Construction of let-7a expression plasmid and its inhibitory effect on k-Ras protein in A549 lung cancer cells.
- Author:
Xiao-yan HE
1
;
Jun-xia CHEN
;
Xi OU-YANG
;
Zheng ZHANG
;
Hui-min PENG
Author Information
- Publication Type:Journal Article
- MeSH: Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Genetic Vectors; Humans; Lung Neoplasms; metabolism; pathology; MicroRNAs; genetics; metabolism; Plasmids; Proto-Oncogene Proteins p21(ras); genetics; metabolism; RNA, Messenger; genetics; Transfection
- From: Journal of Southern Medical University 2010;30(11):2427-2431
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo elucidate the role of let-7a-mediated gene regulation in the pathogenesis of lung cancer.
METHODSTwo template DNA sequences were designed based on hsa-let-7a sequence in miRBase database. The let-7a expression construct and a control plasmid, namely pGenesil-let-7a and pGenesil-control, respectively, were generated by cloning the annealed oligonucleotides into pGenesil-1 and then transfected into A549 cells, which were selected by G418 to establish the lung cancer cell lines stably expressing let-7a-GFP and control-GFP. The living cells were counted by MTT assay and cell growth curves were drawn to analyze the cell proliferation. The k-Ras mRNA level was assessed by semi-quantitative RT-PCR, and the expression of k-Ras protein was determined by Western blotting and immunocytochemistry.
RESULTSThe recombinant vectors were verified by sequencing. The cell growth curves indicated that the proliferation of the cells transfected with pGenesil- let-7a were inhibited significantly compared with that of cells transfected with pGenesil-control and A549 cells. Semi- quantitative RT-PCR analysis showed that the levels of k-Ras mRNA almost remained unchanged in cells with or without the treatments. Western blotting and immunocytochemistry demonstrated a significant decrease of k-Ras protein levels in cells transfected with pGenesil-let-7a, but not in cells transfected with pGenesil-control, when compared to A549 cells.
CONCLUSIONlet-7a over-expression represses the expression of k-Ras protein and significantly inhibits the growth of lung cancer cells.