Inhibitory effects of short hairpin RNA against caspase-8 on apoptosis of murine hepatoma Hepa1-6 cells.

Caspase-8 plays an important role in death-receptor-mediated apoptosis of hepatocytes. We constructed short hairpin RNAs (shRNAs) against caspase-8 and investigated the effects of caspase-8 targeting shRNAs on apoptosis of Hepa1-6 cells induced by TNF-alpha. Oligonucleotides coding for four shRNAs against caspase-8 were cloned into mammalian expression vector Pgenesil-1 containing U6 promoter, which were then introduced into Hepa1-6 cells using liposome-mediated transfection. Effects of caspase-8-shRNAs on apoptosis of Hepa1-6 cells induced by TNF-alpha were detected by PI apoptosis detection kit. Effects of caspase-8-shRNAs on caspase-8 mRNA expression in apoptosis Hepa1-6 cells induced by TNF-alpha were detected by real-time fluorescent RT-PCR. Of the four caspase-8-shRNAs, Pgenesil-caspase-8-1 and Pgenesil-caspase-8-2 were successfully constructed. The apoptosis of Hepa1-6 cells induced by TNF-alpha was significantly inhibited by either Pgenesil-caspase-8-1 or Pgenesil-caspase-8-2 (p < 0.05). Caspase-8 mRNA expression levels in apoptosis Hepa1-6 cells induced by TNF-alpha were significantly decreased by either Pgenesil-caspase-8-1 or Pgenesil-caspase-8-2 (p < 0.05). This study suggested that shRNAs against caspase-8 could effectively inhibit apoptosis of Hepa1-6 cells induced by TNF-alpha by suppressing caspase-8 mRNA expression.