Role of the cell cycle-related gene cdk2 and its associated ceRNA network in sexual size dimorphism of Cynoglossus semilaevis

Abstract

Sexual size dimorphism (SSD) in Cynoglossus semilaevis affects its annual production and restricts aquaculture development. Our previous multi-omics data analysis showed that cell cycle genes and the relevant non-coding RNAs (ncRNAs) were closely involved in SSD regulation. In this study, we analyzed cyclin-dependent kinase 2 (cdk2) gene together with its associated microRNA (miRNA) and long ncRNA (lncRNA) in C. semilaevis, predicting a competing endogenous RNA (ceRNA) regulatory network (MSTRG.24810.1-miR-460-cdk2) and verifying the targeting relationship using dual luciferase reporter assays. Expression profile analysis showed that cdk2 and the lncRNA MSTRG.24810.1 were highly expressed in female gonad and muscle, and their expression levels increased from 3-month-old (3M) to 8M. On the other hand, their negative regulator miR-460-x displayed lower expression in female than in male. After miR-460-x mimic transfection in C. semilaevis ovarian cells, the expressions of cdk2, cyclin E, and MSTRG.24810.1 were significantly decreased and cell cycle transition through G1 to S phase was obviously blocked. In vitro and in vivo experiments also indicated that RNAi-mediated knock-down of cdk2 caused down-regulation of MSTRG.24810.1 and other cell cycle related genes like cyclin E, cyclin A, e2f1, and h2b. Taken together, these results suggested that cdk2 gene and its associated ceRNA network may affect sex growth difference and differentiation of C. semilaevis individuals via regulating cell division and proliferation. The study will not only expand our knowledge on SSD regulatory mechanism, but also help to make an application on promoting growth and development of the fish.