Sphingolipid metabolites including ceramide, sphingosine, and sphingosine 1-phosphate (S1P) have recently emerged as a new class of lipid messengers that regulate cell proliferation, differentiation and survival. Sphingosine kinase 1 (SK1) is a key enzyme to regulate these sphingolipid metabolites. RNA interference (RNAi) is a major new genetic tool for investigating mammalian cells to silence gene expression post-transcriptionally. In this study, we investigated if RNAi for SK1 down-regulates endogenous liver SK1 using “hydrodynamic transfection method” in vivo. We injected either SK1 or scrambled sequence (SCR) small interfering RNA (siRNA) into male CD1 mice through the dorsal penis vein with “hydrodynamic transfection method”. Down-regulation of SK1 was confirmed by levels of mRNA and protein using real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blot, respectively, in livers of mice treated with SK1 siRNA as compared to those in mice treated with SCR siRNA. SK1 siRNA through the dorsal penis vein injection significantly reduced endogenous SK1 mRNA levels in 18 hours and 48 hours after injection (P<0.05 & P<0.01, respectively). Western blot and sphingolipid profile indicated that SK1 siRNA injection reduced levels of SK1 protein and S1P in the livers. Interestingly, livers of mice treated with SK1 siRNA caused apoptosis in hepatocytes whereas no apoptotic cells were observed in livers of mice treated with SCR siRNA (P<0.05 in 48 hours). This apoptosis was related to caspase-3 cleaved by western blot analysis. SK1 siRNA injection through the dorsal penis vein using “hydrodynamic transfection method” reduced endogenous SK1 mRNA and protein levels and SK1 down-regulation result in causing apoptosis in hepatocytes. This is the first evidence that siRNA injection using this delivery system reduces endogenous target gene expression in vivo and SK1 down-regulation may constitute a new chemotherapeutic strategy.
[Proc Amer Assoc Cancer Res, Volume 47, 2006]