Regulation Of PARP1 By MicroRNA-7a In Zebrafish Embryos In Exposure To DEHP At Low Concentrations

Di(2-ethylhexyl)phthalate(DEHP)is a kind of phthalates.This kind of liquid is colorless,viscous,lipophilic and hydrophobic.It is mainly used as a plasticizer to soften plastics.miRNA is a type of biological endogenous non-coding single-stranded smallmolecule RNA.It is in a length of about 20-24 nucleotides.It is involved in regulation of genes in organisms at transcription and post-transcriptional level.Studies have shown that DEHP exposure caused DNA damage and may interfere with cell cycle.Chronic low-dose exposure to a mixture of environmental endocrine disruptors induced miRNAs deregulation in mouse concomitant with intratesticular estradiol reduction.DEHP can induce neurotoxicity through oxidative stress,but the other molecular mechanisms remain to be further studied.miR-7a plays an important role in the development of normal brain tissue,pancreatic β cells,heart tissue,and skin.miR-7a affects the migration and proliferation of mouse neural stem cells by regulating the target gene cdc42,thereby inhibiting peripheral nerve repair.Thus,miR-7a may be involved in DEHP-induced neurotoxicity.In this research,we screened PARP1 as the target gene of miR-7a through bioinformatics analysis.Then we explored whether DEHP affects the normal development of zebrafish embryos by regulating miR-7 and its target gene PARP1.During the experiments,we constructed four recombinant plasmids GL3-PARP1-WT,pGL3-PARP1-MUT,eGFP-PARP1-WT,eGFP-PARP1-MUT.Using dual-luciferase reporter gene assay and dual-fluorescent protein assay,we found that miR-7a can interact with PARP1 3’-UTR.When miR-7a mimic was transfected into zebrafish ZF4 cells,the expression of PARP1 was significantly reduced at m RNA level.When miR-7a inhibitor was transfected,the expression of PARP1 was significantly increased at m RNA level.This demonstrated that miR-7a has a negative effect on PARP1 expression at the post-transcriptional level.As the results of the acute toxicity experiment,the cell viability of zebrafish ZF4 cells was significantly reduced when the DEHP concentration was 50,100,200 mg/L,and the it was even reduced to 67.61% compared with the blank group when the DEHP concentration was 400 mg/L.However,it had no significant effect on the proliferation of zebrafish ZF4 cells exposed to DEHP even if miR-7a overexpressed(mimic)or was inhibited(inhibitor).In addition,exposure to DEHP at low concentrations can also affect early development of zebrafish embryos and reduced their survival rate.In the zebrafish embryos exposed to DEHP at different concentrations,miR-7a expression and DEHP concentration is positively correlated,while PARP1 expression and DEHP concentration is negatively correlated.However,there was no significant correlation between the expression of miR-7a and PARP1.In summary,DEHP can affect the growth and development of zebrafish embryos by regulating the expression of miR-7a.However,the molecular mechanism of how miR-7a regulates its target gene PARP1 remains to be determined.