Abstract
To construct short hairpin RNA (shRNA) eukaryotic expression vectors targeting Livin and Survivin genes, and to explore the impact of co-transfection of Livin and Survivin shRNA expression vectors on the biological behavior of HepG2 cells. shRNA eukaryotic expression vectors pSD11-Livin and pSD11- Survivin were designed and constructed then transfected into HepG2 cells 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. The Livin and Survivin shRNA eukaryotic expression vectors were successfully constructed and transfected into HepG2 cells. The relative mRNA expression levels of Livin and Survivin in HepG2 cells co-transfected with pSD11-Livin and pSD11-Survivin were 0.12 ± 0.02 and 0.33 ± 0.13, respectively, which was significantly lower than levels in cells transfected with either pSD11-Livin or pSD11-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 at 48 h, 60 h, or 72 h 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). Co-transfection with pSD11-Livin and pSD11-Survivin was more efficient than transfection with either vector alone in reducing the mRNA and protein expression of Livin and Survivin genes in HepG2 cells. Co-transfection also inhibited the proliferation of transfected cells more than the other groups, and induced cellular apoptosis more effectively.
Highlights
Liver cancer is one of the most common digestive malignancies
Methods: short hairpin RNA (shRNA) eukaryotic expression vectors pSD11-Livin and pSD11Survivin were designed and constructed transfected into HepG2 cells separately or in combination. mRNA and protein expression in transfected cells was assessed by quantitative fluorescence PCR and Western blotting, respectively
The relative mRNA expression levels of Livin and Survivin in HepG2 cells co-transfected with pSD11-Livin and pSD11-Survivin were 0.12 ± 0.02 and 0.33 ±0.13, respectively, which was significantly lower than levels in cells transfected with either pSD11-Livin or pSD11-Survivin (P
Summary
Liver cancer is one of the most common digestive malignancies. Conventional therapies such as surgical resection and chemotherapy are ineffective, and it is rare to achieve complete remission or cure. Livin and Survivin are members of the Inhibitor of Apoptosis (IAP) 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). 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. These vectors were transfected or co-transfected into hepatocellular carcinoma HepG2 cells, with the aim of exploring the effects of single and combinatorial gene silencing on the biological behavior of HepG2 cells
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