IHNV通过调控虹鳟miR-146a-3p表达影响I型干扰素作用的机制

Infectious hematopoietic necrosis virus (IHNV) is a viral pathogen of cold-water fish, the rainbow trout (Oncorhynchus mykiss), which can cause severe death in fry and fingerling fish. However, there are no efficient methods to control the IHNV infection in China until now. MiR-146a-3p is a member of mature microRNAs. MiR-146a has been proved to regulate the innate immune responses in mammals. Of interest, based on the previous IHNV-inducible miRNAs sequencing, analysis and experiment results, miR-146a-3p was found to be indeed associated with the regulation of type I IFN response and affect the virus replication in rainbow trout. Hence, we intend to experimentally validate the function of miR-146a-3p in the rainbow trout or rainbow trout cell line by real-time PCR, immunoprecipitation, omics analysis, luciferase reporter gene assay, artificial control of gene expression and so on. The roles of miR-146a-3p in the regulation of type I IFN responses during IHNV infection will be revealed from the following aspects: analysis of miR-146a-3p expression profile and biosynthesis pathway during IHNV infection, the effects of miR-146a-3p on the IFN production and signaling, and how miR-146a-3p participates in IHNV-induced type I IFN response through its target genes. These results will be beneficial to gain novel insight into the miRNA mediated regulation of type I IFN function in the antiviral response of fish and provide instructions for the development of efficient and safe drugs for preventing and treating IHNV infection.

传染性造血器官坏死病病毒（IHNV）是冷水性鱼类虹鳟的强致死性病原，但目前我国尚无针对该病毒感染的有效防治方法。miR-146a-3p属微小RNA（miRNA）成熟体家族成员，已知对哺乳动物先天性免疫反应具有调节作用，基于前期我们对IHNV感染虹鳟miRNA测序和深度分析及实验，证实该分子在IHNV感染虹鳟时的确与I型干扰素调节有关，且可影响病毒复制。在此基础上，本项目拟以虹鳟或虹鳟传代细胞系为材料，利用荧光定量、免疫共沉淀、组学分析、荧光报告基因检测、基因表达干预等多种实验手段对miR-146a-3p在IHNV攻毒后的表达模式、诱导产生途径、对干扰素生成和信号转导的影响及调控靶基因发挥作用进行研究，揭示IHNV感染激活miR-146a-3p表达调节鱼体或细胞中I型干扰素反应的方式，从而阐明鱼类抗病毒反应中miRNA对I型干扰素作用的调控机制，为研发高效、安全的抗IHNV感染药物提供支持。

虹鳟是我国冷水性鱼类养殖的主要品种，传染性造血器官坏死病病毒（IHNV）感染可引起虹鳟大量死亡，但目前我国针对该病毒的防治手段尚且不足。干扰素（IFN）系统同样是鱼类抗病毒感染的首道防线。IHNV具有IFN逃逸特性，但miRNA在IHNV感染诱导IFN的作用还不明确。本项目围绕IHNV感染过程中如何通过调控miR-146a-3p实现IFN逃逸这一问题开展研究，取得了以下结果：①构建了IHNV感染和未感染的虹鳟脾脏小RNA表达文库，并进行高通量测序，鉴定到miR-146a-3p为IHNV感染相关miRNA，且预测其具有免疫调节功能。miR-146a-3p在IHNV感染虹鳟和RTG-2细胞中的表达趋势一致，即仅在感染初期呈现显著上调表达，之后下调表达；②用IHNV感染细胞，而后借助IFN通路抑制剂和siRNA干扰确定IHNV感染调控miR-146a-3p表达依赖RIG-I/NF-κB途径，也受I型IFN信号转导的影响；③过表达miR-146a-3p时，IHNV复制增强，抑制病毒诱导的IFN表达，通过降低STAT1磷酸化来抑制IFN效应途径，抑制miR-146a-3p则会降低病毒复制、促进IFN反应；④综合过表达miRNA的转录组测序、生物信息预测和双荧光报告实验结果，初步判断miR-146a-3p的靶基因是Notch、DAAM、CTBP、SOS、Wnt3a和CCND1，且IHNV抑制Wnt3a和CCND1的表达主要通过miR146a-3p来实现；过表达miR-146a-3p后回补靶基因或敲低靶基因后回补miR-146a-3p抑制物，经与对照间的比较，确定miR-146a-3p的确通过调控Wnt3a和CCND1表达来影响IHNV的复制及其诱导IFN反应。本项目的研究结果为阐明IHNV借助宿主miRNA限制IFN反应的分子机制奠定了基础，同时也为抗IHNV感染药物的研发提供思路。