The Roles Of MiR-490-5p In Perfluorooctanoic Acid (PFOA)-induced Developmental Cardiotoxicity In Chicken Embryos

Objective:The aims of this study were to evaluate the developmental cardiotoxicity induced by perfluorooctanoic acid(PFOA)exposure in chicken embryos,and to investigate the potential roles of miR-490-5p in such toxicities with a series of molecular biology experimental means.This study revealed molecular mechanism of PFOA-induced developmental toxicities,provided new information about the toxicological information of PFOA,and contributed to the scientific evidences for associated human health risks.Methods:This study was divided into two sets of experiments.The first set investigated the toxicological effects following exposure to various doses of PFOA,while the second set investigated the effects of miR-490-5p overexpression/silencing lentivirus treatment.The first set of experiments included four groups: vehicle control(sunflower oil),0.5 mg/kg PFOA exposure,1 mg/kg PFOA exposure,and 2 mg/kg PFOA exposure.After hatching,the hatchling chickens were anesthetized to perform electrocardiography and then sacrificed,heart samples were collected.After histological processing,hematoxylin-eosin staining(HE staining)was performed to visualize the pathological changes of heart tissues.Meanwhile,the levels of differentially expressed miRNA/m RNA in PFOA-exposed heart tissues were analyzed with RNA sequencing.With additional quantitative real-time PCR(q RT-PCR)confirmation and bioinformatics analysis,the most significantly differentially altered miRNA miR-490-5p and its downstream target genes synaptic vesicle-associated protein 91(SNAP91)and LY6/PLAUR structural domain protein 6(LYPD6)were identified and selected for further investigation in the follow-up studies.In the miR-490-5p overexpression/silencing lentivirus treatment experimental set,miR-490-5p’s roles in PFOA-induced developmental cardiotoxicity was verified bidirectionally via in ovo lentivirus transfection in chicken embryos.Five groups were included: vehicle control,control lentivirus,2 mg/kg PFOA exposure,miR-490-5p overexpression/silencing lentivirus and PFOA exposure + miR-490-5p overexpression/silencing lentivirus.The developmental cardiotoxicity following miR-490-5p overexpression/silencing were assessed via ECG and HE staining as described in the previous experimental set,and the efficiency of miR-490-5p overexpression/silencing was quantified with q RT-PCR.q RT-PCR and western blotting were performed to investigate the expression levels of miR-490-5p and the m RNA/protein expression levels of its two downstream target genes SNAP91 and LYPD6.Results:The results of pathological and functional analysis on the chicken hearts revealed significant decreases in right ventricular wall thickness and significant increases in heart rate following exposure to 1 or 2 mg/kg PFOA,suggesting developmental cardiotoxicity at such doses.RNA sequencing analysis between vehicle control and 2 mg/kg PFOA exposed samples identified 21 differentially expressed miRNAs and 1142 differentially expressed m RNAs,which were mainly enriched in GO terms such as metabolism and transportation;the most significantly enriched KEGG pathway was the metabolic pathway.Among the differentially expressed miRNAs,miR-490-5p was significantly upregulated in the chicken hearts developmentally exposed to 1 or 2 mg/kg PFOA(48.0% and 64.3% relative to control,respectively)as confirmed with q RT-PCR.Both the results of target gene prediction and bioinformatics analysis of differential miRNAs revealed a strong correlation between miR-490-5p and SNAP91/LYPD6,suggesting SNAP91 and LYPD6 as potential downstream target genes of miR-490-5p.In the miR-490-5p overexpression/silencing lentivirus experimental set,lentivirusmediated miR-490-5p overexpression and silencing efficacy was 78.7% and 66.7%,respectively.miR-490-5p overexpression mimicked PFOA exposure,leading to thinning of right ventricular wall and elevated heart rate in hatchling chickens.Co-treatment with both PFOA and miR-490-5p overexpression resulted in more prominent changes.In contrast,miR-490-5p silencing attenuated the developmental cardiotoxicity induced by PFOA exposure in chicken embryos,abolishing the right ventricular wall thinning and heart rate elevation induced by PFOA exposure.In addition,it was observed that both m RNA and protein expression levels of miR-490-5p potential downstream target genes,SNAP91 and LYPD6,were significantly decreased in heart tissues;similarly,overexpression of miR-490-5p also reduced the expression levels of both target genes;the expression levels were even lower after co-treatment with both PFOA exposure and miR-490-5p overexpression.On the other hand,miR-490-5p silencing increased the expression levels of SNAP91 and LYPD6,which could effectively alleviate the expression downregulation caused by PFOA exposure.It is suggested that miR-490-5p participates in PFOA-induced developmental cardiotoxicity by regulating its downstream targets,SNAP91 and LYPD6.Conclusions:(1)Developmental exposure to PFOA in chicken embryos could lead to developmental cardiotoxicity,mainly manifested in the form of thinned right ventricular wall and elevated heart rate.(2)miRNAs are involved in PFOA-induced developmental cardiotoxicity,among which miR-490-5p is a potential molecular target of PFOA developmental cardiotoxicity,whose expression level is elevated in PFOA-exposed chicken heart tissue samples.(3)SNAP91 and LYPD6 are involved in PFOA-induced developmental cardiotoxicity as miR-490-5p downstream target genes.miR-490-5p/SNAP91/LYPD6 were new molecular targets in PFOA-induced developmental cardiotoxicity.