.05);E-cadherin的mRNA及蛋白表达水平较TGF-β1诱导组和空载体组显著上调(P<0.05)、α-SMA的mRNA及蛋白表达水平较TGF-β1诱导组和空载体组显著下调(P<0.05)。结论运用shRNA-β-catenin干扰载体转染TGF-β1诱导的HKC细胞,能够抑制肾小管上皮细胞-间充质转分化。

Objective To determine the effect of β-catenin gene shRNA on the epithelial mesenchymal transition in the human proximal tubular epithelial cells（HKCs） induced by TGF-β1.Methods shRNA-β-catenin vector was constructed and then transfect into HKC cells induced by TGF-β1.RT-PCR and Western blot analysis were used to detect mRNA and protein expression levels of β-catenin to screen the best inhibitory vector.RT-PCR,immunofluorescence,and Western blot analysis were employed to detect mRNA and protein expression levels of renal tubular epithelial cell differentiation-related proteins,α-SMA and E-cadherin.Results The recombinant plasmid shRNA-β-catenin was confirmed by digestion and sequencing.After HKC cells were transfected with the plasmid,β-catenin expression at the mRNA and protein levels were significantly lower than the empty vector group（P0.05）.Plasmid shRNA-β-catenin-1 was screened,and transfected in HKC cells.β-catenin expression at the mRNA and protein levels were significantly lower in the cells transfected with shRNA-β-catenin-1 than in the cells with TGF-β1 inducement and the cells transfected with empty vector（P0.05）.In the shRNA-β-catenin-1 group,the amount of nuclear transferred β-catenin was significantly reduced compared with TGF-β1-induced group and the empty vector group（P0.05）,while E-cadherin mRNA and protein expression levels were significantly increased than the other 2 groups（P0.05）.On the contrast,compared with TGF-β1-induced group and the empty vector group,α-SMA mRNA and protein expression levels were significantly lower in the group（P0.05）.Conclusion shRNA-β-catenin vector attenuates epithelial mesenchymal transdifferentiation in human proximal tubular epithelial cells induced by TGF-β1.