WIF-1阻断WNT通路对PAX2诱导的肾小管上皮细胞间充质转分化作用的影响

Effects of Blocking WNT Signaling Pathway with WIF-1 on PAX2 Induced Epithelial-mesenchymal Transition

目的体外转染配对盒基因2(PAX2)观察其对WNT通路的激活作用,应用WIF-1阻断WNT信号通路后观察WNT通路对PAX2基因诱导转分化的作用,探讨PAX2基因转分化机制。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,将实验细胞分为转染组、空载组和对照组,采用Western blot和实时PCR对各组进行WNT通路分子WNT4及β-catenin表达检测。加入WIF-1至稳定转染PAX2细胞中,分为WIF-1 5μg/mL组、WIF-1 10μg/mL组、WIF-1 15μg/mL组及稳转组。加药后48 h收集细胞,应用免疫荧光法、Western blot和实时PCR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。结果 Western blot和实时PCR结果显示,转染组WNT4和β-catenin蛋白和mRNA表达水平较空载组及对照组明显增加(P<0.05)。免疫荧光法、Western blot和实时PCR结果显示,WIF-1 5μg/mL组、WIF-1 10μg/mL组和WIF-1 15μg/mL组β-catenin和α-SMA蛋白及mRNA表达较稳转组下调(P<0.05),WIF-1 15μg/mL组下降明显,各浓度WIF-1组E-cadherin蛋白及mRNA较稳转组明显上调(P<0.05),WIF-1 15μg/mL组明显。结论 PAX2基因可以在体外肾小管上皮细胞激活WNT通路。应用WIF-1阻断WNT通路后出现转分化逆转。推测PAX2基因可能通过WNT通路诱导上皮细胞间充质转分化。

Objective To observe the activated effect of WNT pathway after PAX2 transfection,and investigate the effect of PAX2 induced epithelial-mesenchymal transition as blocking WNT signaling pathway with WIF-1 and to discuss the mechanism of PAX2 induced EMT.Methods The pEGFP-PAX2 plasmid was wansfected into rats normal renal tubular epithelial cells line（NRK52E） with lipofectamine 2000.Stable transfected NRK52E with PAX2 was constructed by G418.NRK52E were divided into three groups：control group,empty vector group and transfection group.WNT4 and β-catenin expression were examined by Western blot and real-time PCR.PAX2 stable transfected cells were divided into four group：WIF-1 5 μg/mL,WIF-1 10 μg/mL,WIF-1 15 μg/mL and stable transfection group.β-catenin,E-cadherin and α-SMA were examined by immunofluorescen method,Western blot,and real-time PCR at 48 h in different groups.Results The results of Western blot and real-time PCR showed that the protein and mRNA of WNT4 and β-catenin were significantly increased in transfection group compared with the control group and empty vector group （P ＜ 0.05）.Immunofluorescen,Westem blot and real-time PCR showed that the protein and mRNA expression of β-catenin and α-SMA were decreased in WIF-1 5 μg/mL group,WIF-1 10 μg/mL group and WIF-115 μg/mL group compared with the stable transfection group （P ＜ 0.05）,and significantly in WIF-1 15 μg/mL.E-cadherin protein and mRNA significantly increased in all the WIF-1 groups,compared with the stable transfection group （P ＜ 0.05）,while significantly in WIF-1 15 μg/mL.Condusion PAX2 can activate the WNT pathway.Blocking WNT signaling by WIF-1 can reverse PAX2 induced epithelial-mesenchymal transition.It is speculated that PAX2 can induce the EMT by WNT pathway.