Construction and application of a noval gene-target therapy system in hepatocellular carcinoma / 生物医学工程学杂志

ABSTRACT

This paper is aimed to explore the efficiency of a noval gene-target therapy system in hepatocellular carcinoma (HCC) treatment in vitro, by using survivin gene as the target. A new fusion promoter (AV) driving pcDNA3. 1 (-)AV plasmid, which contained the cytomegalovirus (CMV) enhancer and alpha-fetoprotein (AFP) promoter, was constructed by molecular biologic method. The eukaryotic expression plasmid pcDNA3. 1(-)AVGFP and pcDNA3. 1 (-)AVsiRNA-survivin were constructed by cloning and inserting the green fluorescent protein (GFP) and siRNA-survivin sequence into pcDNA3. 1(-) AV plasmid separately. Then these two plasmids were transfected into HepG2, SMMC-7721 and Hela cells by using nanoparticles of calcium phosphate. The transfection efficiencies were detected by GFP. Reversed transcript polymerase chain reaction (RT-PCR) and western-blot were used to evaluate the knockdown efficiency of siRNA-survivin. The growth curves and cell death of HepG2 cells transfected with or without pcDNA3. 1(-)AVsiRNA-survivin were detected by MTT assay and flow cytometry assay, respectively. The results showed that after transfected with pcDNA3. 1(-)AVGFP vector, only HepG2 cells displayed strong GFP signaling, whereas, no GFP was found in Hela cells, suggesting that AV promoter can specifically drive downstream of gene expression in HCC cells. Furthermore, the mRNA and protein expression levels of survivin in HepG2 cells, but not in Hela and SMMC-7721 cells, were significantly silenced after pcDNA3. 1(-)AVsiRNA-survivin transfection. Finally, compared with the control cells, HepG2 cells, which were transfected with pcDNA3. 1(-) AVsiRNA-survivin plasmid, presented 68.8% cell death, including 38.68% apoptosis and 30.12% necrosis, and significant cell growth inhibition (P < 0.05). These findings indicated that this noval gene-target therapy system could specifically target HCC cells with high efficiency, providing a new gene therapy strategy for HCC.