MAGE-A3基因对宫颈癌细胞增殖及侵袭的影响及其机制探讨

Effects of MAGE-A 3 gene on proliferation and migration of human cervical cancer cells and its possible mechanism

目的:研究黑素瘤相关抗原A3(melanoma-associated antigen-A3,MAGE-A 3)基因对宫颈癌细胞增殖及侵袭能力的影响,并探讨其可能的作用机制。方法:首先采用实时荧光定量PCR及蛋白质印迹法检测宫颈癌HeLa和Siha细胞中MAGE-A3 mRNA和蛋白的表达水平。再分别用携带有MAGE-A3基因的慢病毒(LV-MAGE-A3)感染Siha细胞,构建过表达MAGE-A3基因的细胞株(Siha-MAGE-A3细胞);用携带有靶向MAGE-A3基因的siRNA(siRNA-MAGE-A3)转染HeLa细胞,构建沉默MAGE-A3基因表达的细胞株(HeLa-siRNA-MAGE-A3细胞);实时荧光定量PCR及蛋白质印迹法检测转染效率。采用细胞克隆形成实验和CCK-8法检测过表达或沉默MAGE-A 3基因表达对HeLa和Siha细胞克隆形成和增殖能力的影响;Transwell侵袭实验和细胞划痕愈合实验检测过表达或沉默MAGE-A3基因表达对HeLa和Siha细胞侵袭和迁移能力的影响;实时荧光定量PCR及蛋白质印迹法检测过表达或沉默MAGE-A3基因对宫颈癌细胞上皮-间质转化(epithelial-mesenchymal transition,EMT)标志物E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)和波形蛋白(vimentin)表达的影响;蛋白质印迹法检测磷脂酰肌醇3-激酶(phosphatidylinositol-3-kinase,PI3K)/蛋白激酶B(protein kinase B,PBK,又称AKT)通路关键分子AKT及磷酸化AKT(phospho-AKT,p-AKT)表达水平的变化。结果:MAGE-A3基因在HeLa细胞中的表达被沉默,在Siha细胞中被成功过表达。与对照组和阴性对照组相比,MAGE-A3基因过表达Siha-MAGE-A3细胞的增殖、迁移和侵袭能力明显增强(P值均<0.01),E-cadherin蛋白的表达水平下调(P<0.05),N-cadherin、vimentin和p-AKT蛋白的表达水平上调(P值均<0.01);与此相反,MAGE-A3基因沉默后,HeLa-siRNA-MAGE-A3细胞的增殖、迁移和侵袭能力受到明显抑制(P值均<0.01),E-cadherin蛋白的表达水平上调(P<0.05),N-cadherin、vimentin和p-AKT蛋白的表达水平下调(P值均<0.01)。结论:过表达MAGE-A3基因可以促进宫颈癌细胞的增殖、迁移和侵袭能力,沉默MAGE-A3基因可抑制宫颈癌细胞的增殖、迁移和侵袭;其机制可能涉及EMT和PI3K-AKT信号通路的激活。

Objective:To investigate the effects of melanoma-associated antigen-A3(MAGE-A3)gene on the proliferation and invasion of human cervical cancer cells,and to explore its possible mechanism.Methods:The expression levels of MAGE-A3 mRNA and protein in human cervical cancer HeLa and Siha cells were detected by real-time fluorescent quantitative PCR and Western blotting,respectively.Siha cells were infected with the recombinant lentivirus carrying MAGE-A3 gene(LV-MAGE-A3)to construct the MAGE-A3 gene overexpressed cells.HeLa cells were transfected with the specific siRNA targeting MAGE-A3 gene(siRNA-MAGE-A3)to establish the MAGE-A3 gene silenced cells.The infection or transfection efficiency was detected by real-time fluorescent quantitative PCR and Western blotting.The effects of overexpressing or silencing MAGE-A3 on the colony-forming and proliferation abilities of cervical cancer HeLa and Siha cells were investigated by cell counting kit-8(CCK-8)and colony-forming assay,respectively.The migration and invasive abilities of HeLa and Siha cells were measured by wound healing assay and Transwell chamber assay,respectively.The expression levels of epithelial-mesenchymal transition(EMT)markers including E-cadherin,N-cadherin and vimentin in the transfected or infected cells were detected by real-time fluorescent quantitative PCR and Western blotting,respectively.The expression levels of phosphatidylinositol 3-kinase(PI3K)/protein kinase B(PKB,AKT)signaling pathway key molecules AKT and phospho-AKT(p-AKT)in HeLa and Siha cells were detected by Western blotting.Results:The MAGE-A 3 gene were silenced in HeLa cells transfected with siRNA-MAGEA3,and successfully overexpressed in Siha cells infected with LV-MAGEA3.Compared with the control groups,the proliferation,migration and invasion abilities of Siha cells infected with LV-MAGEA3 were significantly enhanced(all P<0.01).The expression levels of N-cadherin,vimentin,and p-AKT were significantly up-regulated(all P<0.01)in LV-MAGEA3 infected Siha cells,while the expression of E-cadherin was down-regulated(P<0.05).In contrast,after the transfection with siRNA-MAGEA3,the proliferation,migration and invasion abilities of HeLa cells were markedly inhibited(all P<0.01),and the expression levels of N-cadherin,vimentin,and p-AKT were down-regulated(all P<0.01),while the expression of E-cadherin was up-regulated(P<0.05).Conclusion:Over-expression of MAGE-A 3 gene enhances the proliferation,migration and invasion of human cervical cancer cells in vitro,while silencing MAGE-A 3 gene has the opposite effects.The mechanism may involve the activation of EMT and PI3K/AKT signaling pathway.