环境与职业医学2024，Vol.41Issue(3)：323-329,7.DOI:10.11836/JEOM23333
人支气管上皮样细胞中苯并[a]芘代谢物-DNA加合物图谱:基于染色质免疫共沉淀测序技术
Map of benzo[a]pyrene metabolites-DNA adducts in human bronchial epithelial-like cells:Based on chromatin immunoprecipitation followed by sequencing technology
摘要
Abstract
[Background]The active metabolite of benzo[a]pyrene(BaP),7,8-dihydroxy-9,10-epoxybenzo[a]pyrene(BPDE),can form adducts with DNA,but the spectrum of BPDE-DNA adducts is unclear. [Objective]To identify the distribution of BPDE adduct sites and associated genes at the whole-genome level by chromatin immunoprecipitation followed by sequencing(ChIP-Seq),and serve as a basis for further exploring the toxicological mechanisms of BaP. [Methods]Human bronchial epithelial-like cells(16HBE)were cultured to the fourth generation in the logarithmic growth phase.Cells were harvested and added to chromatin immunoprecipitation lysis buffer.The lysate was divided into experimental and control groups.The experimental group received a final concentration of 20 μmol·L-1 BPDE solution,while the control group received an equivalent volume of dimethyl sulfoxide solution.The cells were then incubated at 37 ℃ for 24 h.Chromatin fragments of 100-500 bp were obtained through sonication.BPDE-specific antibody(anti-BPDE 8E11)was used to enrich DNA fragments with BPDE adducts.High-throughput sequencing was conducted to detect BPDE adduct sites.The top 1000 peak sequences were subjected to motif analysis using MEME and DREME software.BPDE adduct target genes at the whole-genome level were annotated,and Gene Ontology(GO)functional analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis of BPDE adduct target genes were con-ducted using bioinformatics techniques. [Results]The high-throughput sequencing detected a total of 842 BPDE binding sites,distributed across various chromosomes.BPDE co-valently bound to both coding and non-coding regions of genes,with 73.9％binding sites located in intergenic regions,19.6％in intronic regions,and smaller proportions in upstream 2 kilobase,exonic,downstream 2 kilobase,and 5'untranslated regions.Regarding the top 1000 peak sequences,four reliable motifs were identified,revealing that sites rich in adenine(A)and guanine(G)were prone to binding.Through the enrichment analysis of binding sites,a total of 199 BPDE-adduct target genes were identified,with the majority located on chromosomes 1,5,7,12,17,and X.The GO analysis indicated that these target genes were mainly enriched in nucleic acid and protein binding,participating in the regulation of catalytic activity,transport activity,translation elongation factor activity,and playing important roles in cell division,differentiation,motility,substance transport,and information transfer.The KEGG analysis revealed that these target genes were primarily enriched in pathways related to cardiovascular diseases,cancer,and immune-inflammatory responses. [Conclusion]Using ChIP-Seq,199 BPDE adduct target genes at genome-wide level are identified,impacting biological functions such as cell division,differentiation,motility,substance transport,and information transfer.These genes are closely associated with cardiovascular diseases,tumors,and immune-inflammatory responses.关键词
7,8-二羟-9,10-环氧苯并[a]芘/人支气管上皮样细胞/染色质免疫共沉淀测序/DNA加合物/生物信息学Key words
7,8-dihydroxy-9,10-epoxybenzo[a]pyrene/human bronchial epithelial-like cell/chromatin immunoprecipitation followed by sequencing/DNA adduct/bioinformatics分类
医药卫生引用本文复制引用
冀婷玉,曹彬,吕懿,佟晓敏,孙宏宇,郑金平..人支气管上皮样细胞中苯并[a]芘代谢物-DNA加合物图谱:基于染色质免疫共沉淀测序技术[J].环境与职业医学,2024,41(3):323-329,7.基金项目
山西省重点研发计划(国际合作)项目(201703D421021) （国际合作）
山西省"1331工程"提质增效项目(2021-5-2-2-B1) （2021-5-2-2-B1）
山西省基础研究计划(自由探索类)青年科学研究项目(20210302124301) This study was funded. （自由探索类）
