建立稳定敲减MAP4K4表达的A549细胞系及初步分析

Establishment and Preliminary Analysis of Lung Cancer Cell Line A549 with Stable MAP4K4 Knockdown

目的分析敲减MAP4K4表达对癌细胞增殖及迁移运动的影响,并探究潜在的分子机理。方法构建稳定敲减MAP4K4表达A549细胞,并综合免疫荧光、细胞增殖与迁移运动等实验方法,检测其亚细胞定位,并分析对照组与敲减组细胞增长及迁移运动变化情况。结果A549细胞中MAP4K4定位于黏着斑及细胞边缘部位,稳定敲减MAP4K4表达诱导癌细胞成簇生长,阻滞细胞周期进程及细胞迁移运动。进一步分析发现,敲减MAP4K4表达诱导E-cadherin积累而下调N-cadherin,扰乱“钙黏蛋白转换”及上皮-间质转换。最终,对照组与敲减组细胞分别联合顺铂(终浓度为5μmol/L)及紫杉醇(终浓度为20 nmol/L)处理,稳定敲减MAP4K4表达可明显增强化疗药物对癌细胞的杀伤效果。结论A549细胞中MAP4K4可通过调控“钙黏蛋白转换”促上皮-间质转化而调控癌细胞恶性表型及化疗耐药。

Objective To analyze the effect of knocking down MAP4K4 expression on the proliferation and migration of cancer cells,and to explore its underlining molecular mechanisms.Methods A stable knockdown MAP4K4cell line was constructed and the subcellular localization of the cells was determined with immunofluorescence,cell proliferation assay and cell migration assay.In addition,the effects of down-regulated MAP4K4 expression were analyzed by examining the difference between the proliferation and migration of cancer cells in the knockdown group and those of the control group.Results MAP4K4 was localized in focal adhesion and cell edges in A549 cells.Stable knockdown of MAP4K4 expression induced cancer cells to grow in clusters and arrested the progression of the cell cycle and cell migration.Further analysis found that knocking down MAP4K4 expression in A549 cells induced the accumulation of epithelial cell marker E-cadherin,and subsequently,the down-regulation of N-cadherin,a mesenchymal cell marker,thereby disrupting the"cadherin switch"and the epithelial-mesenchymal conversion.Then,the control group and the knockdown group both received the combined treatment of cisplatin at a final concentration of 5μmol/L and paclitaxel at a final concentration of 20 nmol/L.The stably knocked down MAP4K4 expressing cells showed significantly enhanced toxicity of chemotherapeutic drugs to cancer cells.Conclusion The study shows that MAP4K4 regulates the malignant phenotypes of cancer cells and chemoresistance by regulating"cadherin switch"to promote epithelial-mesenchymal transition in A549 cells.