MicroRNA在丹酚酸B逆转肾小管上皮细胞转分化时的表达变化

目的:研究丹酚酸B逆转转化生长因子(TGF)-β1诱导的肾小管上皮细胞-间充质细胞转分化时microRNA(miRNA)表达谱的变化。方法:将体外培养的人近端肾小管上皮细胞系(HK-2)细胞分为3组:①对照组:未加入丹酚酸B或者TGF-β1;②TGF-β1组:在细胞培养基中加入TGF-β1(浓度为5ng/mL);③丹酚酸B逆转组:在细胞培养基中先TGF-β1(浓度为5ng/mL),48h后HK-2细胞成功诱导EMT后形成的间充质细胞,再更换为TGF-β1(浓度为5ng/mL)和丹酚酸B(50μmol/L)继续培养72h,采用倒置相差显微镜观察细胞形态学变化;免疫细胞化学染色检测细胞E-cadherin的表达情况;基因芯片检测各组细胞miRNA的表达变化。结果:①丹酚酸B具有逆转HK-2细胞EMT的作用;②与正常HK-2细胞比较,TGF-β1组有25种miRNA的表达出现显著改变,其中15种表达下调2倍以上,10种表达上调2倍以上。③与TGF-β1组比较,丹酚酸B逆转组,有44种miRNA的表达出现显著改变,其中24种表达下调2倍以上,20种表达上调2倍以上。④有10种miRNA表达在两组同时出现显著改变,其中8种在TGF-β1组明显下调,而在丹酚酸B逆转组明显上调,1种在TGF-β1组明显上调,而在丹酚酸B逆转组则明显下调。结论:丹酚酸B具有阻止慢性肾脏疾病进行性发展的潜能,而这一作用与其能有效调控miRNA的表达有关。

Alteration of miRNA Expression in Salvianolic Acid B-reversed Epithelial-mesenchymal Transition of Renal Tubular Epithelial Cells

Objective：To investigate the alteration of microRNA（miRNA）expression patterns during the reversal effects of Salvianolic acid B（Sal B）on epithelial-mesenchymal Transition（EMT）induced by transforming growth factor-β1（TGF-β1）.Methods：In this study,Human kidney proximal tubular cell line（HK-2）was cultured in vitro as the proximal tubular cell model.Cells were divided into three groups as follows：control group,transforming growth factor-β1（TGF-β1）group,Salvianolic acid B（Sal B）reversal group.Epithelial-mesenchymal transition（EMT）was induced by human TGF-β1 for 48h,and then TGF-β1 plus Sal B for 72h,the effects of the Sal B on cytomorphology were observed by inverted phase contrast microscope,and expressions of E-cadherin and miRNAs were detected by immunocytochemistry and gene chips respectively.Results：①Sal B had reversal effects on EMT.②Compared with normal HK-2 cells,25 miRNAs expressions were significantly changed in TGF-β1 group,of which 15 were downregulated and 10 unregulated more than 2 times.③Compared with TGF-β1 group,44 miRNAs were showed differential expressions in Sal B reversal group with 24 downregulated and 20 upregulated more than 2 times.④Among the 10 miRNAs altered in both the two groups,8 were markedly downregulated in TGF-β1 group while upregulated in Sal B reversal group,and 1 dramatically upregulated in TGF-β1 group while downregulated in Sal B group.Conclusion：Salvianolic acid B（Sal B）has potential protect effects on development of chronic renal diseases,miRNAs expressions may be closely associated with the reversal effect.