[Specific inhibition of Raf-1 gene expression in HNE1 by RNA interference].
OBJECTIVE
To silence the expression of Raf-1 gene in HNE1 cells using vector-based RNA interference (RNAi) technique.


METHODS
The vector containing the human U6 promoter was used for targeted gene silencing when a dsDNA oligonucleotide encoding an appropriate shRNA was ligated into the vector, and 67nt oligonucleotide fragment was inserted into the downstream of the U6 promoter. Plasmids containing different Raf-1 target sequences [ (1) pshuttle-Raf-1-a( 225), (2) pshattle-Raf-1-b ( 358) and (3) pshuttle-Raf-1-c(474)], were transfected into HNE1 cells. Expression of Raf-1 mRNA was assayed by RT-PCR. Apoptosis were determined by cytometry.


RESULTS
Vector-based RNAi had advantages over antisense RNA because it could be delivered to the target cell more efficiently, and effect could last longer. Raf-1 expression could be inhibited by plasmid-expressed shRNA. Three different targeting sequences were selected from Raf-1 gene, and the inhibitory effect of pSIREN shuttle-Raf-1-b (358) was biggest.


CONCLUSIONS
Raf-1 expression in HNE1 cells can be inhibited significantly using plasmid-based RNAi.
Liu
Li-Zhong
LZ
Department of Otorhinolaryngology, West China Hospital, Sichuan University, Chengdu 610041, China.
Liang
Chuan-Yu
CY
Dou
Yan-Ling
YL
Tian
Ling
L
Wei
Yu-Quang
YQ
Wen
Yan-Jun
YJ
Li
Jiong
J
Deng
Hong-Xing
HX
Kan
Bing
B
D017209:Apoptosis / Q000235:genetics*; D045744:Cell Line, Tumor; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D009303:Nasopharyngeal Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D011401:Promoter Regions, Genetic; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics*; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D014162:Transfection
D012333:RNA, Messenger; D019908:Proto-Oncogene Proteins c-raf