Key Laboratory for Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, 266071, China; Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266237, China; Shandong Key Laboratory of Marine Fisheries Biotechnology and Genetic Breeding, Qingdao, 266071, China.
Key Laboratory for Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, 266071, China; Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266237, China; College of Fisheries and Life Sciences, Shanghai Ocean University, Shanghai, 200000, China.
Fish Shellfish Immunol. 2023 Mar;134:108606. doi: 10.1016/j.fsi.2023.108606. Epub 2023 Feb 8.
The tumor necrosis factor receptor-associated factor 6 (TRAF6) can act as a fundamental adaptor protein in a chain reaction of signal transduction and cascade events to finish off immune defenses. However, immunomodulatory research on TRAF6 gene is still limited in fish. In this study, a novel miRNA, Cse-miR-33 was identified from the whole genome of Chinese tongue sole (Cynoglossus semilaevis). After separate infections with three different Vibrio strains (V. harveyi, V. anguillarum, V. parahemolyticus) and one virus (nervous necrosis virus, NNV), the expressions of CsTRAF6 and Cse-miR-33 displayed significant time-dependent changes in immune related tissues and the trends were opposite in general. Through target gene prediction and dual luciferase reporter assay, Cse-miR-33 was proven to regulate CsTRAF6 by combining with 3'-UTR sequence of the gene. The results of qRT-PCR and western blotting (WB) analyses showed that Cse-miR-33 blocked the translation of CsTRAF6 protein at post-transcriptional level, rather than degrading the target mRNA. Further experiment indicated that Cse-miR-33 inhibitor largely reduced the death rate of Chinese tongue sole caused by V. harveyi and NNV. The expressions of CsTRAF6-associated immune genes (such as CsIL-1R, CsMYD88, CsIRAK1, CsTNFα, CsIL6 and CsIL8) were also significantly changed in response to Cse-miR-33 agomir and inhibitor. The study suggested that Cse-miR-33 affected the immune response via targeting CsTRAF6 in C. semilaevis, which would provide us deep insights into miRNA-mediated regulatory network and help improve the immunity in fish.
肿瘤坏死因子受体相关因子 6（TRAF6）可以作为信号转导和级联反应链中的基本衔接蛋白，从而完成免疫防御。然而，鱼类中关于 TRAF6 基因的免疫调节研究仍然有限。在本研究中，从中国舌鳎（Cynoglossus semilaevis）的全基因组中鉴定出一种新型 miRNA，Cse-miR-33。在分别感染三种不同的弧菌（V. harveyi、V. anguillarum、V. parahemolyticus）和一种病毒（神经坏死病毒，NNV）后，CsTRAF6 和 Cse-miR-33 的表达在免疫相关组织中呈现出明显的时间依赖性变化，总体趋势相反。通过靶基因预测和双荧光素酶报告基因检测，证明 Cse-miR-33 通过结合基因的 3'-UTR 序列来调节 CsTRAF6。qRT-PCR 和 Western blot（WB）分析结果表明，Cse-miR-33 在转录后水平阻断了 CsTRAF6 蛋白的翻译，而不是降解靶 mRNA。进一步的实验表明，Cse-miR-33 抑制剂在很大程度上降低了中国舌鳎因 V. harveyi 和 NNV 引起的死亡率。CsTRAF6 相关免疫基因（如 CsIL-1R、CsMYD88、CsIRAK1、CsTNFα、CsIL6 和 CsIL8）的表达也因 Cse-miR-33 激动剂和抑制剂而发生显著变化。该研究表明，Cse-miR-33 通过靶向 C. semilaevis 中的 CsTRAF6 影响免疫反应，这将为我们深入了解 miRNA 介导的调控网络提供帮助，并有助于提高鱼类的免疫力。
