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

作　　者：冀婷玉 曹彬 吕懿[1] 佟晓敏 孙宏宇 郑金平[1,2] JI Tingyu;CAO Bin;LYU Yi;TONG Xiaomin;SUN Hongyu;ZHENG Jinping(Department of Hygienic Toxicology,School of Public Health,Shanxi Medical University,Taiyuan,Shanxi 030001,China;Department of Public Health and Preventive Medicine,Changzhi Medical College,Changzhi,Shanxi 046000,China)

机构地区：[1]山西医科大学公共卫生学院卫生毒理学教研室,山西太原030001 [2]长治医学院公共卫生与预防医学系,山西长治046000

出　　处：《环境与职业医学》2024年第3期323-329,共7页Journal of Environmental and Occupational Medicine

基　　金：山西省重点研发计划(国际合作)项目(201703D421021);山西省“1331工程”提质增效项目(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)。

摘　　要：[背景]苯并[a]芘(BaP)的活性代谢产物7,8-二羟-9,10-环氧苯并[a]芘(BPDE)可与DNA加合,但BPDE-DNA加合物图谱尚不明确。[目的]采用染色质免疫共沉淀测序技术(ChIP-Seq),在全基因组水平上鉴定BPDE加合位点分布及加合基因,为深入研究BaP的毒作用机制提供依据。[方法]人支气管上皮样细胞16HBE培养至第四代达对数生长期。收获细胞并加入染色质免疫共沉淀裂解缓冲液,将裂解产物等分为实验组和对照组,实验组添加终浓度20μmol·L^(–1)的BPDE溶液,对照组添加相同体积的二甲基亚砜溶液,在37℃环境下孵育24 h。超声获得100~500 bp的染色质小片段。使用BPDE特异性抗体(anti-BPDE 8E11)富集与BPDE加合的DNA片段,高通量测序检测BPDE加合位点,使用MEME和DREME软件对前1000个峰序列进行模体分析,注释BPDE在全基因组水平的加合靶基因,并通过生物信息学技术对BPDE加合靶基因进行基因本体论(GO)功能分析和京都基因与基因组百科全书(KEGG)通路分析。[结果]高通量测序共检测出842个BPDE结合位点,在各染色体上均有分布。BPDE可以与基因编码区和非编码区共价结合,73.9%的结合位点分布在基因间区,19.6%分布在内含子区,上游2千碱基区域、外显子区、下游2千碱基区域及5′非翻译区也有少量分布。对前1000个峰序列进行分析,寻找到4条可靠的模体,发现富含鸟嘌呤(G)和腺嘌呤(A)的位点易于结合。对结合位点进行富集,共鉴定出199个BPDE加合靶基因,大多分布在1号、5号、7号、12号、17号和X染色体上。GO分析显示,靶基因主要富集于核酸和蛋白质结合,参与调节催化活性、转运活性、翻译延伸因子活性,在细胞分裂、分化、运动、物质运输和信息传递方面发挥重要作用。KEGG分析显示靶基因主要富集于心血管疾病、癌症、免疫炎症反应等相关通路。[结论]利用ChIP-Seq在全基因组水平上共鉴定出199个BPDE加合靶�[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 wholegenome 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 20μmol·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 conducted using bioinformatics techniques.[Results]The high-throughput sequencing detected a total of 842 BPDE binding sites,distributed across various chromosomes.BPDE covalently 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

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 

分 类 号：R11[医药卫生—公共卫生与预防医学]