Study of growth inhibition of lung cancer cells by siRNA targeting mutant K-ras gene in vitro and in vivo.
- Author:
Zhi-ping ZHANG
1
;
Guan-chao JIANG
;
Fan YANG
;
Zu-li ZHOU
;
Jun WANG
Author Information
- Publication Type:Journal Article
- MeSH: Adenocarcinoma; genetics; pathology; therapy; Animals; Apoptosis; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Gene Silencing; Genetic Therapy; Genetic Vectors; Humans; Lung Neoplasms; genetics; pathology; therapy; Mice; Mice, Nude; Plasmids; genetics; Point Mutation; RNA, Messenger; genetics; metabolism; RNA, Small Interfering; genetics; Reverse Transcriptase Polymerase Chain Reaction; Transfection; Xenograft Model Antitumor Assays; ras Proteins; genetics; metabolism
- From: Chinese Journal of Surgery 2007;45(18):1267-1270
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo 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.
METHODSOne 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.
RESULTSThe 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.
CONCLUSIONSsiRNA 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.