YAP promotes the migration and invasion of hepatocellular carcinoma cells through regulating the expression of EEF1A2
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摘要:
目的：研究Yes 相关蛋白(yes-associated protein，YAP)通过调控真核细胞翻译延伸因子1A2(eukaryotic translation elongation factor 1 alpha 2，EEF1A2)的表达促进肝癌细胞迁移和侵袭的机制。方法：通过建立YAP和EEF1A2干扰表达或过表达的SMMC-7721和SK-HEP1肝癌细胞模型，采用Transwell实验检测细胞迁移和侵袭能力的改变；应用反转录和实时定量PCR (reverse transcription-quantitative real-time polymerase chain reaction，RT-qPCR)和Western blot法检测相关基因的mRNA和蛋白表达水平；利用染色质靶向酶切检测(cleavage under targets and release using nuclease，CUT&RUN)-qPCR实验检测YAP能否与 EEF1A2基因的启动子序列结合。结果：EEF1A2在肝癌组织中高表达，并与肝癌细胞的迁移能力呈正相关；EEF1A2可以促进低迁移能力的SMMC-7721细胞和高迁移能力的SK-HEP1细胞迁移和侵袭，调控细胞中上皮间质转化(epithelial-mesenchymal transition，EMT)相关标志物 Snail 和 E - cadherin 的表达；YAP 可以上调 EEF1A2 的表达而促进肝癌细胞的迁移和侵袭； CUT&RUN-qPCR实验结果显示YAP可以与EEF1A2基因的启动子序列结合。结论：YAP可以通过与EEF1A2基因的启动子序列结合，促进EEF1A2的表达，进一步促进肝癌细胞的迁移和侵袭。
Abstract:
Objective：To investigate the mechanism of yes - associated protein(YAP)in promoting the migration and invasion of hepatocellular carcinoma cells through regulating the expression of eukaryotic translation elongation factor 1 alpha 2(EEF1A2). Methods：YAP and EEF1A2 were knockdown or overexpressed in human hepatocellular carcinoma(HCC)cell lines(SMMC-7721 and SK - HEP1)respectively. Transwell assays were performed to examine the changes of the migratory and invasive ability of the HCC cells. Reverse transcription-quantitative real-time polymerase chain reaction(RT-qPCR)and Western blot were conducted to evaluate the expression of the indicated genes in HCC cells. Cleavage under targets and release using nuclease(CUT&RUN)-qPCR assay was performed to evaluate whether YAP interacts with the promoter of EEF1A2. Results：Upregulation of EEF1A2 was shown in HCC tissues，and the expression of YAP and EEF1A2 was positively correlated with the spontaneous migratory potential of HCC cell lines. EEF1A2 facilitated the migration and invasion of the low migratory potential SMMC7721 cells and the high migratory potential SK-HEP1 cells and regulated the expression of Snail and E-cadherin，which are two well-characterized epithelial-mesenchymal transition(EMT) markers. The results further showed that YAP regulated HCC cell migration and invasion by up - regulating EEF1A2. In addition， CUT&RUN-qPCR analysis demonstrated YAP occupancy at the promoter of the EEF1A2 in HCC cells. Conclusion：YAP up-regulates the expression of EEF1A2 through interacting with the sequence on the promoter of the EEF1A2，and subsequently promotes HCC cell migration and invasion.
