Apoptosis induced by nonspecific effects of siRNA in human umbilical vein endothelial cell

RNA interference (RNAi) is a powerful tool used to produce post-translational gene silencing in a sequence-dependent manner. However, sequence-independent activation of interferon systems induced by double-stranded RNA can interfere with the data interpretation of RNAi experiments. In this study, we observed cytokine activation and cytotoxicity caused by small interfering RNA (siRNA) in human umbilical vein endothelial cells (HUVECs). Transfection with different sequences of siRNAs (21-nucleotides) induced apoptosis in HUVECs within 24 hours, in a dose-dependent and sequence-independent manner. These effects could not be achieved by the application of transfection agents nor by siRNA only. In HUVECs, the expression of toll-like receptor 3 (TLR3), a receptor for double-stranded RNA, was significantly increased by the transfection with siRNAs regardless of their sequences. The expressions of interleukin 6, interleukin 8, and interferon-β were also up-regulated by siRNA transfection. The activation of RNA-dependent protein kinase (PKR) peaked within 30 min and slowly decreased 4 hours after transfection with siRNA, which was followed by the phosphorylation of eukaryotic initiation factor 2α(eIF2α). These studies suggest that transfection with double-stranded oligonucleotides in HUVECs shows sequence-independent apoptotic effects associated with the upregulation of TLR3 and cytokines, which should be considered when designing experiments using RNAi.