Dahlgren Cecilia, Wahlestedt Claes, Thonberg Håkan
Department of Cell and Microbiology, Programme for Genomics and Bioinformatics, Karolinska Institutet, Berzelius väg 35, S-171 77 Stockholm, Sweden.
Biochem Biophys Res Commun. 2006 Mar 24;341(4):1211-7. doi: 10.1016/j.bbrc.2006.01.085. Epub 2006 Jan 30.
Gene silencing by RNAi and siRNAs has become a well-used tool for researchers. Because of its relatively small size, siRNA was originally thought to avoid activation of anti-viral responses. Recent reports demonstrating so-called "off-target effects" are therefore alarming. One issue raised is that siRNA induces interferon-regulated genes at the transcriptional level. We characterize the anti-viral responses of synthetic siRNA and in vitro-transcribed siRNA by measuring the mRNA levels of IFN-beta and OAS2 in HeLa cells. Transfections with both traditional and LNA-modified synthetic siRNA cause no anti-viral responses, whereas transfection with either long dsRNA or in vitro-transcribed siRNA leads to greater than 1000-fold induction of these genes. The lack of response was also demonstrated at the level of phosphorylated eIF2alpha, and measuring of IFN-beta by ELISA in cell culture media from the human cell line MCF-7. Altogether, transfection with synthetic siRNA does not induce anti-viral responses in these two cell lines. Our results reinforce the role of siRNA as an effective tool for reverse genetics and strengthen siLNA as a tool for future therapeutic applications.
RNA干扰（RNAi）和小干扰RNA（siRNAs）介导的基因沉默已成为研究人员常用的工具。由于其相对较小的尺寸，siRNA最初被认为可避免激活抗病毒反应。因此，近期有关所谓“脱靶效应”的报道令人担忧。其中一个问题是，siRNA在转录水平诱导干扰素调节基因。我们通过测量HeLa细胞中IFN-β和OAS2的mRNA水平，来表征合成siRNA和体外转录siRNA的抗病毒反应。传统的和经锁核酸（LNA）修饰的合成siRNA转染均未引发抗病毒反应，而长双链RNA（dsRNA）或体外转录siRNA转染则导致这些基因的诱导倍数超过1000倍。在磷酸化真核翻译起始因子2α（eIF2α）水平以及通过酶联免疫吸附测定法（ELISA）检测人细胞系MCF-7细胞培养基中的IFN-β时，也证实了这种无反应情况。总之，合成siRNA转染在这两种细胞系中不会诱导抗病毒反应。我们的结果强化了siRNA作为反向遗传学有效工具的作用，并巩固了锁核酸增强型小干扰RNA（siLNA）作为未来治疗应用工具的地位。
