沉默轴突导向蛋白4D对胃癌细胞SGC-7901生长、自噬及上皮-间质转化的影响

Effects of silencing Sema4D on the growth,autophagy and epithelial-mesenchymal transformation of gastric cancer cells SGC-7901

目的探讨沉默轴突导向蛋白4D(Sema4D)对胃癌细胞SGC-7901生长、自噬及上皮-间质转化的影响。方法将SGC-7901细胞随机分为对照组、shRNA-NC组、Sema4D-shRNA1组、Sema4D-shRNA2组与Sema4D-shRNA3组,构建shRNA Sema4D载体转染至SGC-7901细胞。采用RT-PCR检测各组Sema4D mRNA表达水平;采用克隆形成实验检测细胞克隆形成率;流式细胞仪检测细胞凋亡情况;显微镜观察上皮-间质转化形态学变化;Western blotting检测Ki-67、增殖细胞核抗原(PCNA)、Bax、Bcl-2、caspase-3、cleaved caspase-3、Beclin1、p62、LC3Ⅰ、LC3Ⅱ、E-cadherin、N-cadherin、波形蛋白表达水平。结果 RT-PCR检测结果显示,与对照组比较,Sema4D-shRNA1组、Sema4D-shRNA2组和Sema4D-shRNA3组Sema4D mRNA表达水平均明显降低,且Sema4D-shRNA3组明显低于Sema4D-shRNA1组和Sema4D-shRNA2组(P<0.05),选择Sema4D-shRNA3进行后续试验。克隆形成实验结果显示,与对照组比较,Sema4D-shRNA3组克隆形成率明显降低(P<0.05)。流式细胞仪检测结果显示,与对照组比较,Sema4D-shRNA3组细胞凋亡率明显升高(P<0.05)。Western blotting检测结果显示,与对照组比较,Sema4D-shRNA3组Ki-67、PCNA、p62、N-cadherin、波形蛋白表达水平明显降低,Beclin1、E-cadherin蛋白表达水平明显升高,Bax/Bcl-2、cleaved caspase-3/caspase-3、LC3Ⅱ/LC3Ⅰ比值明显升高(P<0.05)。显微镜观察可见Sema4D-shRNA3组细胞上皮-间质转化受到抑制。结论 shRNA Sema4D可抑制SGC-7901细胞的增殖、自噬、上皮-间质转化,促进其凋亡。

Objective To investigate the effects of silencing axon guide protein 4D (Sema4D) on the growth,autophagy and epithelial-mesenchymal transformation of gastric cancer cell SGC-7901.Methods The cells were randomly divided into control group,shRNA-NC group,Sema4D-shRNA1 group,Sema4D-shRNA2 group and Sema4D-shRNA3 group,and transfected into SGC-7901 with shRNA Sema4D vector.The expression of Sema4D mRNA was detected by RT-PCR.The clone formation rate was determined by colony formation assay.Flow cytometry was used to detect apoptosis.Morphological changes of epithelial-mesenchymal transformation were observed microscopically.Western blotting was used to detect the expression levels of Ki67,proliferating cell nuclear antigen (PCNA),Bax,Bcl-2,caspase-3,cleaved caspase-3,Beclin1,p62,LC3Ⅰ,LC3Ⅱ,E-cadherin,N-cadherin,and vimentin proteins.Results The results of RT-PCR showed that compared with the control group,the mRNA levels of Sema4D-shRNA1 group,Sema4D-shRNA2 group and Sema4D-shRNA3 group were significantly lower,and the mRNA level of Sema4D-shRNA3 group was significantly lower than that of Sema4D-shRNA1 group and Sema4D-shRNA2 group (P<0.05).Sema4D-shRNA3 was selected for subsequent experiments.The results of colony forming assay showed that the colony forming efficiency of Sema4D-shRNA3 group was significantly lower than that of the control group (P<0.05).Flow cytometry results showed that compared with the control group,the apoptosis rate of Sema4D-shRNA3 group was significantly increased (P<0.05).Western blotting results showed that compared with the control group,the expression levels of Ki67,PCNA,p62,N-cadherin and vimentin proteins in the Sema4D-shRNA3 group were significantly lower,Beclin1 and E-cadherin proteins were significantly higher,and the ratios of Bax/Bcl-2,cleaved caspase-3/caspase-3 and LC3Ⅱ/LC3Ⅰ were significantly higher (P<0.05).Microscopically,the epithelial-mesenchymal transformation was inhibited in the Sema4D-shRNA3 group.Conclusion shRNA Sema4D can inhibit proliferation,autophagy and epithelial-mesenchymal transformation of SGC-7901 cells and promote apoptosis.