miR-27 a调控Sfrp1对肾小管上皮细胞EMT的影响

Effect of the regulation of Sfrp1by miR-27a on EMT of renal tubular epithelial cells

目的探讨高糖培养肾小管上皮细胞中miR-27 a调控分泌型卷曲相关蛋白1(Sfrp1)对细胞向间质细胞转分化(EMT)的影响。方法将NRK-52E细胞分为正常糖(NG组)和高糖(HG组)两组,利用RT-qPCR检测NRK-52E细胞中miR-27 a、Sfrp 1的表达情况,Western blot检测NRK-52E细胞中Sfrp1、E-钙粘蛋白(E-cadherin)、α平滑肌肌动蛋白(α-SMA)、Ⅳ型胶原(col-Ⅳ)的蛋白表达;将NRK-52E细胞分为野生型Sfrp 1-3′UTR+miR-27 a mimics阴性对照组(wt Sfrp 1-3′UTR+miR-27 a mnc组)、野生型Sfrp 1-3′UTR+miR-27 a mimics组(wt Sfrp 1-3′UTR+miR-27 a m组)、突变型Sfrp 1-3′UTR+miR-27 a mimics阴性对照组(mt Sfrp 1-3′UTR+miR-27 a mnc组)以及突变型Sfrp 1-3′UTR+miR-27 a mimics组(mt Sfrp 1-3′UTR+miR-27 a m),通过双荧光素酶报告基因检测系统检测萤火虫荧光值与海肾荧光值的比值,验证miR-27 a对Sfrp1表达的影响;在高糖诱导下,利用细胞转染实验将NRK-52E细胞设miR-27 a mimics阴性对照组(miR-27 a mnc组)、miR-27 a mimics组(miR-27 a m组)、miR-27 a inhibitor阴性对照组(miR-27 a inc组)以及miR-27 a inhibitor组(miR-27 a i组),通过RT-qPCR检测各组细胞中miR-27 a、Sfrp 1 mRNA的表达,Western blot检测各组细胞中Sfrp1、E-cadherin、α-SMA、col-Ⅳ蛋白的表达水平。结果RT-qPCR结果显示,与NG组比较,HG组miR-27 a表达水平增高(P<0.05),Sfrp 1 mRNA表达水平降低(P<0.05);Western blot结果显示,与NG组比较,HG组α-SMA、col-Ⅳ蛋白水平增高(P<0.05),Sfrp1、E-cadherin蛋白表达水平降低(P<0.05);荧光素酶报告基因显示,wt Sfrp 1-3′UTR+miR-27 a m组荧光素酶活性明显低于wt Sfrp 1-3′UTR+miR-27 a mnc组(P<0.05),而mt Sfrp 1-3′UTR+miR-27a mnc组与mt Sfrp 1-3′UTR+miR-27 a m组荧光素酶活性未见明显变化(P>0.05);细胞转染RT-qPCR结果显示,与miR-27 a mnc组比较,miR-27 a m组miR-27 a表达增加(P<0.05),而Sfrp 1 mRNA表达降低(P<0.05);与miR-27 a inc组比较,miR-27 a i组miR-27 a表达降低(P<0.05),而Sfrp 1 mRNA表达增加(P<0.05);Western blot结果显示,与miR-27 a mnc组比较,miR-27 a m组α-SMA、col-Ⅳ蛋白表达增加(P<0.05),Sfrp1、E-cadherin蛋白表达降低(P<0.05);与miR-27a inc组比较,miR-27 a i组α-SMA、col-Ⅳ蛋白表达降低(P<0.05),Sfrp1、E-cadherin蛋白表达增加(P<0.05)。结论miR-27 a可以通过抑制Sfrp1的表达促进肾小管EMT,参与糖尿病肾病肾纤维化的发生。

Objective To investigate the effect of the regulation of secreted frizzled-related protein1(Sfrp1)by miRNA-27 a on epithelial-mesenchymal transition(EMT)of renal tubular epithelial cells.Methods NRK-52E cells were divided into two groups:normal glucose group(NG)and high glucose group(HG,30 mmol/L).RT-qPCR was applied to detect the expression of miR-27 a and Sfrp 1 in NRK-52E cells.Western blot was performed to detect the protein expression levels of Sfrp1,E-cadherin,α-smooth muscle actin(α-SMA),and collagen-Ⅳ(col-Ⅳ)in NRK-52E cells.Based on the transfected reagents,NRK-52E cells were classified into 4 groups:wild type Sfrp 1-3′UTR+miR-27 a mimics negative control(wt-mnc group),wild type Sfrp 1-3′UTR+miR-27 a mimics(wt-m group),mutated Sfrp 1-3′UTR+miR-27 a mimics negative control(mt-mnc group),and mutated Sfrp 1-3′UTR+miR-27 a mimics(mt-m group).Dual-luciferase reporter assay was used to detect firefly luciferase activity and Renilla luciferase activity,respectively.The ratio of firefly luciferase activity to Renilla luciferase activity was calculated to examine whether miR-27 a regulates Sfrp1 expression.According to transfected reagents,NRK-52E cells cultured in high glucose were divided into miR-27 a mimics negative control group(miR-27 a mnc),miR-27 a overexpression group(miR-27 a m),miR-27 a inhibitor negative control group(miR-27 a inc),miR-27 a inhibitor group(miR-27 a i).The expression of miR-27 a and Sfrp 1 in each group was detected by RT-qPCR.The expression levels of Sfrp1,E-cadherin,α-SMA,and col-Ⅳin each group were detected by Western blot.Results RT-qPCR results showed that miR-27 a expression level in HG group was increased relative to NG group(P<0.05),while Sfrp 1 mRNA expression level was decreased(P<0.05).Western blot results revealed that the expression levels ofα-SMA and col-Ⅳwere upregulated in HG group(P<0.05),while the expression levels of Sfrp1 and E-cadherin were downregulated relative to NG group(P<0.05).Dual Luciferase assay showed the luciferase activity in mt-m group was remarkably lower than that in wt-mnc group(P<0.05),while the luciferase activity in mt-m group was not obviously different from that in mt-mnc group(P>0.05).RT-PCR analysis demonstrated that miR-27 a expression was upregulated in miR-27 a m group relative to miR-27 a mnc group(P<0.05),but Sfrp 1 mRNA expression was downregulated(P<0.05).miR-27 a expression was decreased in miR-27 a I group relative to miR-27 a inc group(P<0.05),while Sfrp 1 mRNA expression was increased(P<0.05).Western blot results showed that when compared with the miR-27 a mnc group,the protein expressions ofα-SMA and col-IV in miR-27 a m group were increased(P<0.05),while the protein expression levels of Sfrp1 and E-cadherin were decreased(P<0.05).The protein expression levels ofα-SMA and col-Ⅳin the miR-27a i group were decreased relative to miR-27a inc group(P<0.05),while the protein expression levels of Sfrp1 and E-cadherin were increased(P<0.05).Conclusion miR-27a can promote EMT of renal tubular epithelial cells by inhibiting Sfrp1 expression and involve in the occurrence of renal fibrosis in diabetic nephropathy.