Table_8_Integrated lncRNA and mRNA Transcriptome Analyses in the Ovary of Cynoglossus semilaevis Reveal Genes and Pathways Potentially Involved in Reproduction.XLSX

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.

已有研究表明，长链非编码RNA（long non-coding RNAs，lncRNAs）参与多种生物学过程。然而，长链非编码RNA在半滑舌鳎（Cynoglossus semilaevis）繁殖过程中的作用尚不明确，尤其是在驱动卵巢发育与排卵的分子调控机制方面。因此，为探究调控繁殖过程的mRNA与长链非编码RNA调控机制，本研究收集了三个发育阶段的半滑舌鳎卵巢组织进行RNA测序。在IV期与V期卵巢的比较组中，本研究共鉴定出312个差异表达（differentially expressed，DE）mRNA以及58个差异表达长链非编码RNA；在V期与VI期卵巢的比较组中，共鉴定出1059个差异表达mRNA以及187个差异表达长链非编码RNA。基因本体（Gene Ontology，GO）与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）富集分析结果显示，差异表达mRNA显著富集于ECM-受体相互作用、卵母细胞减数分裂以及类固醇激素生物合成通路。此外，本研究还开展了基因集富集分析（gene set enrichment analysis，GSEA），以进一步筛选潜在的繁殖相关通路，例如脂肪酸代谢与视黄醇代谢通路。基于富集分析结果，本研究筛选出具有潜在繁殖调控功能的差异表达mRNA，并将其分为六大类：信号转导、细胞生长与死亡、免疫应答、代谢、转运与分解代谢以及细胞连接。研究人员基于反义、顺式以及反式调控机制，预测了差异表达长链非编码RNA与mRNA之间的相互作用关系，并构建了内源竞争RNA（competing endogenous RNA，ceRNA）调控网络。本研究预测多个长链非编码RNA可调控与繁殖相关的基因，包括cyp17a1、cyp19a1、mmp14、pgr以及hsd17b1。对上述长链非编码RNA靶基因进行功能富集分析后发现，这些靶基因参与了多条信号通路，包括TGF-beta信号通路、Wnt信号通路以及MAPK信号通路；同时还参与了多项繁殖相关通路，例如孕酮介导的卵母细胞成熟、卵母细胞减数分裂以及GnRH信号通路。实时定量荧光PCR（RT-qPCR）分析结果显示，两个长链非编码RNA（XR_522278.2与XR_522171.2）主要在卵巢组织中表达。双重荧光原位杂交实验结果显示，XR_522278.2与XR_522171.2分别与其靶基因cyp17a1和cyp19a1在卵泡细胞层中共定位。上述结果进一步证实，长链非编码RNA可能通过调控基因表达参与相关生物学过程。综上，本研究构建了半滑舌鳎卵巢组织的长链非编码RNA表达谱，并为解析长链非编码RNA在半滑舌鳎繁殖调控过程中的作用提供了新的见解。