Detection Of BPDE-added Target Genes In 16HBE Cells By ChIP-Seq

Objective:Benzo[a]pyrene(B[a]P)undergoes metabolic activation to form the final carcinogen7,8-dihydroxy-9,10-epoxybenzo[a]pyrene(BPDE),affinity site with DNA Covalent binding to form a BPDE-DNA adduct-induced gene mutation is one of the important mechanisms of benzo[a]pyrene(B[a]P)-induced tumors in lung cancer,but its target gene for adduct formation is currently Research is not yet comprehensive.This study aimed to comprehensively analyze the BPDE addition gene,its function and signaling pathway by chromatin immunoprecipitation sequencing(ChIP-Seq),and further study B[a]P for further searching for B[a]P target gene.The carcinogenic mechanism provides a scientific basis.Methods:Human bronchial epithelioid cell line 16HBE cells were selected for culture.After formaldehyde cross-linking,the cells were collected,the cell concentration was adjusted to2?10~7/ml,and the corresponding volume of cell lysis buffer was added.The lysate was divided into BPDE incubation group and DMSO solvent control group,and mixed with BPDE stock solution and equal volume DMSO,respectively.After homogenization and incubation at 37°C for 24 h,the cells were disrupted by sonication,and the chromatin was fragmented into a detectable size(100-500 bp),and the fragment size was detected by agarose gel electrophoresis.The BPDE-DNA complex was enriched using a standard immunoprecipitation method using Anti-BPDE body(Clone-8E11)and A/G magnetic beads.The BPDE-DNA complex was purified by elution and reverse cross-linking.After the PCR product was verified by PCR,the second generation sequencing technology was used to detect the BPDE addition gene.GO enrichment analyzes gene function,and KEGG analyzes genes involved in signaling pathways.Results:1.The distribution of BPDE binding sites in the whole genome:the number of binding sites detected is 842,the average length is 496 bp,and the distribution of BPDE binding sites on each chromosome,there are more binding sites on the 1,5,12,16th and X chromosomes.The BPDE binding site was mostly in the intergenic region(73.9%)and the intron region(19.6%),but also distributed in the exon region,2kb upstream of the transcription start site and 2kb downstream of the transcription start site.2.Characterization of BPDE-binding DNA sequences:Motif(motif)analysis,using MEME and DREME software to detect the significant Motif sequences in the top 1000 Peak sequences with the most significant enrichment,and predicting the four most likely Motif.The adenine(A)and guanine(G)content in the motif is higher,especially the highest content of guanine(G),suggesting that BPDE tends to bind to the DNA sequence enriched by guanine(G)and adenine(A).3.Distribution of BPDE-binding genes on chromosomes:218 of 842 binding sites are annotated as gene fragments,covering 199 genes,and each chromosome has a distribution of BPDE-binding genes,and there are more genes on 1,5,7,12 and X chromosomes.4.Functional analysis of BPDE binding gene:GO enrichment analysis,screening criteria is p-value<0.05,obtaining the first 10 GO terms at each level of molecular function,biological process,and cellular components.The BPDE-binding gene is mainly enriched in nucleic acid binding at the molecular functional level,including DNA bindingand RNA binding,as well as catalytic activity,molecular function regulation,transcriptional regulator activity and transport activity;biological process level mainly includes cell migration,cell cycle regulation,cell process and metabolic process,etc.;the cell component level mainly includes membrane components and organelles.5.Pathway analysis of BPDE binding gene:KEGG analysis,with p-value<0.05 as the screening standard,obtained the most significant 40 pathways involved in BPDE binding gene.Among them,the genes enriched in tumor pathways are the most,including small cell lung cancer,non-small cell lung cancer,renal cell carcinoma,endometrial cancer,gastric cancer,etc.The gene pathways enriched in tumor-related pathways include proteoglycans in tumors and tumors.Transcriptional disorders,chemical carcinogenesis,MAPK signaling pathway,apoptosis,sputum metabolic pathway,calcium signaling pathway,p53 signaling pathway,Ras signaling pathway,cytochrome P450 metabolic xenogeneic pathway.Mainly involved in ADH,PTEN,EEF2,GNB5,TIAM1,RRAS2,RAB37,CACNG3,TAOK1,Rap1,ACTG1,XRCC2,ALK,MCM9,ACACB,ACTB and other genes.Conclusion:1.The binding site of BPDE on human genomic DNA is distributed on each chromosome.The identified 199 additive genes are mainly located on chromosomes 1,5,7,12,17 and X.The binding sites of BPDE are mainly distributed in the intergenic and intron regions,and tend to bind to the DNA sequences enriched by guanine(G)and adenine(A).2.BPDE addition gene mainly affects cell cycle,apoptosis,migration,adhesion and other biological functions,and plays a certain role in signal transduction and biological regulation.Addition of genes to tumor-associated signaling pathways,including p53 signaling pathway,Ras signaling pathway,cytochrome P450 metabolic xenogeneic pathway,MAPK signaling pathway,apoptosis and the like,involving XRCC2,ALK,MCM9,TAOK1,RAB37 and the like.