MiRNA-194 targets MyD88a to regulate immune response in Cyprinus carpio after recovery from gossypol toxicity

Cottonseed meal has been one of the predominant protein feedstuffs used in aqua feed, which contains gossypol. It is well known that gossypol, a natural polyphenolic compound found in seeds, can be toxic to fish. The toxicity of gossypol persists even after a recovery period; however, the underlying mechanism remains largely unknown. To investigate the toxic mechanism of gossypol after the recovery period, Cyprinus carpio were administered a continuous oral dose of 20 mg/kg center dot d gossypol (gossypol acetate dose/fish weight center dot d) for one week, followed by a two-week recovery period. The spleen index, spleen morphology, lysozyme (LZM) activity, acid phosphatase (ACP) activity and nitric oxide (NO) concentration were obviously changed in the spleens of C. carpio after two weeks of recovery. Then, miRNA sequencing identified 22 differentially expressed miRNAs in the spleens of C. carpio two weeks after the gossypol treatment. The mRNA expression levels of immune-associated genes were significantly increased in the spleens after recovery. MiR-194 was chosen for future study through a search of existing literature and an analysis of miRNA and mRNA expression levels. To further characterize the potential target of miR-194, we identified putative miR-194 binding sites in the 3 ' UTR of myeloid differentiation factor 88a (MyD88a). Dual-luciferase reporter assays revealed that miR-194 downregulated the expression level of MyD88a through binding to seed-matching sequence in the 3 ' UTR of MyD88a. Moreover, C. carpio were intraperitoneally administered with miR-194 antagomir. Furthermore, down-regulation of miR-194 increased the expression levels of MyD88a and some pro-inflammatory cytokines and anti-inflammatory cytokines. Altogether, the results demonstrate miR-194 can target MyD88a to regulate the immune response of C. carpio after a two-week recovery period following gossypol administration.