Study of growth inhibition of lung cancer cells by siRNA targeting mutant K-ras gene in vitro and in vivo / 中华外科杂志

ABSTRACT

<p><b>OBJECTIVE</b>To study the inhibitory effect of mutant K-ras gene depletion by small interfering RNA on the growth of lung cancer cell line-H441 cells in vitro and in vivo.</p><p><b>METHODS</b>One pair of 63 bp reverse repeated sequence targeting mutant K-ras(V12) mRNA spaced by 9 bp nucleotide were synthesized and inserted into plasmid pSilencer3.1 eukaryotic expression vector. After transient and stable transfection into H441 cells, the mutant K-ras mRNA and protein level were measured by RT-PCR and Western blotting, and the H441 cells proliferation was measured by MTT method, and the apoptosis rate was detected by flow-cytometry. H441 cells treated with pSilencer3.1-K-ras(V12) were transplanted subcutaneously in nude mice and their tumorigenesis ability was observed.</p><p><b>RESULTS</b>The recombinant plasmid pSilencer3.1-K-ras(V12) was successfully constructed by sequencing. The introduction of pSilencer3.1-K-ras(V12) was showed to efficiently and specifically inhibit the expression of K-ras(V12) gene according to the results of RT-PCR and Western blotting (P < 0.01, as compared with controls). The inhibitory effect on cell proliferation was confirmed by MTT test (P < 0.05, as compared with controls). Apoptosis rate of H441 cells treated with pSilencer3.1-K-ras(V12) was significantly higher than that of the control cells (P < 0.01). The test in vivo showed that downregulation of K-ras(V12) expression in H441 cells apparently affected their ability to form tumors in nude mice.</p><p><b>CONCLUSIONS</b>siRNA targeting mutant K-ras mRNA can specifically suppress the expression of mutant K-ras gene in H441 cells, and therefore has a substantially inhibitory effect on cell proliferation in vitro and in vivo, it provides a new method and material to the gene therapy of lung cancer.</p>