Identification of functional ncRNAs in the liver reveals a novel regulatory mechanism associated with the dynamic balance of growth and reproduction in Cynoglossus semilaevis

Non-coding RNAs (ncRNAs) play crucial roles in the growth and reproduction of fish. However, the involvement of ncRNAs in the growth and reproductive balance of Chinese tongue sole (Cynoglossus semilaevis) from the late vitellogenesis stage to postovulation stage remains unexplored. Thus, to elucidate the regulatory mechanisms underlying the growth and reproductive balance regulated by ncRNAs, we performed whole transcriptome RNA sequencing of liver tissue at three ovarian stages, resulting in a total of nine libraries. In stages IV vs. V, 48 differentially expressed (DE) long non-coding RNAs (lncRNAs), 119 DE circular RNAs (circRNAs), 249 DE miRNAs, and 1797 DE mRNAs were identified. In stages IV vs. VI, we identified 209 DE lncRNAs, 417 DE circRNAs, 360 DE miRNAs, and 6760 DE mRNAs. In stages V vs. VI, we identified 133 DE lncRNAs, 376 DE circRNAs, 272 DE miRNAs, and 5129 DE mRNAs. Enrichment analysis revealed a significant enrichment of 116 DE mRNAs related to growth or reproduction in various pathways, including those related to arginine biosynthesis, steroid biosynthesis, steroid hormone biosynthesis, arginine and proline metabolism, and the insulin signalling pathway. Gene set enrichment analysis (GSEA) was further conducted to identify potential pathways related to growth-reproduction balance including the arginine biosynthesis pathway and steroid biosynthesis pathway. By utilizing antisense-, cis-, and trans-regulatory mechanisms, we have predicted that lncRNAs play regulatory roles in growth and reproduction-related genes, including odc1-a, hsd11b2, fdft1, mapk8, irs2, sms, cyp24a1, crkl, flot2a, inppi1a, prkab1, prkaca, prkar2a, gls, pik3cg, glul, cyp1a1, and soat2. By leveraging the network mechanism of competitive endogenous RNAs (ceRNAs), our study successfully predicted the intricate interactions among DE lncRNAs, DE circRNAs, DE miRNAs, and DE mRNAs including acacb, amd1, cyp27b1, faxdc2, glul, pik3cg, prkab1, and rps6kb2. A dual-fluorescence in situ hybridization assay revealed the colocalization of XR_522197.2 and glul in the liver parenchyma and around the central vein. The dual-luciferase reporter assay was employed to further demonstrate the regulatory relationships within the axis composed of TCON_00029307, novel_circ_008472, novel-m0618-5p, and cyp27b1. The comprehensive findings of this study outline the expression patterns of lncRNAs, circRNAs, and miRNAs in the liver of female C. semilaevis, providing a novel perspective that these three types of ncRNAs play critical roles in regulating the dynamic balance between growth and reproduction in this species.