miR-150-5p缓解肾小管上皮细胞-间质转化及纤维化的作用机制探讨

The mechanism of miR-150-5p alleviating renal tubular epithelial-mesenchymal transition and fibrosis

目的探究miR-150-5p对转化生长因子-β(TGF-β)诱导肾小管上皮细胞间质纤维化的作用及机制。方法利用TGF-β1诱导HK-2细胞构建肾间质纤维化HK-2细胞模型,通过RT-qPCR检测HK-2细胞中miR-150-5p的表达水平;构建的肾间质纤维化HK-2细胞模型转染miR-150-5p mimic和inhabitor后,通过RT-qPCR和Western blot检测各组HK-2细胞中E-cadherin、平滑肌肌动蛋白(α-SMA)、Vimentin和Snail的表达;通过RT-qPCR和ElISA分别检测各组细胞及培养液中Col-Ⅰ、Col-Ⅲ和FN的表达;使用targetscan预测miR-150-5p靶基因,并通过双荧光素酶报告基因实验验证;构建的肾间质纤维化HK-2细胞模型转染miR-150-5p mimic和pcDNA/β-catenin后,通过Western blot和ElISA检测各组细胞中I型胶原蛋白(Col-Ⅰ)、Ⅲ型胶原蛋白(Col-Ⅲ)和细胞纤连蛋白(FN)的表达。结果在TGF-β1诱导的HK-2细胞中miR-150-5p的表达显著降低(P<0.05);间质纤维化HK-2细胞模型转染miR-150-5p mimic后,miR-150-5p的表达显著升高(P<0.05),E-cadherin mRNA和蛋白表达也显著升高(P<0.05),α-SMA,Vimentin和Snail的mRNA与蛋白表达显著降低(P<0.05);间质纤维化HK-2细胞模型转染miR-150-5p inhabitor后,结果与mimic相反。间质纤维化HK-2细胞转染miR-150-5p mimic后,Col-I、Col-Ⅲ和FN的表达水平显著降低(P<0.05);转染miR-150-5p inhabitor后,Col-Ⅰ、Col-Ⅲ和FN的表达升高(P<0.05)。经双荧光素酶报告验证β-catenin是miR-150-5p的靶基因;间质纤维化HK-2细胞模型共同转染miR-150-5p和pcDNA/β-catenin后,细胞中Col-I,Col-Ⅲ和FN的蛋白表达水平显著降低(P<0.05)。结论miR-150-5p通过靶向β-catenin基因,调控TGF-β1诱导的HK-2细胞的上皮间质转化(EMT)过程和细胞外基质(ECM)的积累。

Objective To explore the mechanism of miR-150-5p on TGF-βrenal tubular epithelial cell interstitial fibrosis.Methods HK-2 cell model of renal interstitial fibrosis was constructed by using HK-2 cells induced by TGF-β1.The expression level of miR-150-5p in HK-2 cells was detected by RT-PCR.After transfecting miR-150-5p mimic and inhabitor into the constructed HK-2 cell model of renal interstitium fibrosis,E-cadherin,α-SMA,Vimentin and Snail in HK-2 cells of each group were detected by RT-PCR and Western blot.The expressions of Col-Ⅰ,Col-Ⅲand FN in cell medium of each group were detected by RT-PCR and Elisa.miR-150-5p target genes were predicted by targetscan and verified by double luciferase reporter gene assay.The constructed renal interstitial fibrosis HK-2 cell model was transfected with miR-150-5p mimic and pcDNA/β-catenin,and CollagenⅠ,Col-Ⅲand cellular fibronectin in the cells were detected by WB and Elisa.Results The expression of miR-150-5p was significantly increased in HK-2 cells induced by TGF-β1(P<0.05).The expression of 5p was significantly increased(P<0.05),the mRNA and protein expressions of E-cadherin were significantly increased(P<0.05),and the mRNA and protein expressions ofα-SMA,Vimentin and Snail were significantly decreased(P<0.05);after transfection of miR-150-5p inhabitors in the fibrotic HK-2 cell model,the results were opposite to mimic.β-catenin was verified as the target gene of miR-150-5p by dual luciferase report;after co-transfection of miR-150-5p and pcDNA/β-catenin in the interstitial fibrosis HK-2 cell model,Col-I in the cells,the protein expression levels of Col-Ⅲand FN were significantly decreased(P<0.05).Conclusion miR-150-5p can regulate TGF-β1-induced EMT process and ECM accumulation in HK-2 cells by targetingβ-catenin gene.