叉头框蛋白O3a表达下调对人肾小管上皮细胞EMT的影响

Effects of FoxO3a Knocking Down on the Epithelial-to-mesenchymal Transition of HK-2 Human Renal Proximal Tubular Cells

目的:研究叉头框蛋白O3a(forkhead box protein O3a,Fox O3a)下调对人肾小管上皮细胞(HK-2)上皮-间质转化(EMT)的影响和机制。方法:体外培养HK-2细胞,10 ng/m L TGF-β1诱导HK-2细胞建立EMT细胞模型,Western blot检测E-cadherin、α-SMA表达,证明细胞EMT模型建立成功。Western blot检测在EMT细胞和正常细胞中转录因子Fox O3a的表达变化。敲低正常HK-2细胞中的Fox O3a后,Western blot检测细胞的EMT程度。Western blot检测EMT细胞与低表达Fox O3a细胞中β-Catenin的表达变化。结果:TGF-β1处理HK-2细胞作用后E-cadherin表达量显著减少(P<0.01),α-SMA蛋白表达量明显增加(P<0.05),证明EMT细胞模型建立成功。与正常HK-2细胞相比,EMT细胞中转录因子Fox O3a显著减少(P<0.01)。敲低HK-2细胞中的Fox O3a后,E-cadherin表达量显著减少(P<0.01),α-SMA蛋白表达量明显增加(P<0.05),出现EMT现象。相比于正常细胞,EMT细胞的β-Catenin表达增加(P<0.05);Fox O3a敲低的细胞中β-Catenin表达也显著增加(P<0.01)。结论:HK-2细胞通过降低转录因子Fox O3a的表达,从而上调β-Catenin,促进细胞上皮-间质转分化。

Objective：To explore the effect and mechanism of forkhead box protein O3 a knocking down on the epithelial-to-mesenchymal transition of HK-2 human renal proximal tubular cells.Method：HK-2 cells cultured in vitro,10 ng/m L TGF-β1 induced EMT of HK-2 cells,The expression levels of E-cadherin,α-SMA were determined by Western blot and immunofluorescence assay.The expression of Fox O3 a in EMT of HK-2 cells and HK-2 cells were determined by Western blot.Knocking down Fox O3 a in the HK-2 cells,the levels of EMT marker proteins were determined by Western blot.The expression level of β-catenin in the EMT of HK-2 cells and low expression Fox O3 a of the cells were determined by Western blot.Result：Compared with HK-2 cells incubated with free TGF-β1,the protein expression of E-cadherin significantly decreased（P〈0.01）,and the expression levels ofα-SMA markedly increased in HK-2 cells（P〈0.05）.The expression levels of Fox O3 a markedly diminished（P〈0.01）in HK-2 cells by the inducement of TGF-β1.In knocking down Fox O3 a of HK-2 cells,the protein expression of E-cadherin significantly decreased（P〈0.01）,and α-SMA markedly increased.The expression level of β-catenin in the EMT of HK-2 cells and low expression Fox O3 a of the cells significantly increased by Western blot.Conclusion：Down-regulation of Fox O3 a could promote EMT of human renal proximal tubular epithelial cells by increasing the expression levels of β-catenin.