Specific inhibition of Raf-1 gene expression in HNE1 by RNA interference.
- Author:
Li-Zhong LIU
1
;
Chuan-Yu LIANG
;
Yan-Ling DOU
;
Ling TIAN
;
Yu-Quang WEI
;
Yan-Jun WEN
;
Jiong LI
;
Hong-Xing DENG
;
Bing KAN
Author Information
- Publication Type:Journal Article
- MeSH: Apoptosis; genetics; Cell Line, Tumor; Gene Expression; Genetic Vectors; Humans; Nasopharyngeal Neoplasms; genetics; metabolism; pathology; Promoter Regions, Genetic; Proto-Oncogene Proteins c-raf; genetics; RNA Interference; RNA, Messenger; genetics; Transfection
- From: Chinese Journal of Otorhinolaryngology Head and Neck Surgery 2006;41(11):805-808
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo silence the expression of Raf-1 gene in HNE1 cells using vector-based RNA interference (RNAi) technique.
METHODSThe 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.
RESULTSVector-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.
CONCLUSIONRaf-1 expression in HNE1 cells can be inhibited significantly using plasmid-based RNAi.