VEPH1介导TGF-β信号通路调控黑色素瘤细胞EMT和增殖的研究

The research of VEPH1 regulates epithelial mesenchymal transition and proliferation of melanoma cells through the TGF-βsignaling pathway

目的探讨VEPH1通过TGF-β信号通路对人皮肤黑色素瘤(CM)细胞的上皮-间质转化(EMT)和增殖的调控作用。方法体外培养黑色素瘤细胞,将黑色素瘤细胞分别体外转染VEPH1、TGF-β过表达或干扰质粒,或经TGF-β通路抑制剂SB-431542处理后,运用实时荧光定量PCR(qRT-PCR)和蛋白免疫印迹法(Western blot)分别检测VEPH1、TGF-β、EMT等相关基因及蛋白表达,以及对CM细胞EMT不同蛋白表达和增殖的影响。结果qRT-PCR结果显示,VEPH1在人皮肤黑色素瘤B16-BL6、B16和A375细胞中的表达明显低于正常人皮肤细胞(HaCaT),在A375细胞中表达最低,故选用A375细胞进行后续实验。转染VEPH1过表达质粒或SB-431542处理后,A375细胞中E-钙黏蛋白的mRNA和蛋白表达量显著上升(P<0.05),TGF-β、Smad4、N-钙黏蛋白和波形蛋白的mRNA和蛋白表达量均显著降低(P<0.05),细胞增殖显著降低。与VEPH1载体组相比,VEPH1载体+SB-431542组TGF-β、Smad4、N-钙黏蛋白和波形蛋白的mRNA和蛋白表达量均显著降低(P<0.05),E-钙黏蛋白表达升高,细胞增殖亦显著降低(P<0.05);转染TGF-β过表达质粒或si-VEPH1质粒后,A375细胞TGF-β、Smad4、波形蛋白和N-钙黏蛋白的mRNA和蛋白表达增加,E-钙黏蛋白的表达减少,细胞增殖明显增强,VEPH1载体与TGF-β载体共转染后,TGF-β、Smad4、N-钙黏蛋白和波形蛋白表达增强,E-钙黏蛋白表达下降,细胞增殖亦明显增强;转染si-VEPH1质粒后A375细胞VEPH1表达量明显下降,而SB-431542组及TGF-β载体组VEPH1表达量无明显下降趋势。结论VEPH1可以通过抑制TGF-β信号通路来抑制人类CM,揭示了其作为CM诊断、预后指标及治疗靶点的潜力。

Objective To investigate the intervention effect of ventricular zone expressed PH domain-containing 1(VEPH1)on epithelial mesenchymal transition(EMT)and proliferation of human cutaneous melanoma(CM)cells based onthe transforming growth factor-β(TGF-β)signaling pathway.Methods The melanoma cells were cultured in vitro.After transfecting the melanoma cells with overexpression or interference plasmids of VEPH1 or TGF-βoverexpression plasmids,or treating the cells with SB-431542(TGF-βpathway inhibitor),we detected the expression of genes and proteins relevant to VEPH1,TGF-β,and EMT by quantitative reverse transcription polymerase chain reaction(qRT-PCR)and Western blot to observe the effect of these proteins on CM cell proliferation.Results qRT-PCR results showed that the expression of VEPH1 in melanoma cells(B16-BL6,B16 and A375 cells)was significantly lower than that in HaCaT cells,and the lowest expression was found in A375 cells,so A375 cells were selected for follow-up experiments.After transfection with VEPH1 overexpression plasmid or SB-431542,the mRNA and protein expression of E-cadherin in A375 cells were significantly increased,the mRNA and protein expressions of TGF-β,Smad4,N-cadherin and vimentin were significantly decreased,and the cell proliferation was significantly decreased(P<0.05).Compared with the VEPH1 vector group,the expression of TGF-β,Smad4 and N-cadherin in the VEPH1 vector+SB-431542 group was significantly reduced(P<0.05);the expression of E-cadherin was increased,and the cell proliferation was also significantly decreased(P<0.05).The expression of TGF-β,Smad4,N-cadherin and vimentin were increased after co-transfection with VEPH1 vector,while the expression of E-cadherin was decreased,and the cell proliferation was also enhanced(P<0.05).The expression of VEPH1 in A375 cells was significantly decreased after transfection with si-VEPH1 plasmid,while that in SB-431542 and TGF-βvector group was not significantly decreased.Conclusions VEPH1 can inhibit human CM cells by the intervention on TGF-βsignaling pathway.This study reveals the potential of VEPH1 as a diagnostic,prognostic and therapeutic target for CM.