哈维氏弧菌诱导鮸鱼microRNA-375表达并发挥抑炎作用的分子机制研究

The innate immune system plays a major role in pathogen recognition and immune response in fish, pattern recognition receptor recognizes PAMPs to activate signal pathway and produce cytokines to clear pathogens and damaged cells. The excessive inflammatory response can cause severe damage to the body, so the innate immune pathway is tightly regulated to keep the body in a relatively stable state. Vibrio harveyi is an important pathogen of marine fish, we found that the increase of V. harveyi will induce miR-375 expression in Miichthys miiuy, overexpression of miR-375 could inhibit the production of cytokines, suggesting that miR-375 may inhibit the excessive inflammatory reaction in bacterial infection. In this project, we first predict the candidate target genes of miR-375 from immune genes; second, using the dual luciferase reporter to verify the real target gene. We produce polyclonal antibody of the target protein, western blot and qRT techniques were used to study the regulation method, the signaling pathways regulated by miR-375 were elucidated by the expression detection of downstream molecules and cytokines. Then, we used in vivo experiments to verify the effect of miR-375 on target gene and its inhibitory effect, get the influence of miR-375 on the survival status of Miichthys miiuy. We finally understand the molecular regulation mechanism of miR-375 to play a role in inhibiting inflammation, enrich the understanding of innate immune signal pathway regulation and disease prevention in fish.

先天性免疫系统在鱼类的病原识别和免疫应答中发挥主要作用，模式识别受体识别PAMPs后激活信号通路，产生细胞因子引发炎症反应以清除病原体和受损的细胞。而持续过度的炎症反应会造成严重损伤，因此先天性免疫通路受到严密调控以保持机体的稳态。哈维氏弧菌是海水鱼类重要的致病菌，申请人发现哈维氏弧菌会诱导鮸鱼miR-375表达上调，过表达miR-375会抑制炎症细胞因子产生，推测miR-375可能在细菌感染后抑制过度炎症反应。本项目首先预测miR-375候选调控的基因，利用双荧光素酶报告系统验证靶基因；Western blot和qPCR揭示对靶基因的调控方式；通过对靶基因下游分子及细胞因子的检测阐明miR-375所调控的免疫信号通路；体内实验证明调控的作用方式和抑制效果，以及miR-375对鮸鱼生存状况的影响，明确miR-375发挥抑炎作用的分子机制，丰富对鱼类先天性免疫信号通路调控和疾病防控的认识。

鮸鱼是东海海域重要的经济养殖鱼种，日益严重的细菌性疾病阻碍了养殖产业的发展，而该物种的免疫学研究还相对滞后，亟需要深入开展分子免疫学的相关研究。本项目以鮸鱼为研究对象，详尽地对十多个microRNA开展了靶基因的验证及分子调控机制的研究工作，主要研究内容如下：（1）对miRNA-132，miRNA-101a，miRNA-144，miRNA-148，miRNA-1388，miRNA-2187，miRNA-181b，miRNA-20，miRNA-21，miRNA-210，miRNA-3570，miR-375等十多个microRNA开展了生物信息学预测靶基因，并利用双荧光报告技术验证了miRNA与靶基因的对应关系；（2）利用Western blot和qPCR技术揭示了上述microRNA对靶基因的调控方式；（3）利用qPCR和双荧光报告基因技术研究了上述microRNA对先天性免疫反应和炎症信号通路的调控作用。上述研究内容丰富了对鱼类先天性免疫信号通路调控和疾病防控的理解。