AIM:To investigate the inhibitory effect of hepatitis C virusinternal ribosome entry site (HCV IRES) specific inhibitor RNA(IRNA) on gene expression mediated by HCV IRES in vivo.METHODS:By using G418 screening system,hepatomacells constitutively expressing IRNA or mutant IRNA (mIRNA)were established and characterized,and HCV repliconscontaining the 5’ untranslated region (5’UTR) wereconstructed by using the same method.Cotransfection ofpCMVNCRIuc containing HCV 5’UTR-Iuc fusion genes andeukaryotic vector of IRNA into human hepatic carcinomacells (HepG2) was performed and the eukaryotic expressionplasmid of IRNA was transfected transiently into HCVreplicons,pCMVNCRIuc or pCDNA-Iuc was cotransfected withpSV40-β Gal into IRNA expressing hepatoma cells by usinglipofectamine 2000 in vitro.Then the reporting geneexpression level was examined at 48 h after transfection byusing a luminometer and the expressing level of HCV Cantigen was analysed with a confocal microscope.RESULTS:Transient expression of IRES specific IRNA couldsignificantly inhibit the expression of reporter gene and viralantigen mediated by HCV IRES by 50% to 90% in vivo,butmIRNA lost its inhibitory activity completely.The luciferasegene expression mediated by HCV IRES was blocked inthe HHCC constitutively expressing IRNA.At 48h aftertransfection,the expression level of reportor gene descreasedby 20%,but cap-dependent luciferase gene expression wasnot affected.IRNA could inhibit the HCV replicon expression24h after transfection and the highest inhibitory activitywas 80% by 72h,and the inhibitory activity was notincreased until 7d after transfection.CONCLUSION:IRNA can inhibit HCV IRES mediated geneexpression in vivo. AIM: To investigate the inhibitory effect of hepatitis C virus internal ribosome entry site (HCV IRES) specific inhibitor RNA (IRNA) on gene expression mediated by HCV IRES in vivo. METHODS: By using G418 screening system, hepatoma cells constitutively expressing IRNA or mutant IRNA mRNA) were established and characterized, and HCV replicons were ligated to the 5 ’untranslated region (5’UTR) were constructed by using the same method. Detection of pCMVNCRIuc containing HCV 5’UTR-Iuc fusion genes andeukaryotic vector of IRNA into human hepatic carcinoma cells (HepG2) was performed and the eukaryotic expression of midsmid of IRNA was transfected transiently into HCVreplicons, pCMVNCRIuc or pCDNA-Iuc was cotransfected with pSV40-β Gal into IRNA expressing hepatoma cells by using Lipofectamine 2000 in vitro. The reporting gene expression level was examined at 48 h after transfection by using a luminometer and the expressing level of HCV Cantigen was analyzed with a confocal microscope .RESULTS: Transient exp ression of IRES specific IRNA could be given for the expression of reporter gene and viralantigen mediated by HCV IRES by 50% to 90% in vivo, butmIRNA lost its inhibitory activity completely. The luciferase gene expression mediated by HCV IRES was blocked in HHCC constitutively expressing IRNA. At 48h aftertransfection, the expression level of reportor gene descreasedby 20%, but cap-dependent luciferase gene expression was not affected. IRNA could inhibit the HCV replicon expression24h after transfection and the highest inhibitory activitywas 80% by 72h, and the inhibitory activity was not created by 7d after transfection. CONCLUSION: IRNA can inhibit HCV IRES mediated gene expression in vivo.