Inhibition of tumorigenesis and invasion of hepatocellular carcinoma by siRNA-mediated silencing of the livin gene

Abstract

Livin, a member of the inhibitor of apoptosis protein family, is overexpressed in a variety of solid tumors and cancer cell lines. Livin overexpression has been reported in hepatocellular carcinoma (HCC), suggesting the biological significance of livin in HCC progression. However, the mechanisms of livin and the consequence of its down-regulation in HCC have not been fully elucidated. A small interfering RNA (siRNA) eukaryotic expression vector specific to livin was constructed by gene recombination, and the nucleic acid was sequenced. It was then transfected into the human HCC cell line SMMC-7721. RT-PCR and Western blotting were used to validate the gene-silencing efficiency of livin in SMMC-7721 cells. Stable clones were obtained by G418 screening. Using multiple cellular and molecular approaches, such as an apoptosis assay, MTT assay, flow cytometry, Western blotting and a migration assay, the effects of livin inhibition on cell growth, migration and the induction of apoptosis in SMMC-7721 cells were observed. The siRNA eukaryotic expression vector specific to livin was constructed by gene recombination, and efficiently decreased the mRNA and protein expression of livin. The targeted inhibition of livin strongly sensitized SMMC-7721 cells to pro-apoptotic stimuli, and was associated with caspase-3 activation. In addition, the MMT assay indicated that the silencing of livin inhibited the cell growth of SMMC-7721 cells by specifically inhibiting cell mitosis. The results also showed that the silencing of livin strongly reduced the invasive capacity of SMMC-7721 cells. The findings suggest that livin expression not only provides HCC cells with increased resistance to apoptotic stimuli, but also contributes significantly to the proliferation and invasive capacity of HCC cells. Inhibition of livin may be a potential targeted approach for the treatment of HCC.