肝癌细胞株-乙型肝炎病毒DNA整合事件的高通量靶向捕获测序分析  

Analysis of HBV DNA integration events in hepatocellular carcinoma cell lines by high throughput targeted capture sequencing

作　　者：羊泽锐 邓海君[1] 胡源[1] YANG Zerui;DENG Haijun;HU Yuan(Key Laboratory of Molecular Biology on Infectious Diseases of Ministry of Education,Chongqing Medical University,Chongqing,400016,China)

机构地区：[1]重庆医科大学感染性疾病分子生物学教育部重点实验室,重庆400016

出　　处：《陆军军医大学学报》2024年第2期110-117,共8页Journal of Army Medical University

基　　金：科技部重点研发计划(2018YFE0107500);重庆市教委科学技术研究项目(KJQN202100422);重庆市渝中区自然基金(20210116)。

摘　　要：目的 采用探针捕获和高通量测序技术研究HepG2.2.15与HepAD38 2个乙型肝炎病毒(hepatitis B virus, HBV)肝癌细胞系中病毒DNA在宿主基因组中的整合位点特征。方法 提取HepG2.2.15与HepAD38细胞基因组DNA,采用探针捕获和高通量测序策略对2个肝癌细胞株中整合的HBV DNA进行捕获测序,使用Trimmomatic、BBAP、BWA(Burrows-Wheeler Aligner)等生物信息学软件进行数据分析;最后通过聚合酶链反应(polymerase chain reaction, PCR)及克隆测序对病毒DNA整合特征进行验证。结果 利用探针捕获和高通量测序技术分别在HepG2.2.15与HepAD38细胞系中检测出12、7个HBV DNA整合事件,随机分布在chr1、chr2、chr7、chr9、chr16、chr17、chr19、chrX染色体上,整合位点上下游基因出现频率较高的有DPP7、TRIM56、GPC3等。结论 成功建立基于探针捕获和高通量测序技术检测HBV DNA在宿主基因组上的整合片段的方法,HepG2.2.15与HepAD38细胞系中HBV整合事件在染色体上随机发生,大部分整合位点发生在HBV基因组上的X基因区域。Objective To investigate the integration characteristics of hepatitis B virus(HBV)viral DNA in the host genome of hepatocellular carcinoma HepG2.2.15 cells and HepG2-derived cell line HepAD38 by probe-based capture and high-throughput sequencing technology.Methods After the genomic DNA of HepG2.2.15 and HepAD38 cells were extracted,probe-based capture technique was used to capture the integrated DNA fragments of HBV DNA in the obtained genomic sequences,and the intergrated fragments were further studied with high-throughput sequencing technology.The bioinformatics software such as Trimmatic,BBAP,and BWA(Burrows-Wheeler Aligner)were empolyed for data analysis.Finally,the characteristics of integrated DNA were validated through PCR and cloning and sequencing.Results The probe-based capture and high-throughput sequencing technology detected 12 and 7 integrated DNA fragments in HepG2.2.15 and HepAD38 cells,respectively.These integration events were randomly distributed on chromosomes chr1,chr2,chr7,chr9,chr16,chr17,chr19,and chrX.DPP7,TRIM56 and GPC3 appeared with higher frequencies at the upstream and downstream of the integration sites.Conclusion We successfully establish a method to detect and analyze the integrated DNA fragments of HBV DNA in host genome based on probe-based capture and high-throughput sequencing technology.HBV integration events occur randomly on the chromosomes in HepG2.2.15 and HepAD38 cells,and most of integration sites are in the X gene region of the HBV genome.

关 键 词：乙型肝炎病毒 HBV整合 探针捕获 高通量测序 克隆测序 

分 类 号：R373.21[医药卫生—病原生物学] R394.3[医药卫生—基础医学] R735.7