Effects Of FoxO3a Knock Down On The Epithelial-to-mesenchymal Transition Of Human Renal Proximal Tubular Cells

Objective: 1.To examine whether Fox O3 a transcription factor is involved in human renal tubular cells EMT process;2.To determine whether the Fox O3 a regulates ?-Catenin effect on the epithelial-to-mesenchymal transition of HK-2 human renal proximal tubular cells.Methods: 1.EMT was induced in HK-2 cells by 10 ng/ml TGF-?1,the expression levels of E-cadherin,Vimentin,?-SMA were determined by Western blot;2.Expression of Vimentin,?-SMA and the morphology of cells was analyzed by immunofluorescence staining;3.The expression of Fox O3 a and p-Fox O3 a in EMT of HK-2 cells were determined by Western blot;4.Confocal laser scanning microscope were used to detect the distribution of Fox O3 a on EMT of HK-2 cells;5.Knocking down of Fox O3 a in the HK-2 cells by si RNA,the levels of EMT marker proteins were determined by Western blot;6.The expression of ?-Catenin in the EMT of HK-2 cells and expression Fox O3 a of the cells were determined by Western blot and immunofluorescence assay.Result 1.EMT cell model was established by 10ng/ml TGF-?1 induced of HK-2 cells.Compared with HK-2 cells incubated without TGF-?1,the protein expression of E-Cadherin significantly decreased(P<0.01),and the expression levels of Vimentin,?-SMA markedly increased in HK-2 cells(P<0.05);the level of Vimentin,?-SMA in HK-2 cells by immunofluorescence assay and the cells adopted a more fibroblast-like morphology.2.The expression levels of Fox O3 a markedly diminished(P<0.01)in HK-2 cells by TGF-?1treatment,and the expression of p-Fox O3 a markedly increased(P<0.05).Fox O3 a was reduced in renal tubular EMT process.3.TGF-?1 regulates Fox O3 a phosphorylation,nuclear export and retention in the cytoplasm.4.Western blotting and RT-PCR results shows that knocking down Fox O3 a in HK-2 cells,decreased the protein expression of E-cadherin significantly(P<0.01),and increased ?-SMA markedly(P<0.05).These data suggest that Fox O3 a can regulate epithelial-to-mesenchymal transition of HK-2 human renal proximal tubular cells.5.The expression of ?-Catenin in the EMT of HK-2 cells significantly increased and expression Fox O3 a decreased by Western blot and immunofluorescence assay.Conclusion: 1.Fox O3 a is involved in EMT process.2.Fox O3 a can regulate epithelial-to-mesenchymal transition of HK-2 human renal proximal tubular cells.These data suggest down-regulation of Fox O3 a could promote EMT of human renal proximal tubular epithelial cells by increasing the expression levels of ?-Catenin.