The identification of miRNAs that regulate ovarian maturation in Cynoglossus semilaevis

MicroRNAs (miRNAs) participate in reproduction by regulating the expression of target genes; however, the regulation of target mRNAs by miRNAs along the brain-pituitary-gonad (BPG)-liver axis in fish reproduction and ovarian maturation has not been investigated, but a better understanding of these processes would have significant value. In this study, we performed comprehensive miRNA sequencing of BPG-liver organs at ovarian stages IV, V and VI in Cynoglossus semilaevis, yielding a total of thirty-six small RNA libraries. In total, 3224 miRNAs were identified, including 1,945 known miRNAs and 1279 novel miRNAs. The number of miRNAs downregulated from stage IV to stage VI in all tissues was higher than the number upregulated. The key identified miRNAs (miR-34-x, miR-186-x, miR-2188-x, miR-153-x, miR-204-x, miR-211-x, miR-2188-y, miR-223-y, miR-724-x, miR-144-y, novel-m0050-3p, and novel-m0017-5p) were found to possibly simultaneously regulate several mRNAs and participate in different biological functions at various ovarian stages. Based on target gene relationships, we screened important mRNAs related to reproduction (igf1r, hsd3b, igf2r, pgr, ghr, sstr2, cyp19a1, lhcgr, AR, cyp17a1, sst2, gpr54, pgrmc1, esr1, and paqr7b). Through KEGG pathway analysis, we found that the following signaling pathways were enriched: steroid hormone biosynthesis, oocyte meiosis, progesteronemediated oocyte maturation, focal adhesion, endocytosis, neuroactive ligand-receptor interaction, cytokine-cytokine receptor interaction, and Jak-STAT signaling. The expression patterns of the nine most differentially expressed miRNAs (miR-211-x, miR-153-x, miR-2188-x, miR-223-y, miR-2188-y, miR-186-x, miR-724-x, miR221-x, and miR-21-y) were assayed with RT-qPCR. The expression trends for all the miRNAs validated by RTqPCR were consistent with the sequencing data. Moreover, the miR-186-x binding sites in igf2r of C. semilaevis were verified for the first time. We speculated that miR-186-x directly regulates igf2r expression, modulates the reproductive endocrine system in C. semilaevis through the BPG-liver axis, and affects ovary maturation. Overall, this study reports a comprehensive analysis of transcriptomic modulation of the BPG-liver axis related to C. semilaevis ovary maturation.