CONSTRUCTION OF SIRNA EXPRESSION VECTOR DRIVING BY PROMOTER OF HUMAN ALPHAFETOPROTEIN GENE AND THE PRELIMINARY IDENTIFICATION OF ITS RNAI EFFECT IN HEPG2

Objective:To explore the possibility of RNA interference of target gene medieted by a siRNA expression vector driving by human alpha-fetoprotein gene promoter,thus offer a primary study for fulfilling the targeting RNAi therapy of hepato- cellular carcinoma.Method:The promoter of human AFP gene was amplified with PCR and inserted into pSilencer1.0-U6 to re- place its U6 promoter.DNA template of siRNA targeting human hLRH-1 was designed according to a formerly selected RNAi site.After synthesized in vitro and annealed,the formed double-stranded DNA template was subclonted to construct the siRNA ex- pression vector targeting hLRH-1.The recombinant was introduced into HepG2 mediating by Lipofectamine.Real-time RT- PCR was carried out to analyse the RNAi effect in AFP expressing cells induced by the established RNAi system,as well as the al- terations of downstream target genes of hLRH-1.Results:A siRNA expression vector pSiAFP driving by promoter of human AFP gene was obtained in this study.By using human hLRH-1 as a report gene,the established RNAi system was shown to in- duce a knockdown of target gene in HepG2 by up to 85 %,and this inhibition in turn resulted in down-regulated expression of Cy- clinE1 and Oct4.Conclusion:The siRNA expression vector driving by promoter of human AFP gene could mediate RNAi effect of target gene in AFP expressing cell HepG2,this offer preliminary experimental data for clinical hepatocellular carcinoma cell-target- ing and gene-specific RNAi therapy.