Laboratory of Human Retrovirology, Clinical Services Program, Applied and Developmental Directorate, SAIC-Frederick, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA, Laboratory of Proteomics and Analytical Technologies, Advanced Technology Program, SAIC-Frederick, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA and Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Nucleic Acids Res. 2014 Jan;42(1):583-98. doi: 10.1093/nar/gkt844. Epub 2013 Sep 18.
In addition to silencing specific genes, small interfering RNA (siRNA) transfection is also associated with the non-specific induction of inflammatory cytokines and type I interferon. Those so-called "off-target" effects have considerable implications for the interpretation of in vitro studies and clinical application of siRNA. The present study attempted to develop a better understanding of the mechanism involved in these off target effects. Synthesized siRNA significantly enhances DNA-mediated interferon lambda-1 response (IFN-λ1/IL-29), a newly characterized antiviral interferon in non-immune or primary immune cells. This enhancement was most pronounced by double-stranded siRNA with at least a 2-nucleotide overhang at one 3' terminus in a dose-dependent manner, while the presence of DNA was indispensable. A pull-down assay using biotinylated siRNA- or DNA-conjugated beads indicated that retinoic acid-inducible gene I (RIG-I) and interferon gamma-inducible protein 16 (IFI16) were involved in the sensing of siRNA and DNA, respectively. Co-immunoprecipitation analysis further revealed that RIG-I and IFI16 formed a complex via siRNA, and the dissociation of IFI16 from this complex in the presence of DNA activated the downstream STING-TBK1-IRF3 (stimulator of interferon genes - tank-binding kinase 1 - interferon regulatory factor 3) pathway, shedding light on a new physiological signalling pathway to activate innate immunity. Collectively, these findings may provide rational information for siRNA-induced innate immunity, with important implications for developing siRNA-based reagents to control human diseases.
除了沉默特定的基因外，小干扰 RNA（siRNA）转染还与非特异性诱导炎症细胞因子和 I 型干扰素有关。这些所谓的“脱靶”效应对体外研究的解释和 siRNA 的临床应用具有重要意义。本研究试图更好地了解这些脱靶效应的机制。合成的 siRNA 显著增强了 DNA 介导的干扰素 lambda-1 反应（IFN-λ1/IL-29），这是一种新发现的非免疫或原代免疫细胞中的抗病毒干扰素。这种增强作用在双链 siRNA 中最为明显，其至少在一个 3'末端有 2 个核苷酸突出，呈剂量依赖性，而 DNA 的存在是必不可少的。使用生物素化 siRNA 或 DNA 偶联珠的下拉测定表明，视黄酸诱导基因 I（RIG-I）和干扰素γ诱导蛋白 16（IFI16）分别参与 siRNA 和 DNA 的检测。共免疫沉淀分析进一步表明，RIG-I 和 IFI16 通过 siRNA 形成复合物，而在 DNA 存在下 IFI16 从该复合物中的解离激活了下游 STING-TBK1-IRF3（干扰素基因刺激物 - 衔接激酶 1 - 干扰素调节因子 3）途径，揭示了一种新的生理信号通路来激活先天免疫。总之，这些发现可能为 siRNA 诱导的先天免疫提供合理的信息，对于开发基于 siRNA 的试剂来控制人类疾病具有重要意义。
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