microRNA-145对TGF-β1诱导的人肾小管上皮细胞上皮间充质转化的影响

Effects of microRNA-145 on epithelial-mesenchymal transition of TGF-β1-induced human renal proximal tubular epithelial cells

摘要:

目的 研究microRNA-145（miR-145）对转化生长因子（TGF）-β1诱导人肾小管上皮细胞（HK-2）上皮间充质转化（EMT）的影响。方法 人工合成miR-145基因序列，构建真核重组质粒pCMV-miR-145。以未处理HK-2细胞为对照组；以TGF-β1处理HK-2细胞为TGF-β1组；以pCMV-myc空白质粒转染后经TGF-β1处理HK-2细胞为TGF-β1空白质粒组；以pCMV-miR-145质粒转染后经TGF-β1处理HK-2细胞为TGF-β1+miR-145组。采用实时荧光定量PCR检测miR-145表达。采用Western blot检测TGF-β/Smad信号传导蛋白TGF-β1、Smad3、Smad2/3、p-Smad2/3，以及EMT生物标记物蛋白α-平滑肌肌动蛋白（α-SMA）、纤维连接蛋白（FN）和I型胶原蛋白（ColⅠ）的表达水平。采用ELISA法检测培养细胞上清液中FN和Col I的含量。结果 miR-145表达质粒构建成功，重组质粒有效转染TGF-β1诱导HK-2细胞。与对照组比较，TGF-β1+miR-145组细胞中miR-145表达上调（P < 0.01）；与对照组和TGF-β1+miR-145组比较，TGF-β1组和TGF-β1空白质粒组细胞中miR-145表达均下降（P < 0.01）。与TGF-β1组和TGF-β1空白质粒组比较，TGF-β1+miR-145组细胞内TGF-β1、Smad3、Smad2/3和p-Smad2/3蛋白表达量减少（P < 0.05）；α-SMA、FN、ColⅠ蛋白表达亦明显减少（P < 0.05）；TGF-β1+miR-145组培养液上清中FN、ColⅠ含量减少（P < 0.05）。结论 miR-145参与TGF-β1处理HK-2细胞EMT的调控。miR-145可能通过抑制TGF-β依赖的Smad信号通路活性，从而抑制肾小管EMT。

Abstract:

Objective To investigate the effects of microRNA-145 (miR-145) on epithelial-mesenchymal transition (EMT) of TGF-β1-induced human renal proximal tubular epithelial (HK-2) cells. Methods The gene sequence of miR-145 was synthesized and cloned into pCMV-myc to construct recombinant plasmid pCMV-miR-145. HK-2 cells were divided into four groups:control (untreated), TGF-β1 (treated with TGF-β1), blank+TGF-β1 (treated with TGF-β1 after HK-2 cells transfected with blank plasmid) and miR-145+TGF-β1 (treated with TGF-β1 after HK-2 cells transfected with pCMV-miR-145 recombinant plasmid). Expression of miR-145 was detected by real-time PCR (RT-PCR). TGF-β1, Smad3, Smad2/3, p-Smad2/3, α-SMA, FN and type I collagen (Col I) protein levels were detected by Western blot. Concentrations of fibronectin (FN) and Col I in cell culture supernatants were measured using ELISA. Results pCMV-miR-145 recombinant plasmid was successfully transfected into HK-2 cells. Compared with the control group, the miR-145+TGF-β1 group showed a significant up-regulation in the expression level of miR-145 (P < 0.01). However, the TGF-β1 and blank+TGF-β1 groups showed a significant down-regulation in the expression level of miR-145 compared with that in the control and miR-145+TGF-β1 groups (P < 0.01). Compared with the TGF-β1 and blank+TGF-β1 groups, the miR-145+TGF-β1 group showed significantly reduced levels of the signal proteins TGF-β1, Smad3, Smad2/3 and p-Smad2/3 (P < 0.05), as well as significantly reduced levels of the biomarkers α-SMA, FN and Col I (P < 0.05). Meanwhile, concentrations of FN and Col I in cell culture supernatants also decreased (P < 0.05). Conclusions miR-145 modulates the EMT of HK-2 cells treated with TGF-β1, possibly by inhibition of the activation of TGF-β-dependent Smad signaling pathway.