A novel miRNA, Cse-miR-33, functions as an immune regulator by targeting CsTRAF6 in Chinese tongue sole (Cynoglossus semilaevis)

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.