Impact Of Co-transfection With Livin And Survivin ShRNA Expression Vectors On Biological Behavior Of HepG2Cells

Background and Objective:Hepatocellular carcinoma (HCC) is one of the most common digestive malignancies and it claims an increasing incidence. Conventional therapies such as surgical resection and TACE are ineffective, and it is rare to achieve complete remission or cure. Application of RNA interference (RNAi) to reduce oncogene expression and thereby change the biology of tumor cells is one of the most important methods in tumor therapy. Livin and Survivin are both members of the Inhibitor of Apoptosis (LAP) family of proteins, and are highly expressed in hepatocellular carcinoma cells. These two genes exert their anti-apoptotic effects through the IAP repeat domains (baculovirus-IAP repeat, BIR). Knockdown of either Livin or Survivin can manifest therapeutic effects against liver cancer. However, it remains unknown as to whether the combined inhibition of these two genes shows any synergistic effects on cellular proliferation and apoptosis in human liver cells. In the present study, we designed and constructed eukaryotic expression vectors that encode Livin and Survivin shRNAs. These vectors were transfected or co-transfected into hepatocellular carcinoma HepG2cells, with the aim of exploring the effects of single and combinatorial gene silencing on the biological behavior of HepG2cells.Methods:ShRNA eukaryotic expression vectors pSD11-Livin and pSD11-Survivin were designed and constructed then transfected into HepG2cells separately or in combination. mRNA and protein expression in transfected cells was assessed by quantitative fluorescence PCR and Western blotting, respectively. Cell proliferation was measured by MTT assay and cell apoptosis by TUNEL assay.Results: The Livin and Survivin shRNA eukaryotic expression vectors were successfully constructed and transfected into HepG2cells. The relative mRNA expression levels of Livin and Survivin in HepG2cells co-transfected with pSDll-Livin and pSDll-Survivin were significantly lower than levels in cells transfected with either pSD11-Livin or pSDll-Survivin (P<0.05). The relative protein expression levels of Livin and Survivin in the co-transfected cells were also significantly decreased compared to single-transfection (P<0.05). The inhibition rate of cell growth in the co-transfection group was higher than that in the single-transfection groups at48h,60h, or72h after transfection (P<0.01). The apoptotic rate increased to the greatest extent in the co-transfection group relative to any other group (P<0.05).Conclusions: Expression of Livin and Survivin gene in HepG2cells may be independent of each other, and separate inhibition of the expression of a gene in the IAPs may not induce the change of expression of any other gene of IAP family. Compared with the single gene suppression, Co-transfection of Livin and Survivin gene expression vectors can reduce the expression of Livin and Survivin gene in HepG2cells more effectively, which may shows synergistic effects between Livin and Survivin gene on the anti-apoptosis mechanism of HepG2cells. Compared with the single gene suppression, Co-transfection also inhibited the proliferation of transfected cells more than the other groups, and induced cellular apoptosis more effectively.