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

Abstract

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.