Construction of let-7a expression plasmid and its inhibitory effect on k-Ras protein in A549 lung cancer cells / 南方医科大学学报
Journal of Southern Medical University
;
(12): 2427-2431, 2010.
Article
in Chinese
| WPRIM
| ID: wpr-323644
ABSTRACT
<p><b>OBJECTIVE</b>To elucidate the role of let-7a-mediated gene regulation in the pathogenesis of lung cancer.</p><p><b>METHODS</b>Two 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>let-7a over-expression represses the expression of k-Ras protein and significantly inhibits the growth of lung cancer cells.</p>
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Pathology
/
Plasmids
/
RNA, Messenger
/
Transfection
/
Gene Expression Regulation, Neoplastic
/
Proto-Oncogene Proteins p21(ras)
/
MicroRNAs
/
Cell Line, Tumor
/
Genetic Vectors
/
Genetics
Limits:
Humans
Language:
Chinese
Journal:
Journal of Southern Medical University
Year:
2010
Type:
Article
Similar
MEDLINE
...
LILACS
LIS