参与抗病毒固有免疫调控的lncRNAs的筛选及相关作用机制研究

RIG-I signaling pathway is very important for anti-viral immune response. The studies on the regulatory roles and the related mechanisms of differentially expressed lncRNAs induced by RIG-I activation are very rare and limited.This study is an extension of previous works of our group. We will focus on the lncRNAs regulated by RIG-I signaling. We first screened the differentially expressed lncRNAs between pre- and post-viral infection by lncRNA genechip. By informatical analysis, we will have a clear understanding of differentially expressed lncRNAs regulated by RIG-I signaling. The preliminary data have been demonstrated by Real-time PCR. Based on these data, we will extend our studies to the regulatory roles of these lncRNAs in anti-viral immune response by overexpression, knockdown, knockout techniques. We will figure out the related regulatory mechanisms by dual-luciferase reporter assay, Western Blot, RIP, ChIRP techniques. We will also monitor the anti-viral immune response in lncRNAs transgenic mice or gene knockout mice. By these studies, hopefully we will obtain a clear landscape of differentially expressed lncRNAs and have a deep understanding of the contribution of novel lncRNAs in the regulation of anti-viral immune responses and the related mechanisms. This study will provide a theoretical basis for understanding the pathogenesis of related diseases and provide new possible drug targets for the intervention of related diseases.

RIG-I信号通路参与机体抗RNA病毒免疫应答，关于其诱导的差异表达lncRNAs及lncRNAs在抗病毒免疫中的调控和机制研究还非常有限。本研究在课题组多年工作基础上，从RIG-I信号通路调控的lncRNAs入手，通过基因芯片筛选RNA病毒感染后表达差异的lncRNAs。在前期已经验证表达差异基础上，通过过表达、基因沉默、基因敲除等多种实验手段证实这些lncRNAs在抗病毒固有免疫应答中的调控作用；并通过双荧光素酶报告系统、WB、RIP以及ChIRP等技术手段探讨其发挥作用的相关机制；并在体内通过lncRNAs转基因小鼠或基因敲除小鼠探讨其对抗病毒固有免疫反应的影响。通过本研究以期对RIG-I信号调控的差异lncRNAs有一整体认识，并对关键未知功能的lncRNAs所发挥的调控作用及作用机制进行深入研究，为理解相关疾病的发病机制提供理论依据，为相关疾病的干预提供了新的可能的药物靶标。

本研究前期通过芯片筛选的方法鉴定出RNA病毒感染后上调或下调的lncRNAs，后续对芯片结果进行了实验验证，并对关键lncRNAs 的功能进行了系统研究。以LINC02605为例，我们发现其是一新的ISGs,表达上调依赖NF-κB和JAK-STAT信号通路。病毒刺激后LINC02605主要分布于细胞核中。功能上发现过表达LINC02605可以促进RNA病毒诱导的I型干扰素的产生、增强病毒对ISRE启动子的激活、抑制病毒的复制。而敲低LINC02605则导致抗病毒免疫反应减弱，病毒复制水平增高。机制上，我们发现LINC02605可以miRNA hsa-miR-107对PTEN的抑制作用。利用hsa-miR-107的模拟物和抑制剂都证明hsa-miR-107不仅能抑制PTEN的表达，且负向调控抗病毒固有免疫反应。若敲低LINC02605可使PTEN的mRNA、蛋白质水平降低，过表达LINC02605则起相反的作用。进一步研究发现LINC02605通过调节PTEN水平影响干扰素调节因子3（interferon regulatory factor 3, IRF3）的Ser97位磷酸化水平，进而改变IRF3的核转位信号,调控IRF3转运入核，并最终上调I型干扰素及下游炎性因子。核浆分离实验证实敲低LINC02605抑制IRF3的核转位激活，使细胞更易受到病毒侵犯，过表达则起到相反的效果。总之，本课题首先证明了lncRNA LINC02605作为一个新ISG，对抗病毒固有免疫发挥正向调控。这些发现为深入理解lncRNAs与抗病毒固有免疫反应的联系提供新的科学依据，以期对相关疾病的干预提供新靶标。