[Inhibition of HCV IRES controlled reporter gene expression by RNA interference].
OBJECTIVE
To develop a RNAi approach that specifically targets the HCV IRES sequence by vector-expressed short hairpin RNA (shRNA) in vitro, and to assess the inhibitory effect of the shRNA on reporter gene expression.


METHODS
Eukaryotic expressing plasmids, pIRES-GFP and p5' UTR-Luc containing GFP or luciferase gene controlled by HCV IRES were cotransfected into HepG2 cells with either a RNAi plasmid pshRNA-HCV or a control plasmid pTZU6+1. At 24, 48, 72 hours post transfection, the fluorescence in the transfected cells was studied using fluorescence microscopy. The levels of GFP RNA were determined using RT-PCR and those of protein were determined using Western blot. The activities of luciferase were assayed using a dual luciferase assay system.


RESULTS
The introduction of RNAi plasmid efficiently and specifically down-regulated the expression of the reporter gene. RT-PCR showed that the RNAs of GFP gene were distinctly reduced (about 60%) when the pIRES-GFP was cotransfected with pshRNA-HCV, whereas the control vector did not exhibit inhibitory effect on the mRNA level, according to Western blot assay. The luciferase activity also decreased by 60%-70% in comparison to the control plasmid.


CONCLUSIONS
Our results demonstrate that the shRNA targeting HCV IRES shows a strong inhibitive effect on the expression of the reporter gene controlled by this sequence, suggesting that RNAi-based anti-HCV strategy may represent a potential approach in the therapy of HCV infection.
Chen
Wei-xian
WX
Key Laboratory of Infectious Diseases, Institute for Viral Hepatitis, Chongqing University of Medical Sciences, Chongqing, 400016, China.
Shan
Lin-na
LN
Chen
Juan
J
Zhang
Zhen-zhen
ZZ
Zhang
Bing-qiang
BQ
Huang
Ai-long
AL
D005786:Gene Expression Regulation; D017930:Genes, Reporter; D015316:Genetic Therapy; D005822:Genetic Vectors; D056945:Hep G2 Cells; D016174:Hepacivirus / Q000235:genetics*; D006526:Hepatitis C / Q000628:therapy; D006801:Humans; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics*; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D012333:RNA, Messenger