WNT4基因沉默对PAX2诱导肾小管上皮细胞间充质转分化的影响  

Influence of WNT4 siRNA in PAX2-induced epithelial-mesenchymal transition in normal renal tubular epithelial cells

作　　者：李里[1,2] 南晓娟[1] 吴玉斌[1] 

机构地区：[1]中国医科大学附属盛京医院小儿肾脏风湿免疫科,辽宁沈阳110004 [2]辽宁医学院附属第一医院儿科,辽宁锦州121000

出　　处：《吉林大学学报（医学版）》2013年第4期720-725,I0002,I0003,共8页Journal of Jilin University：Medicine Edition

基　　金：辽宁省沈阳市科学技术计划项目资助课题(F10-205-1-29);中国医科大学附属盛京医院博士优秀课题基金资助课题(ma26)

摘　　要：目的:观察体外转染配对盒基因2(PAX2)对WNT4通路的激活作用及WNT4通路对PAX2诱导转分化的作用,探讨PAX2转分化机制。方法:应用脂质体介导将重组质粒pEGFP-PAX2转染入体外培养的大鼠肾小管上皮细胞,经G418筛选,建立稳定表达PAX2细胞系。细胞分为稳定转染PAX2组、空载组和对照组。Western blotting法和实时荧光定量(Real-time)PCR法检测各组细胞WNT4和β-catenin表达。应用靶向WNT4基因的siRNA沉默稳定转染PAX2细胞的WNT4,应用荧光显微镜观察转染效率,进行沉默鉴定筛选出最佳干扰链。将最佳干扰链WNT4siRNA转染至稳定转染PAX2细胞。在沉默24、48、72和96h应用Westernblotting法和Real-time PCR法检测β-catenin、正常肾小管上皮细胞表型标志物E-钙黏蛋白(E-cadherin)和成纤维细胞表型标志物α-肌动蛋白(α-SMA)表达,同时筛选出沉默最佳时间点。应用免疫组织化学染色法检测沉默最佳时间点组E-cadherin和α-SMA蛋白表达。结果:Western blotting和Real-time PCR检测,稳定转染PAX2细胞组WNT4和β-catenin蛋白及mRNA表达水平较空载组和对照组明显增加(P<0.05)。阴性siRNA转染入稳定转染PAX2细胞24h后荧光显微镜观察,转染效率为47.46%±4.48%。3条WNT4干扰链分别转染至稳定转染PAX2细胞,Real-time PCR和Western blotting结果显示第3干扰链沉默效果最佳,WNT4mRNA表达抑制率为66.9%±3.8%(P<0.05);WNT4蛋白表达抑制率为63.7%±2.5%(P<0.05)。将最佳干扰链转染至稳定转染PAX2细胞,干扰24、48、72和96h后,各时间点细胞中的β-catenin mRNA及蛋白表达水平较对照组下调,72h组下调明显(P<0.05);各时间点细胞中E-cadherin mRNA及蛋白表达水平较对照组明显上调,72h组上调明显(P<0.05);各时间点细胞中α-SMA mRNA及蛋白表达水平较对照组明显下调,72h下调明显(P<0.05)。结论:PAX2转染至肾小管上皮细胞后激活了WNT4通路,PAX2在体外诱导正常肾小管上皮细胞间充质转分化可能�Objective To observe the activational effect of PAX2transfection on Wnt4pathway and the effect of WNT4gene on PAX2-induced epithelial-mesenchymal transition,and to discuss the mechanism of PAX2 transition.Methods The pEGFP-PAX2plasmid was transfected into rat normal renal tubular epithelial cells（NRK52E）with lipofectamine 2000.The cell line NRK52Estable expressing PAX2 was constructed by G418.NRK52E were divided into control group,empty vector group and stable transfection PAX2 group.The expressions of WNT4andβ-catenin were examined by Western blotting and Real-time PCR in various groups.The WNT4gene siRNA silencing was stably transfected into WNT4of PAX2cells and evaluated through fluorescence detection,and the best chain was chosen.After the best WNT4siRNA chain was transfected into NRK52E,the expressions ofβ-catenin,E-cadherin andα-SMA were examined by Western blotting and Real time-PCR at 24,48,72,and 96hafter silencing.Results The Western blotting and Real-time PCR results showed that the protein and mRNA expressions of WNT4andβ-catenin in stable transfection PAX2group were significantly increased compared with control group and empty vector group（P0.05）.The transfection efficiency of negtive siRNA was（47.46± 4.48）%.The third WNT4transfection chain was the best transfection chain detected by Real-time PCR and Western blotting.The inhibitory rate of WNT4mRNA was（66.9±3.8）%（P0.05）,and the inhibitory rate of WNT4 protein was（63.7±2.5）%（P0.05）.24,48,72,and 96hafter WNT4siRNA transfection,compared with control group,the mRNA and protein expressions ofβ-catenin were decreased,especially at 72h（P0.05）;the mRNA and protein expressions of E-cadherin were increased,especially at 72h（P0.05）;the mRNA and protein expressions ofα-SMA were decreased,especially at 72h（P0.05）.Conclusion PAX2could activate WNT4/βcatenin pathway.PAX2may induce epitheial-mesenchymal transition in normal renal tubular epithelial cells through WNT4/β-catenin pathway.

关 键 词：WNT4 配对盒基因2 肾小管上皮细胞 转分化 RNA干扰 

分 类 号：R586[医药卫生—内分泌]