Construction of overexpression lentiviral vector and its expression in lung cancer A549 cells of AMPactivated protein kinase

Abstract

Abstract:ObjectiveTo establish a stable lung cancer A549 cell line transfected by AMPactivated protein kinase (AMPK) expression vector, and to observe the effect of AMPK on proliferation as well as on the invasive ability of A549 cells. MethodsFulllength of AMPK gene was amplified and its target gene was digested, then inserted into the GV358 plasmid. Cotranfected 293T cells were subjected to the lentivirus equipment package. Subsequently, we collected the lentivirus supernatant to infect the A549 cells and establish a stably, overexpressed cell line A549. The mRNA and protein of AMPK were examined by realtime quantitative reverse transcriptase polymerase chain reaction (qRTPCR) and Western blotting. The proliferation and invasion abilities of A549 cells were detected by methyl thiazolyl thiazolium (MTT) and Transwell assay. ResultsGV358AMPK lentivirus vectors was successfully constructed by restrictive enzyme digestion and plasmid sequencing. There were significantly increased expressions of AMPK protein (5.87 times, P=0.002) and mRNA (16.12 times, P＜0.001) after transfected with GV358AMPK compared with the Vector group. Meanwhile, AMPK overexpression showed significantly lower proliferation (the forth day: 0.53±0.03 vs. 0.64±0.05, P=0.021; the fifth day: 0.58±0.04 vs. 0.80±0.07, P=0.002) and weaken invasive ability ［(1.6±0.5)×105 vs. (3.4±0.3)×105, P=0.004］ of A549 cells. ConclusionThe lentiviral AMPK expression vector and its A549 cell line is successfully constructed. AMPK overexpression inhibits the proliferation and invasive ability of A549 cells.

Key words:Carcinoma, nonsmallcell lung,Lentivirus,Cell proliferation,AMPactivated protein kinase

Zhang Xiangmin, Liu Lianbin, Zeng Wen, Zhou Maohua, Ye Guilin, Ye Yongqiang, Wang Gang, Li Shaojin. Construction of overexpression lentiviral vector and its expression in lung cancer A549 cells of AMPactivated protein kinase[J]. Journal of International Oncology, 2017, 44(10): 721-726.

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