Study On The Mechanism Of MiR-193b-5p Regulating Acrylamide-induced Cell Cycle And Cell Proliferation In Rat BRL-3A Cells By Targeting FoxO3

Acrylamide(AA)is an important by-product of Maillard reaction,which is genotoxic and carcinogenic agent.AA can induce hepatotoxicity by inducing cell apoptosis.Cell apoptosis or necrosis and cell proliferation are often in a dynamic balance in organisms,that is,after cells entering an apoptotic state,its proliferation ability may also be affected,and cell proliferation is closely related to the cell cycle.However,although in recent years,studies have shown that AA can induce cell cycle arrest and cell proliferation inhibition,its underlying mechanism remains unclear.In addition,miRNAs play a vital role in cell process,which can participate in a variety of cell processes,including apoptosis,cell cycle,cell proliferation,etc.Therefore,the study aims to explore the effect of AA on the cell cycle and cell proliferation,clarify the potential mechanism of miRNA in the process of AA-induced cell cycle arrest and cell proliferation inhibition,supply the hepatotoxic mechanism of AA from the level of miRNA,and provide new ideas for the prevention of AA hepatotoxicity.The main research contents and results of this paper are as follows:(1)First,the rat hepatocyte BRL-3A was treated with AA,and the AA-induced BRL-3A cells were analyzed by Small RNA sequencing technology to find miRNAs with significant difference.On the basis of determining the credibility of the sequencing results,through GO biological function enrichment analysis and KEGG pathway enrichment analysis,the FoxO signaling pathway involved in cell cycle and cell proliferation process regulation and miR-193b-5p that target the FoxO3 gene are selected for in-depth study.(2)Secondly,analyze the potential mechanism of AA-induced cell cycle arrest.FoxO signaling pathway is mainly involved in the regulation of cell cycle and cell proliferation process.The downstream functional proteins of FoxO3,p21 and Cyclin D1,are mainly involved in the regulation of cell cycle G1 phase.In BRL-3A cells induced by AA,the expression of FoxO3 and p21 was upregulated and Cyclin D1 was downregulated.After AA treatment,the expression of p21 in the cell was upregulated.In addition,through the analying of cell cycle by flow cytometry,it was indicated that AA can induce the arrest of BRL-3A cell cycle in G1 phase.In order to further explore the underlying mechanism of the key gene FoxO3 in the process of AA-induced cell cycle G1 arrest,the experiments of knockdown or overexpressing FoxO3 were performed on BRL-3A cells.In AA-treated cells,FoxO3 can also regulate the levels of Cyclin D1 and p21.Therefore,it was shown that FoxO3 can participate in the cell cycle G1 phase arrest induced by AA.(3)Finally,analyze the potential mechanism of miR-193b-5p in AA-induced cell cycle arrest and cell proliferation inhibition.The arrest of the cell cycle G1 phase often influences the process of cell proliferation.Therefore,by exploring how miR-193b-5p induces cell proliferation in BRL-3A cells in AA,the regulation of AA potential mechanism in AA hepatotoxicity can be further clarified.Transfection of miR-193b-5p mimics in BRL-3A cells could downregulate the level of FoxO3 in the cells,while miR-193b-5p inhibitor could inhibit the level of miR-193b-5p and increase the level of FoxO3.In addition,the wild and mutant FoxO3 plasmids were constructed according to the predicted target sites,and the target relationship between miR-193b-5p and its predicted target gene FoxO3 was further confirmed through the dual-luciferase reporter gene experiment.On the basis of determining the targeting relationship,transfecting miR-193b-5p mimics or miR-193b-5p inhibitor into BRL-3A cells further proved the potential mechanism of miR-193b-5p involved in regulating AA-induced BRL-3A cell cycle and cell proliferation inhibition.Finally,cotransfection experiments about miR-193b-5p inhibitor or miR-193b-5p mimics with siFoxO3 or vector-FoxO3 further indicated the effect of the interaction between miR-193b-5p and FoxO3 on cell cycle and cell proliferation.Therefore,it is indicated that miR-193b-5p can regulate AA-induced cell cycle G1 arrest and cell proliferation inhibition by targeting and regulating the expression of FoxO3.In summary,this study starts from the miRNA level and analyzes the mechanism of hepatotoxicity of AA by studying the regulation of miRNA on mRNA.It was found that AA can induce cell cycle G1 arrest and cell proliferation inhibition in rat hepatocyte BRL-3A cells,by inhibiting the expression of miR-193b-5p,and promoting the expression of FoxO3.And miR-193b-5p can regulate AA-induced cell cycle G1 arrest and cell proliferation inhibition by targeting FoxO3,and miR-193b-5p plays an important role in reducing AA liver toxicity.This study explored the potential mechanism of AA cell cycle and cell proliferation toxicity,opened up a new way for in-depth study of the mechanism of AA hepatotoxicity,and provided new ideas for the prevention of AA toxicity.