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

作　　者：田平平 邹琴 卢雨微 郭兵 石明隽 TIAN Pingping;ZOU Qin;LU Yuwei;GUO Bing;SHI Mingjun(School of Nursing,Sanquan College of Xinxiang Medical University,Xinxiang 453003,Henan,China;Department of Pathophysiology,School of Basic Medicine Sciences,Guizhou Medical University,Guiyang 550025,Guizhou,China)

机构地区：[1]新乡医学院三全学院护理学院,河南新乡453003 [2]贵州医科大学基础医学院病理生理学教研室,贵州贵阳550025

出　　处：《贵州医科大学学报》2022年第9期1007-1013,共7页Journal of Guizhou Medical University

基　　金：国家自然科学基金(82060142、81860135)。

摘　　要：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�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 gro

关 键 词：miR-27 a 分泌型卷曲相关蛋白1 NRK-52E 上皮细胞向间质细胞转分化 肾脏纤维化 糖尿病肾病 

分 类 号：R34[医药卫生—基础医学]