Institute of Virology, University Hospital of Essen, University Duisburg-Essen, Essen, Germany.
PLoS One. 2013 May 20;8(5):e64708. doi: 10.1371/journal.pone.0064708. Print 2013.
Our previous results showed that the knockdown of woodchuck hepatitis virus (WHV) by RNA interference (RNAi) led to upregulation of interferon stimulated genes (ISGs) in primary hepatocytes. In the present study, we tested the hypothesis that the cellular signaling pathways recognizing RNA molecules may be involved the ISG stimulation by RNAi.
Primary murine hepatocytes (PMHs) from wild type mice and WHV transgenic (Tg) mice were prepared and treated with defined siRNAs. The mRNA levels of target genes and ISGs were detected by real-time RT-PCR. The involvement of the signaling pathways including RIG-I/MDA5, PKR, and TLR3/7/8/9 was examined by specific inhibition and the analysis of their activation by Western blotting.
In PMHs from WHV Tg mice, specific siRNAs targeting WHV, mouse β-actin, and GAPDH reduced the levels of targeted mRNAs and increased the mRNA expression of IFN-β, MxA, and IP-10. The enhanced ISG expression by siRNA transfection were abolished by siRNA-specific 2'-O-methyl antisense RNA and the inhibitors 2-AP and chloroquine blocking PKR and other TLR-mediated signaling pathways. Furthermore, Western blotting revealed that RNAi results in an increase in PKR phosphorylation and nuclear translocation of IRF3 and NF-êB, indicating the possible role of IRF3 in the RNAi-directed induction of ISGs. In contrast, silencing of RIG-I and MDA5 failed to block RNAi-mediated MxA induction.
RNAi is capable of enhancing innate immune responses through the PKR- and TLR-dependent signaling pathways in primary hepatocytes. The immune stimulation by RNAi may contribute to the antiviral activity of siRNAs in vivo.
我们之前的研究结果表明，通过 RNA 干扰（RNAi）敲低土拨鼠肝炎病毒（WHV）可导致原代肝细胞中干扰素刺激基因（ISGs）的上调。在本研究中，我们检验了这样一个假设，即识别 RNA 分子的细胞信号通路可能参与了 RNAi 诱导的 ISG 刺激。
从野生型小鼠和 WHV 转基因（Tg）小鼠中制备原代鼠肝细胞（PMHs），并用特定的 siRNA 处理。通过实时 RT-PCR 检测靶基因和 ISGs 的 mRNA 水平。通过特异性抑制和 Western blot 分析其激活来研究包括 RIG-I/MDA5、PKR 和 TLR3/7/8/9 在内的信号通路的参与。
在 WHV Tg 小鼠的 PMHs 中，靶向 WHV、小鼠 β-肌动蛋白和 GAPDH 的特异性 siRNA 降低了靶标 mRNA 的水平，并增加了 IFN-β、MxA 和 IP-10 的 mRNA 表达。siRNA 特异性 2'-O-甲基反义 RNA 和 PKR 抑制剂 2-AP 和氯喹阻断其他 TLR 介导的信号通路后，siRNA 转染增强的 ISG 表达被消除。此外，Western blot 显示 RNAi 导致 PKR 磷酸化和 IRF3 和 NF-êB 的核易位增加，表明 IRF3 在 RNAi 指导的 ISG 诱导中可能起作用。相反，沉默 RIG-I 和 MDA5 不能阻断 RNAi 介导的 MxA 诱导。
RNAi 能够通过原代肝细胞中 PKR 和 TLR 依赖性信号通路增强先天免疫反应。RNAi 的免疫刺激可能有助于 siRNA 在体内的抗病毒活性。
