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

作　　者：刘华[1] 何小解[2] 李国君[1] 丁庆雄 梁晚霞[1] 樊娟[1] 

机构地区：[1]深圳市南山区妇幼保健院儿科,广东深圳518052 [2]中南大学湘雅二医院儿童医学中心,湖南长沙410011

出　　处：《中国当代儿科杂志》2017年第6期712-718,共7页Chinese Journal of Contemporary Pediatrics

摘　　要：目的研究micro RNA-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p CMVmiR-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p CMV-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p CMV-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。Objective To investigate the effects ofmicroRNA-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-rniR-145 recombinant plasmid）. Expression ofmiR-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 offibronectin （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 supematants also decreased （P〈0.05）. Conclusions miR- 145 modulates the EMT of HK-2 ceils treated with TGF-β1, possibly by inhibition of the activation of TGF-β-dependentSmad signaling pathway.

关 键 词：microRNA-145 TGF-Β/SMAD信号通路 上皮间充质转化 人肾小管上皮细胞 

分 类 号：R692[医药卫生—泌尿科学]