实验研究
金亮
2019, 43(10): 0-0.
[摘要] 目的 探讨转录因子激活蛋白2E（Transcription factor activator protein 2E, TFAP2E）对人肝癌细胞系HepG2和HCC-LM3细胞增殖和迁移能力的影响及其可能的分子机制。方法 在人肝癌细胞系HepG2和HCC-LM3中使用慢病毒包装的方法转染入TFAP2E过表达的重组质粒，通过筛选得到稳定过表达TFAP2E的肝癌细胞系。通过转染小干扰RNA（Small interfering RNA，siRNA）抑制人肝癌细胞系HepG2和HCC-LM3中TFAP2E的表达。通过蛋白免疫印迹法（Western blotting，WB）在肝癌细胞系中检测TFAP2E蛋白的表达水平，以验证TFAP2E的过表达和敲低效果。通过Cell Counting Kit-8（CCK-8）法评价TFAP2E对肝癌细胞系增殖能力的影响。通过Transwell实验检测TFAP2E对肝癌细胞系迁移能力的影响。通过NetworkAnalyst方法鉴定TFAP2E高、低表达组间的差异表达基因；基于上述的显著差异表达基因，利用Enrichr软件进行通路富集分析，以提示TFAP2E在肝癌细胞中的作用机制。采用Log-rank检验比较TFAP2E的表达水平对肝癌患者生存期的影响。结果 过表达TFAP2E可显著促进HepG2和HCC-LM3细胞的增殖和迁移能力，而敲低TFAP2E可显著抑制HepG2和HCC-LM3细胞的增殖和迁移能力。生物信息学分析提示TFAP2E可能参与TGF-β信号通路的调控。随后的实验证实，在HepG2细胞系中过表达TFAP2E可显著上调SMAD2的磷酸化水平，而敲低TFAP2E则显著下调SMAD2的磷酸化水平。临床相关性分析显示，TFAP2E在肝癌组织中的表达显著高于癌旁组织，且TFAP2E的高表达与肝癌患者的不良预后显著相关。结论 TFAP2E可能通过促进TGF-β信号通路的激活，促进肝癌细胞的增殖和迁移，从而发挥癌基因的功能。
[关键词] 肝癌；TFAP2E；细胞增殖；细胞迁移；TGF-β信号通路
[Abstract] Objective To assess the effects of transcription factor activator protein 2E (TFAP2E) on cells proliferation and migration of hepatocellular carcinoma (HCC) cell lines HepG2 and HCC-LM3, and the potential underlying molecular mechanism. Methods The TFAP2E overexpression recombinant plasmid was transfected into the HepG2 and HCC-LM3 cells, respectively, by lentivirus packaging. The small interfering RNA (siRNA) was used to knock-down the endogenous TFAP2E in HepG2 and HCC-LM3 cells. Western blotting assay was used to detect the protein levels of TFAP2E in HCC cells. Cell Counting Kit-8 (CCK-8) and Transwell assays were used to assess the abilities of HCC cells proliferation and migration, respectively. NetworkAnalyst was used to evaluate the differentially expressed genes between the high and low TFAP2E expression groups, and pathway enrichment analysis was performed by Enrichr to suggest the molecular mechanisms of TFAP2E in HCC cells. Log-rank test was used to assess the overall survival difference between the HCC patients with high and low expression levels of TFAP2E. Results Overexpression of TFAP2E could significantly promote the HepG2 and HCC-LM3 cells proliferation and migration; whereas knockdown of TFAP2E significantly inhibits the HepG2 and HCC-LM3 cells proliferation and migration. Bioinformatics analysis revealed that TFAP2E might be involved in the TGF-β signaling pathway. Consistently, overexpression of TFAP2E induced the p-SMAD2 levels in HepG2 cells; whereas knockdown of TFAP2E led to the downregulation of p-SMAD2 levels. Of note, TFAP2E was significantly upregulated in HCC tissues compared to the non-tumor liver tissues, and the higher expression levels of TFAP2E were significantly associated with poor outcomes of HCC patients. Conclusion TFAP2E might play oncogenic roles in HCC cells, by activating the TGF-β signaling pathway.
[Key words] Hepatocellular carcinoma, TFAP2E, Proliferation, Migration, TGF-β pathway.
