| Objective:Non-alcoholic fatty liver disease(NAFLD)has become a widespread epidemic.We designed experiments to validate the altered m6A methylation levels of CYP450 family member CYP2B6 in a model of NAFLD and explored the regulatory mechanisms that modulate the m6A methylation modifications occurring in CYP2B6 to provide direction for the pathogenesis as well as the treatment of NAFLD.Methods:1.Establishment of a mouse model of hyperlipidemia and insulin resistance.2.The liver tissue of the mice that was left to be retained was HE-stained to determine the success of the high-fat diet model.Further use of epigenome gene chip technology to determine the differences in the m6A methylation modification map of the differential gene.The potential role of these differentiated genes in fatty liver formation and the potential regulatory pathways of these genes are predicted by means of bio-letter analysis.3.The group of genes with the highest relevance in regulating m6A methylation changes was selected and the m6A modification changes of this family of genes were mapped using gene microarray technology.4.MeRIP-qPCR assay was performed to verify the elevated level of m6A methylation of mouse Cyp2b10 gene in steatotic livers.Liver tissue RNA from experimental and control groups was extracted for qPCR experiments and paraffin sections were made for immunohistochemical experiments to determine the expression level of the target gene in fatty liver.5.Steatosis of human normal hepatocytes LO2 was induced with a 2:1 mixture of oleic palmitic acid by oil red staining and oil red semi-quantitative assay to determine the optimal concentration to induce steatosis in hepatocytes.The optimal concentration for induction was also screened by combining MTT assay to see the magnitude of the toxic effects of different concentrations of fatty acids on hepatocytes.6.Hepatocyte steatosis was induced with the optimal concentration of oleic palmitic acid mixture for 24 hours,RNA was extracted,and qPCR experiments were performed.And proteins were collected and Western Blotting experiments were performed to verify the changes in expression of target genes in the in vitro hepatic steatosis model.7.RNA and protein were extracted from steatotic liver of mice,and qPCR and Western Blotting experiments were performed to identify METTL3,a methylesterase that regulates the occurrence of high m6A methylation changes in fatty liver,by screening the expression levels of m6A methylesterase(Writers),demethylase(Erasers),respectively.8.According to the m6A site diagram of the CYP2B6 gene,the primer of different sites is designed,and the site of m6A methylation modification of the CYP2B6 gene is verified by MeRIP-qPCR technology.The plasmids included wild or mutant genes of regulatory sites are designed,and the regulatory effect of the sites is verified by dual luciferase reporter gene assays.9.The regulatory effect of METTL3 on the CYP2B6 gene was verified by infecting cells with lentivirus based on LV3 and LV5,interfering with or overexpressing METTL3,respectively.Extracting proteins,detecting changes in expression levels of β-ACTIN,METTL3 and CYP2B6,respectively.10.The METTL3 gene was knocked out with small interference RNA,and fatty acids were used to induce steatosis in liver cells,grouped into control group,simple fat change group,simple METTL3 interference group,fat variant and METTL3 interference group.The cells were oil-red O-stained and the differences of the fat droplets forming between the groups were compared to verify the role of CYP2B6 m6A methylation modification induced by METTL3 gene in fatty liver.11.The experiment was divided into control group,simple steatosis group,simple METTL3 interference group,steatosis combined METTL3 interference group,to extract membrane protein and cytoplasmic protein respectively,and then measure the expression of GLUT4.12.The experiment was divided into control group,simple steatosis group,simple METTL3 interference group,steatosis combined METTL3 interference group.Extracted the total protein of each group,using Western Blotting experiment to verify the expression level of the IRS,PI3K,ERK,AKT and their phosphorylation level in the insulin signaling pathway,and explored whether the m6A methylation modification regulated by METTL3 had an effect on insulin resistance in NAFLD patients.13.The experiment was grouped into simple METTL3 interference group,steatosis combined METTL3 interference group,METTL3 interference and CYP2B6 overexpression group,steatosis combine METTL3 interference and CYP2B6 overexpression group,extracted total proteins from each group.Using Western Blotting experiment to verify the expression level of the IRS,PI3K,ERK,AKT and their phosphorylation level in the insulin signaling pathway.Investigating whether the reversal effect of CYP2B6 on m6A methylation modification had an effect on insulin resistance in NAFLD patients.14.The cultured LO2 was divided into control group,simple steatosis group,simple CYP2B6 overexpression group,steatosis and CYP2B6 overexpression group,extracted total proteins from each group,using Western Blotting experiment to verify the expression level of the factor of insulin signaling pathway.Results:1.The mice in the HFD group had a significant increase in body weight compared with the control group,and significant lipid droplets showed in the HFD mice liver.A pathological state of insulin resistance also shown,which means the NAFLD model was successfully constructed.The HFD group showed changes in the expression profile of m6A methylation modifications.GO/pathway analysis showed significant enrichment into metabolic pathway and insulin resistance signaling pathway.2.In mice,Cyp2b10,which is homologous to the human CYP2B6 gene,underwent elevated levels of m6A methylation and differential overexpression.3.In vitro experiments,the best induction was achieved with a 1 mM PA:OA mixture.The expression level of CYP2B6 was elevated in the steatosis LO2 cells.It was consistent with the results of mouse model experiments.4.Methylation enzyme METTL3 showed a significant differential overexpression,and the changes were statistically significant.METTL3 acts as a methylation transferase that directly regulates CYP2B6 to undergo changes in m6A methylation.The results of MeRIP-qPCR showed that the sites regulating the occurrence of m6A methylation in CYP2B6 were located at position 66 and position 1291.5.METTL3-regulated m6A methylation change of CYP2B6 do not play a significant role in fat formation in NAFLD,but rather inhibit insulin sensitivity by regulating molecules of the insulin signaling pathway such as insulin receptor substrates(IRS),phosphoinositide 3-kinase(PI3K),serine/threonine kinase(AKT),and glucose transporters(GLUT4).-kinase(PI3K),serine/threonine kinase(AKT),and glucose transporters(GLUT4)to inhibit insulin sensitivity.Conclusion:METTL3-regulated m6A methylation modification of CYP2B6 contributes to insulin resistance in NAFLD patients by regulating the IRS/PI3K/AKT/GLUT4 signalling pathway. |
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