The Regulation Of Cyb561d2by MicroRNA-155in Zebarifsh In Response To Fipronil Exposure

MiRNAs, a class of small (20～24nt) non-coding RNAs, exist widely in eukaryotes, whichare tissue specific and without open reading frame (ORF). MiRNAs can regulate gene expressionat the transcriptional or translational level, involving in cell growth, differentiation and apoptosis,and closely relating to the development of inflammation and cancer. Environmental toxicologicalstudies showed that expression of miRNAs would be changed when exposure to environmentalchemicals, and thus resulting in expression change of their target genes. Therefore, it isnecessary to elucidate the relationship among environmental chemicals, miRNAs and their targetgenes. For environmental toxicological studies and environmental monitoring, miRNAs are thepotential biomarkers for identifying genotoxicity and carcinogenicity of chemicals, and also canbe used to predict the toxicity of environmental chemicals.The toxicological effects of formulations of triazophos and fipronil and their mixture on adultzebrafish (Danio rerio) were evaluated using miRNA microarray in previously project by ourgroup. The results showed that totally21miRNAof219miRNAin the zebrafish were differentlyexpressed. Amomg these miRNA, miR-155was significantly downregulated due to thetreatments with fipronil.In this project, using bioinformatics methods, we predicted that cyb561d2is a target gene ofmiR-155, which is a member of the cytochrome b561(cyt b561) family involved in electrontransfer, cell defence, chemical stress, and has not been experimentally validated to date. Wetherefore exposed zebrafish to fipronil in gradient concentration and the expression of miR-155and cyb561d2were measured by qRT-PCR. Compared with the negative control, the fold changein miR-155expression was0.96after exposure to fipronil at a concentration of0.015mg/L; itsfold change significantly decreased to0.24after the exposure concentration was increased to0.48mg/L. Concurrently, the fold change in cyb561d2expression increased from1.94to4.17when the exposure concentration increased from0.015mg/L to0.480mg/L. Meanwhile, Westernblot analysis showed that the expression of the cyb561d2protein displayed the same pattern asits mRNAwith the increasing fipronil concentrations; its fold change increased from1.46to3.45when the exposure concentration increased from0.015mg/L to0.480mg/L.In order to test the prediction results of bioinformation methods and explain the co-expressionresult, we constructed four types of pRL-TK vectors for luciferase assays:1) vector contain fulllength cyb561d23’-UTR sequence;2) vector contain the putative miR-155-binding site and thesurrounding±30nt sequence;3) vector contain three head-tail tandem repeats of the miR-155predicted interaction site sequence;4) vector contain full length3’-UTR sequence but thepredicted interaction site deleted, then the plasmids were individually co-transfected with the pGL3-control vector into293T cells, along with miR-155mimic.The Dual luciferase reporterassay results demonstrated that cyb561d2is a target of miR-155.ZF4cells was utilised to verify whether the ectopic expression of miR-155could influencethe expression levels of cyb561d2mRNA and its protein. The miR-155mimic, miR-155inhibitor, were chemically synthesised and transfected into ZF4cells respectively to cause theaberrant expression of miR-155. The expression of cyb561d2was reduced in both mRNA (0.29)and protein (0.61) levels when ZF4cells were transfected with an miR-155mimic, whereas itsexpression levels of both mRNA (7.71) and protein (2.78) were increased when endogenousmiR-155was inhibited by transfection with an miR-155inhibitor, both compared with NTC; inorder to evaluated the effects of fipronil on miR-155and cyb561d2in zebrafish and investigatedwhether miR-155could mediate cell viability and proliferation by targeting cyb561d2.MTT assay was used to determine tothe IC50(17.1mg/L) of ZF4cells expose to fipronil, andthe cell viability and proliferation assays demonstrated that ZF4cell proliferation and survivalrate could be regulated by miR-155under IC50fipronil stress, miR-155significantly decreasedthe proliferation of ZF4cells, whereas the miR-155inhibitor increased cell proliferation andsurvival rate. Therefore, miR-155presents a novel fipronil response that could be used as atoxicological biomarker.miRNA and its target genes is not a one-to-one relationship, according to in silico analysis,miR-499, miR-429, miR-216b, miR-200b, miR-200c and miR-194a also target cyb561d2.