半滑舌鳎miR-33调控TRAF6表达的分子机制和免疫功能研究

Half-smooth tongue sole (Cynoglossus semilaevis) is an important marine fish species in China. However, frequent outbreaks of diseases have led to catastrophic economic losses and seriously threaten sustainable development of aquaculture. Understanding immune defense mechanism, seeking effective molecular breeding technology and improving disease resistance are useful to disease control and health management in fish aquaculture. Previously, we found that the target of C. semilaevis miR-33 was TRAF6, an important immune gene. In view of above, the present project focuses on detecting the expressions of TRAF6 mRNA and protein in head kidney cells in vitro and different immune tissues in vivo after respectively adding miR-33 agomir and antagomir, to clarify the regulation mode of miR-33 on TRAF6; observing their pathological changes after miRNA transfection and nervous necrosis virus infection, which reveals the effect of miR-33 regulation on immune functions of the cells and individuals. Transcriptome sequencing data will be then analyzed to screen and validate differentially expressed immune related genes and signaling pathways. Finally, molecular regulation mechanism of miR-33 on TRAF6 and its influence on immune response of C. semilaevis will be studied. The project provides a theoretical basis for promoting research on immune regulation of miRNA and breeding new stock with enhanced disease resistance in fish.

半滑舌鳎是我国重要的海水养殖品种，然而其病害频发，不仅造成巨大经济损失，同时严重威胁水产养殖的可持续发展。开展半滑舌鳎免疫防御机理研究，寻求抗病分子育种技术，提高鱼体抗病免疫力，对病害防治和产业绿色发展意义重大。前期研究发现半滑舌鳎miR-33的靶基因是重要免疫相关基因TRAF6。本项目拟利用miRNA过表达(模拟物)/抑制表达(抑制物)，在半滑舌鳎体外头肾细胞和体内不同免疫组织中检测TRAF6 mRNA和蛋白表达，阐明miR-33对TRAF6的调控方式；进一步进行神经坏死病毒感染，检测miR-33转染后的半滑舌鳎细胞和个体的病变和死亡情况，同时利用转录组分析手段筛选并验证差异表达的免疫基因和信号通路。最终揭示半滑舌鳎miR-33调控TRAF6表达的分子机制和对免疫反应的影响。本项目将为推动鱼类miRNA免疫研究、促进抗病品种改良奠定基础。

半滑舌鳎是我国重要海水养殖物种，然而其病害频发，不仅造成巨大经济损失，同时严重威胁养殖业的可持续发展。MicroRNA作为一类重要的非编码小分子RNA，是近年来广受关注的表观调节因子之一。本项目从半滑舌鳎组学数据中鉴定了miR-33和miR-462，发现他们可分别与靶基因TRAF6和IL-1β的3’-UTR相结合，通过抑制蛋白的方式进行转录后负调控；多种弧菌和病毒感染后，这2个miRNA及其靶基因在肝脏、脾脏、头肾和肠等免疫相关组织中的表达均随时间变化且呈现出大致相反的表达趋势；利用miR-33抑制物解除对TRAF6的负调控作用，可使IL-1R、MYD88、IRAK1、TNFα、IL6、IL8等多个免疫通路相关基因的表达上调，同时有助于提高半滑舌鳎抗感染力，V. harveyi和NNV感染后死亡率明显降低；将miR-462抑制导致MYD88、TRAF6、c-Jun、NF-κB、p38、TNF-β、IL-6 和L-8等免疫基因表达上调，同时可提高半滑舌鳎溶菌酶活性和炎症细胞因子(IL-1β、IL-6、IL-8)蛋白浓度。本研究表明半滑舌鳎miR-33&TRAF6、miR-462&IL-1β在抗细菌和病毒免疫反应中的重要作用，为推动鱼类miRNA免疫研究、提高抗病力奠定基础。