Construction of lentiviral vectors carrying small interference RNA of alpha-fetoprotein gene and its efficiency of target gene silencing

Objective To construct lentiviral vectors carrying small interference RNA (siRNA) of alpha-fetoprotein(AFP) followed by transfection into hepatocellular carcinoma cells and to assess the efficiency of AFP gene silencing.Methods The AFP-specific positive siRNA and non-specific negative siRNA were designed and constructed prior to insertion to the recombinant lentiviral vectors carrying green fluorescent protein (GFP) gene.This was followed by transfection into human hepatocellular carcinoma cells HepG2 and subsequent screening of cell strains with GFP expression,and were assigned into lentivirustransfected positive siRNA group and lentivirus-transfected negative siRNA group respectively.Furthermore,liposome-transfected positive siRNA group,liposome-transfected negative siRNA group,siRNA group which treated with AFP siRNA but without transfection reagent,and blank control group were established simultaneously.The efficiency of transfection was assessed under fluorescent microscope,and the relative expression of AFP mRNA and protein in HepG2 cells was determined via fluorescent quantitative polymerase chain reaction and immunoblotting assay.In addition,the inhibition rate of AFP gene expression was compared among all groups.Results Lentiviral vectors were associated with effective transfection of siRNA into HepG2 cells.The siRNA transfected with lentivirus yielded considerably higher inhibition rate of AFP mRNA than that with liposomes,as evidenced by fluorescent quantitative polymerase chain reaction (92.1％vs 74.3％,P＜0.05).Immunoblotting assay showed that higher inhibition rate of AFP protein was in favor of siRNA transfected with lentivirus,but not with liposomes (88.2％ vs 63.7％,P＜0.05).Conclusion AFP siRNA transfected with lentivirus is associated with more effective inhibition of AFP expression in HepG2 cells. Key words: Carcinoma, hepatocellular;  alpha-Fetoproteins;  RNA interference;  Gene expression regulation;  Lentivirus