Construction of overexpression lentiviral vector and its expression in lung cancer A549 cells of AMP-activated protein kinase
10.3760/cma.j.issn.1673-422X.2017.10.001
- VernacularTitle:AMPK过表达慢病毒载体的构建及稳定转染肺癌A549细胞株的建立
- Author:
Xiangmin ZHANG
1
,
2
;
Lianbin LIU
;
Wen ZENG
;
Maohua ZHOU
;
Guilin YE
;
Yongqiang YE
;
Gang WANG
;
Shaojin LI
Author Information
1. 341000赣州,赣南医学院第一临床医学院
2. 赣南医学院附属肿瘤医院肿瘤防治研究所
- Keywords:
Carcinoma,non-small-cell lung;
Lentivirus;
Cell proliferation;
AMP-activated protein kinase
- From:
Journal of International Oncology
2017;44(10):721-726
- CountryChina
- Language:Chinese
-
Abstract:
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) ×l05 vs.(3.4±0.3) ×105,P=0.004] ofA549 cells.Conclusion The lentiviralAMPK expression vector and its A549 cell line is successfully constructed.AMPK overexpression inhibits the proliferation and invasive ability of A549 cells.
