Integrated lncRNA and mRNA Transcriptome Analyses in the Ovary of Cynoglossus semilaevis Reveal Genes and Pathways Potentially Involved in Reproduction.

Abstract

Long non-coding RNAs (lncRNAs) have been reported to be involved in multiple biological processes. However, the roles of lncRNAs in the reproduction of half-smooth tongue sole (Cynoglossus semilaevis) are unclear, especially in the molecular regulatory mechanism driving ovarian development and ovulation. Thus, to explore the mRNA and lncRNA mechanisms regulating reproduction, we collected tongue sole ovaries in three stages for RNA sequencing. In stage IV vs. V, we identified 312 differentially expressed (DE) mRNAs and 58 DE lncRNAs. In stage V vs. VI, we identified 1,059 DE mRNAs and 187 DE lncRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that DE mRNAs were enriched in ECM-receptor interaction, oocyte meiosis and steroid hormone biosynthesis pathways. Furthermore, we carried out gene set enrichment analysis (GSEA) to identify potential reproduction related-pathways additionally, such as fatty metabolism and retinol metabolism. Based on enrichment analysis, DE mRNAs with a potential role in reproduction were selected and classified into six categories, including signal transduction, cell growth and death, immune response, metabolism, transport and catabolism, and cell junction. The interactions of DE lncRNAs and mRNAs were predicted according to antisense, cis-, and trans-regulatory mechanisms. We constructed a competing endogenous RNA (ceRNA) network. Several lncRNAs were predicted to regulate genes related to reproduction including cyp17a1, cyp19a1, mmp14, pgr, and hsd17b1. The functional enrichment analysis of these target genes of lncRNAs revealed that they were involved in several signaling pathways, such as the TGF-beta, Wnt signaling, and MAPK signaling pathways and reproduction related-pathways such as the progesterone-mediated oocyte maturation, oocyte meiosis, and GnRH signaling pathway. RT-qPCR analysis showed that two lncRNAs (XR_522278.2 and XR_522171.2) were mainly expressed in the ovary. Dual-fluorescence in situ hybridization experiments showed that both XR_522278.2 and XR_522171.2 colocalized with their target genes cyp17a1 and cyp19a1, respectively, in the follicular cell layer. The results further demonstrated that lncRNAs might be involved in the biological processes by modulating gene expression. Taken together, this study provides lncRNA profiles in the ovary of tongue sole and further insight into the role of lncRNA involvement in regulating reproduction in tongue sole.