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

Objective To 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. Methods Full-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. Results GV358-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)×105vs. (3.4±0.3)×105,P=0.004] of A549 cells. Conclusion The 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