miRNA-216 and miRNA-499 target cyb561d2 in zebrafish in response to fipronil exposure ☆

Abstract MicroRNA (miRNA) can regulate the expression of its target gene by mediating mRNA cleavage or by translational repression at a post-transcriptional level. Usually, one miRNA may regulate many genes as its targets, while one gene may also be targeted by many miRNAs. We previously demonstrated that cyb561d2 , whose protein product is involved in cell defense, and chemical stress, is targeted by miR-155 in adult zebrafish ( Danio rerio ) when exposed to fipronil (5-amino-1-[2,6-dichloro-4-(trifluoromethyl) phenyl]-4-[(trifluoromethyl) sulphinyl]-1H-pyrazole-3-carbonitrile). Microcosm Targets prediction showed that the cyb561d2 gene is also highly possibly targeted by miR-194a , miR-216b , miR-429 , and miR-499 . These interactions need to be further validated experimentally. In this study, we evaluated the effects of fipronil on miR-194a , miR-216b , miR-429 , miR-499 and cyb561d2 in zebrafish and investigated whether these four miRNAs c ould regulate the expression of cyb561d2 in both mRNA and protein levels. The expression of cyb561d2 was upregulated in both mRNA and protein level in a dose-dependent manner upon stimulation of fipronil, and miR-216b and miR-499 were downregulated concurrently, whereas there was no significant changes were observed in the expression level of miR-194a and miR-429 . The dual luciferase report assay demonstrated that miR-216 b and miR-499 interacted with cyb561d2 3′-untranslated regions (3′-UTR), miR-194a and miR-429 did not stimulate degradation of cyb561d2 mRNA. The expression of cyb561d2 was reduced in both mRNA and protein level when ZF4 cells were transfected with miR-499 mimic, whereas expression level of both mRNA and protein was increased when endogenous miR-499 was inhibited by transfection with miR-499 inhibitor. Likewise, the mRNA and protein level of cyb561d2 was affected by treatment with the mimics and the inhibitor of miR-216b . In contrast, when ZF4 cells were transfected with a mimic of miR-194a or miR-429 , the expression of cyb561d2 mRNA was not significantly changed. As a result, cyb561d2 is targeted by miR-155 , miR-216b and miR-499 upon fipronil exposure, and miR-194a and miR-429 can not target cyb561d2 . The expression pattern of these 3 miRNAs presents novel fipronil responses that could be used as a toxicological biomarker.