title: [Inhibition of HCV IRES controlled reporter gene expression by RNA interference].
abstract: 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.
authors.affiliation: Key Laboratory of Infectious Diseases, Institute for Viral Hepatitis, Chongqing University of Medical Sciences, Chongqing, 400016, China.
authors.forename: Wei-xian
authors.initials: WX
authors.lastname: Chen
authors.forename: Lin-na
authors.initials: LN
authors.lastname: Shan
authors.forename: Juan
authors.initials: J
authors.lastname: Chen
authors.forename: Zhen-zhen
authors.initials: ZZ
authors.lastname: Zhang
authors.forename: Bing-qiang
authors.initials: BQ
authors.lastname: Zhang
authors.forename: Ai-long
authors.initials: AL
authors.lastname: Huang
mesh_terms: 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
chemical_list: D012333:RNA, Messenger