携带人MST4或人dnMST4转基因的肺癌细胞株构建

Construction of Non-Small Cell Lung Cancer (NSCLC) Cell Lines Stably Overexpressing MST4 or dnMST4 Transgenes

摘要: 目的：利用慢病毒感染分别建立稳定过表达MST4和dnMST4的人肺癌细胞株。方法：分别以pcDNA3.1-MST4、pcDNA3.1-MST4 T178A质粒为模板，PCR分别扩增MST4、MST4 T178A序列(均为1.251 kb)，片段两侧分别添加EcoR I/BamH I和Xba I/BamH I酶切位点，分别In-Fusion克隆至pCDH-RFP和pHEZG中，次日挑选单克隆菌落，提取质粒并进行测序和酶切鉴定；所构建的载体分别命名为pCDH-MST4-RFP和pHEZG-dnMST4。按标准程序进行慢病毒包装，并确认是否成功生产慢病毒；用携带MST4和RFP基因的慢病毒分别感染人肺癌细胞A549和HCC827，用携带dnMST4和GFP基因的慢病毒分别感染人肺癌细胞A549和HCC827，以建立稳定过表达MST4和dnMST4的人肺癌细胞系。应用qRT-PCR和Westernblot检测所建立肺癌细胞系中MST4和dnMST4在RNA层面和蛋白层面的表达水平。结果：测序和酶切鉴定的结果均证实成功构建了pCDH-MST4-RFP和pHEZG-dnMST4，按标准程序生产的携带MST4和RFP基因以及携带dnMST4和GFP基因的慢病毒均能成功感染人肺癌细胞。结论：成功构建稳定过表达MST4或dnMST4的人肺癌细胞株，为进一步研究MST4在肺癌恶性进程中的生物学功能及机制打下了良好基础。

Abstract: Objective: To establish non-small cell lung cancer (NSCLC) cell lines stably overexpressing MST4 and dnMST4 Transgenes by lentivirus infection, respectively. Methods: The fragment of human MST4 gene was amplified by PCR from the template of pcDNA3.1-MST4, and subsequently cloned into the plasmid of pCDH-RFP to obtain the final vector of pCDH-MST4-RFP, which was used to produce lenti-viruses carrying MST4 and RFP genes. The fragment of human dnMST4 gene was also amplified by PCR from the template of pcDNA3.1-MST4 T178A, and subsequently cloned into the plasmid of pHEZG to obtain the final vector of pHEZG-dnMST4, which was used to produce lentiviruses carrying dnMST4 and GFP genes. To establish human NSCLC cell lines stably overexpressing MST4 or dnMST4, lentiviruses harboring MST4 and RFP genes were employed to infect A549 and HCC827 cells, and lentiviruses harboring dnMST4 and GFP genes were also employed to infect A549 and HCC827 cells. The expression levels of MST4 and dnMST4 in the infected lung cancer cell lines at RNA and protein levels were detected by Western blot and qRT-PCR. Results: The lentivirus vector of pCDH-MST4-RFP and pHEZG-dnMST4 were successfully constructed. NSCLC cell lines A549 and HCC827 harboring MST4 and RFP transgenes were generated showing the expression of both MST4 and RFP transgenes. And the cell lines harboring dnMST4 and GFP transgenes were also generated showing the expression of both dnMST4 and GFP transgenes. Conclusion: NSCLC cell lines A549 and HCC827 stably expressing MST4 and dnMST4 transgenes were generated, which had laid a great foundation for further research on the biological function and mechanism of MST4 in the malignant process of lung cancer.