Sex-biased miR-456 targeting spire1/sox11 provides insights into the ceRNA-network of sexual regulation in fish

Competing endogenous RNAs (ceRNAs) are a novel epigenetic regulatory mechanism implicated in sex determination and differentiation in teleosts, which exhibit a diversity of sex-determining mechanisms. In this study, based on whole transcriptome sequencing data, a ceRNA regulatory network composed of sex-inclined miRNAs (miR-456), lncRNAs ( ASTR ), circRNAs ( circklhl29 ), and target mRNAs ( spire1 and sox11 ) was uncovered in the gonadal tissues of Seriola dumerili , an oceanic species with lagged gonadal development. MiRNA-456 was identified as the core of the ceRNA regulatory network and suppressed the expression of spire1 and sox11 , interacting with the ncRNAs including circklhl29 and ASTR . Co-localization of these sex-biased ncRNAs and mRNAs in the gonads suggests that this ceRNA network modulated the gonadal differentiation in both sexes of S. dumerili . In vivo injection in the gonads of S. dumerili suggested the miR-456 agonist could significantly inhibit expression of sox11 and spire1 in male testes, while significant regulatory effects of the miR-456 agonist and antagonist on sox11 and spire1 targets were not observed in female ovaries. The conserved binding sites for sequences of miR-456 and spire1/sox11 targets in various fish species were aligned and dual luciferase reporter gene experiments clarified the universality of the mechanism by which miR-456 bound to inhibit sox11/spire1 targets in large yellow croaker ( Larimichthys crocea ), half-smooth tongue sole ( Cynoglossus semilaevis ), and zebrafish ( Danio rerio ). These results further support the notion that ceRNA networks may be a universal regulatory system in teleosts despite their highly divergent sex regulation programs. • Sex-biased ncRNAs participated in gonad differentiation with sex-related genes by ceRNAs regulatory network • miR-456 targeted spire1 and sox11, while circklhl29, ASTR acted as sponges upregulated the expression of spire1 and sox11. • The ceRNA regulatory network provided a theoretical basis for the sex regulation by ncRNAs in fish.