虹鳟鱼(Oncorhynchusmykiss)是一种典型的冷水水产养殖鱼类,是一种高端水产品。当水温超过其最佳范围12-18°C时,虹鳟鱼的免疫系统变得虚弱和不平衡。夏季高温和全球变暖严重影响了虹鳟鱼产业。本研究的重点是探索高温胁迫下虹鳟鱼免疫反应的调节机制,并确定解释高温抗性的分子元件。在这项研究中,在高温胁迫实验中筛选出单个鱼,并将其分为抗性(R)和敏感(S)组。肝脏转录组测序和mRNA和microRNA的分析,S,和对照组显示,S组(9259)的差异表达基因(DEGs)的数量高于R组(5313)。此外,S和R组之间差异表达的1233个基因主要富集在免疫相关通路中,包括细胞因子-细胞因子受体相互作用,TNF信号和IL-17信号。在这些DEGs中,miR-301b-5p及其编码活化T细胞核因子的两个相互作用蛋白(nfatc2ip)的靶基因。双荧光素酶报告系统和免疫荧光实验验证了miR-301b-5p和nfatc2ip之间的关系。我们还发现,在高温胁迫下,虹鳟鱼肝脏中miR-301b-5p和nfatc2ip的表达水平显着负相关。通过进行功能实验,我们发现,在高温胁迫下,miR-301b-5p表达的激活或nfatc2ip表达的抑制刺激了经典核因子κB和丝裂原活化蛋白激酶通路中p65,p38和JNK的磷酸化.这些操作最初促进了促炎因子IL-1β的分泌,然后增加了IL-6,IL-12和TNF-α的水平。此外,miR-301b-5p表达的激活或nfatc2ip表达的抑制通过激活虹鳟鱼肝脏的炎症反应,刺激高温应激引起的肝脏超微结构损伤的修复。
Rainbow trout (Oncorhynchus mykiss) is a typical cold-water aquaculture fish and a high-end aquatic product. When water temperature exceeds its optimal range of 12-18 °C, the immune system of rainbow trout becomes weakened and unbalanced. High temperature in summer and global warming severely impact rainbow trout industry. The focus of this study was to explore the mechanisms regulating the immune response of rainbow trout under high temperature stress and identify molecular elements that account for resistance to high temperature. In this study, individual fish were screened in a high temperature stress experiment and divided into resistant (R) and sensitive (S) groups. The hepatic transcriptome sequencing and analysis of mRNAs and microRNAs of the R, S, and control groups showed that the number of the differentially expressed genes (DEGs) in the S group (9259) was higher than that in the R group (5313). Furthermore, the 1233 genes differentially expressed between S and R groups were mainly enriched in immune-related pathways, including cytokine-cytokine receptor interaction, TNF signaling and IL-17 signaling. Among these DEGs were miR-301b-5p and its target gene that encodes nuclear factor of activated T cells two interacting protein (
nfatc2ip). The dual-luciferase reporter system and immunofluorescence experiments verified the relationship between miR-301b-5p and
nfatc2ip. We also showed that expression levels of miR-301b-5p and
nfatc2ip significantly negatively correlated in the liver of rainbow trout under high temperature stress. By performing functional experiments, we showed that activation of miR-301b-5p expression or inhibition of
nfatc2ip expression stimulated the phosphorylation of p65, p38, and JNK in the classical nuclear factor kappa-B and mitogen-activated protein kinase pathways under high temperature stress. These manipulations initially promoted the secretion of the pro-inflammatory factor IL-1β and then increased the levels of IL-6, IL-12, and TNF-α. In addition, activation of miR-301b-5p expression or inhibition of
nfatc2ip expression stimulated the repair of the hepatic ultrastructural damage caused by high temperature stress by activating the inflammatory response in rainbow trout liver.