Abstract:

Abstract:Objective To observe the activational effect of   PAX2 transfection on Wnt4 pathway and  the effect of WNT4 gene on PAX2-induced epithelial- mesenchymal transition,and to discuss the mechanism of PAX2 transition.  Methods The pEGFP-PAX2 plasmid was transfected into rat normal renal tubular epithelial cells(NRK52E) with lipofectamine 2000.The cell line NRK52E stable expressing PAX2 was constructed by G418.NRK52E were divided into control group,empty vector group and stable transfection PAX2 group.The expressions of WNT4 and β-catenin were examined by Western blotting and Real-time PCR in various groups.The WNT4 gene siRNA silencing  was stably transfected into WNT4 of  PAX2 cells  and evaluated through fluorescence detection,and the best chain was chosen.After the best WNT4 siRNA chain was transfected into NRK52E,the expressions of β-catenin,E-cadherin and α-SMA were examined by Western blotting and Real time-PCR at 24,48,72, and 96 h after silencing.Results The Western blotting and Real-time PCR results showed that the protein and mRNA expressions of WNT4 and β-catenin  in stable transfection PAX2 group  were significantly increased  compared with control group and empty vector group(P<0.05).The transfection efficiency of negtive siRNA was(47.46±4.48)％.The third WNT4 transfection chain  was the best transfection chain detected by Real-time PCR and Western blotting. The inhibitory rate of WNT4 m RNA was (66.9±3.8)％（P<0.05),and the inhibitory rate of WNT4 protein was （63.7±2.5）％（P<0.05). 24,48,72, and 96 h after WNT4 siRNA transfection,compared with control group,the mRNA and protein expressions of β-catenin were decreased,especially at 72 h(P<0.05);the mRNA and protein expressions of E-cadherin were increased,especially at 72 h(P<0.05);the mRNA and protein expressions of α-SMA were decreased,especially at 72 h(P<0.05).Conclusion PAX2 could activate WNT4/β-catenin pathway.PAX2 may induce epitheial-mesenchymal transition in normal renal tubular epithelial cells through  WNT4/β-catenin pathway.
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Key words: WNT4, paired box gene 2, renal tubular epithelial cells;transition, RNAi