[Study of growth inhibition of lung cancer cells by siRNA targeting mutant K-ras gene in vitro and in vivo].
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Zhang
Zhi-ping
ZP
Department of Thoracic Surgery, Peking University People's Hospital, Beijing 100044, China.
Jiang
Guan-chao
GC
Yang
Fan
F
Zhou
Zu-li
ZL
Wang
Jun
J
D000230:Adenocarcinoma / Q000235:genetics / Q000473:pathology* / Q000628:therapy; D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D020868:Gene Silencing; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics / Q000473:pathology* / Q000628:therapy; D051379:Mice; D008819:Mice, Nude; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D023041:Xenograft Model Antitumor Assays; D018631:ras Proteins / Q000235:genetics* / Q000378:metabolism
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D018631:ras Proteins