The Molecular Mechanism Of BaP Regulating FGF-21 To Affect Liver Lipid Metabolism Based On Bioinformatics Was Investigated

Objective:In this study,bioinformatics and in vitro experiments were used to verify the role and molecular mechanism of regulating FGF-21 by BaP on liver lipid metabolism.Methods:（1）The differentially expressed genes and Hub genes of Bap-induced liver cells were searched in GEO database and enrichment analysis.（2）Search for NAFLD-related targets in GeneCards database,and enrichment analysis of screened genes which was carried out based on Protein Coding.（3）The intersection of BaP differential genes and NAFLD screening genes was used to obtain candidate genes,and than the ROC curve and immune correlation analysis of candidate genes were performed.（4）Docking key genes of candidate genes with AhR protein and bioinformatics analysis,meanwhile to find interacting proteins.（5）HepG2 cells were given different concentrations of BaP(0,1,2,4,8,16,32,64,128,256,512μmol·L^-1)to detect the effects of BaP on the growth viability of HepG2 cells by MTT.（6）According to the concentration screened by MTT,the follow-up test was conducted,the key factors screened by bioinformatics and lipid metabolism genes to were detected by Western Blot and qPCR.Results:（1）Compared with the control group,the BaP group had 393 up-regulated genes,59 down-regulated genes and 20 Hub genes.GO enrichment was mainly related to the regulation of cell death,growth factor receptors,plasma membrane and cell membrane and so on.KEGG enrichment was related to RAS signaling pathway,cancer signaling pathway and other signaling pathways.（2）1163 NAFLD target genes were obtained,and 1042 were obtained after screening.GO enrichment was mainly related to the normal regulation of metabolism and protein complex binding and so on.KEGG enrichment was related to the PPAR pathway,the metabolic pathway of cytochrome P450 heterologous species and other signaling pathways.（3）Four candidate genes,FGF-21,CCL5,CCL11 and LPA,were obtained by intersection of BaP differential genes and NAFLD target genes.ROC curve and immunoinfiltration correlation indicated that candidate genes could be used as biomarkers of NAFLD and liver cancer progression in normal liver.FGF-21 was the central target of candidate genes.（4）Ten docking predicted structures of AhR and FGF-21 proteins were obtained.Twelve miRNAs related to FGF-21 were obtained.Four co-expressed genes with FGF-21 were obtained.The prognosis of FGF-21 high expression group was better than that of low expression group.FGF-21 was highly correlated with macrophage M0,macrophage M1 and monocytes in tumor microenvironment and immune infiltration.The interaction of FGF-21 proteins predicted PPARα,PPARγ,C/EBPα,UCP1,MLXIPL,ACACA,ACOX1,CPT-1α,FASN,SIRT1,SCD1,SREBF1 and PGC1α.（5）MTT results showed that after 24 hours of treatment,BaP concentration at 0-64μmol·L^-1have no effect for the proliferation of HepG2 cells.When BaP concentration was 64-512μmol·L^-1,HepG2 cell proliferation was inhibited.So that 15,30,and 60μmol·L^-1were selected as subsequent experimental concentrations.（6）HepG2 cells given 15,30 and 60μmol·L^-1BaP,compared with control group,mRNA and protein expression of FGF-21 in 30μmol·L^-1BaP group were increased,the differences were statistically significant（P<0.01）.The mRNA and protein levels of FGF-21 were inhibited in 60μmol·L^-1BaP group,and the difference was statistically significant（P<0.05）.（7）The mRNA and protein expressions of CPT-1α,PPARα,PGC1α,PPARγ,SCD1 and C/EBPαin 30μmol·L^-1BaP group were increased compared with those in control group.The difference was statistically significant（P<0.05,P<0.01）.Compared with 30μmol·L^-1BaP group,60μmol·L^-1BaP group inhibited the mRNA and protein levels of CPT-1α,PPARα,PGC1α,PPARγ,SCD1 and C/EBPα.The difference was statistically significant（P<0.05）.Conclusion:According to the bioinformatics screening results,FGF-21,CCL5,CCL11 and LPA can be used as the key factors affecting the occurrence and development of NAFLD by BaP,in which FGF-21 is the central gene.In vitro experiments showed that the regulation of AhR by BaP affects the expression of FGF-21,which in turn affects the expression of lipid metabolism-related genes in HepG2 cells,interferes with lipid metabolism in vivo,and ultimately affects the occurrence and development of related diseases.