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

Zerui YANG; Haijun DENG; Yuan HU

doi:10.16016/j.2097-0927.202306066

您当前的位置：

首页 >

文章列表页 >

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

Basic Medicine | 更新时间：2026-01-09

- Analysis of HBV DNA integration events in hepatocellular carcinoma cell lines by high throughput targeted capture sequencing
- Journal of Army Medical University Vol. 46, Issue 2, Pages: 110-117(2024)
- 作者机构：
  
  400016 重庆, 重庆医科大学感染性疾病分子生物学教育部重点实验室
- 作者简介：
- 基金信息：
  
  the Key Research and Development Program of Ministry of Science and Technology(2018YFE0107500);the Project of Science and Technology Research Program of Chongqing Education Commission(KJQN202100422);the Project of Science and Technology Plan of Yuzhong District, Chongqing(20210116)
- DOI：10.16016/j.2097-0927.202306066
  CLC： R373.21;R394.3;R735.7
- Received：19 June 2023，
  
  Revised：2023-07-06，
  
  Published：30 January 2024
- 稿件说明：
移动端阅览
Zerui YANG, Haijun DENG, Yuan HU. Analysis of HBV DNA integration events in hepatocellular carcinoma cell lines by high throughput targeted capture sequencing[J]. Journal of Army Medical University, 2024, 46(2): 110-117.
DOI：

Zerui YANG, Haijun DENG, Yuan HU. Analysis of HBV DNA integration events in hepatocellular carcinoma cell lines by high throughput targeted capture sequencing[J]. Journal of Army Medical University, 2024, 46(2): 110-117. DOI： 10.16016/j.2097-0927.202306066.

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

DPP

7、

TRIM

56、

GPC

3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。

Abstract

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.

DPP

TRIM

56 and

GPC

3 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

Keywords

references

COLLABORATORS P O. Global prevalence, treatment, and prevention of hepatitis B virus infection in 2016: a modelling study[J]. Lancet Gastroenterol Hepatol, 2018, 3(6): 383-403. DOI: 10.1016/S2468-1253(18)30056-6.

HUANG H C, REN Z G, GAO X X, et al . Integrated analysis of microbiome and host transcriptome reveals correlations between gut microbiota and clinical outcomes in HBV-related hepatocellular carcinoma[J ] . Genome Med, 2020, 12(1): 102. DOI: 10.1186/s13073-020-00796-5.

POLLICINO T, CAMINITI G. HBV-integration studies in the clinic: role in the natural history of infection[J]. Viruses, 2021, 13(3): 368. DOI: 10.3390/v13030368.

MASON W S, JILBERT A R, LITWIN S. Hepatitis B virus DNA integration and clonal expansion of hepatocytes in the chronically infected liver[J]. Viruses, 2021, 13(2): 210. DOI: 10.3390/v13020210.

RAMIREZ R, VAN BUUREN N, GAMELIN L, et al . Target- ed long-read sequencing reveals comprehensive architecture, burden, and transcriptional signatures from hepatitis B virus-associated integrations and translocations in hepatocellular carcinoma cell lines[J ] . J Virol, 2021, 95(19): e0029921. DOI: 10.1128/jvi.00299-21.

MURAKAMI Y, SAIGO K, TAKASHIMA H, et al . Large scaled analysis of hepatitis B virus (HBV) DNA integration in HBV related hepatocellular carcinomas[J ] . Gut, 2005, 54(8): 1162-1168. DOI: 10.1136/gut.2004.054452.

SAITTA C, TRIPODI G, BARBERA A, et al . Hepatitis B virus (HBV) DNA integration in patients with occult HBV infection and hepatocellular carcinoma[J ] . Liver Int, 2015, 35(10): 2311-2317. DOI: 10.1111/liv.12807.

MASON W S, GILL U S, LITWIN S, et al . HBV DNA integration and clonal hepatocyte expansion in chronic hepatitis B patients considered immune tolerant[J ] . Gastroenterology, 2016, 151(5): 986-998. e4. DOI: 10.1053/j.gastro.2016.07.012.

FUJIMOTO A, FURUTA M, TOTOKI Y, et al . Whole-genome mutational landscape and characterization of noncoding and structural mutations in liver cancer[J ] . Nat Genet, 2016, 48(5): 500-509. DOI: 10.1038/ng.3547.

WANG A Q, WU L C, LIN J Z, et al . Whole-exome sequencing reveals the origin and evolution of hepato-cholangio -carcinoma[J ] . Nat Commun, 2018, 9(1): 894. DOI: 10.1038/s41467-018-03276-y.

赵杨静, 李国力, 张恒辉, 等. 应用HBV-Alu-PCR研究肝癌细胞株中乙型肝炎病毒整合[J]. 中华实验和临床感染病杂志(电子版), 2015, 9(5): 134-138. DOI: 10.3877/cma.j.issn.1674-1358.2015.05.027.

ZHAO Y J, LI G L, ZHANG H H, et al . Identification of hepatitis B virus integration in human hepatocellular carcinoma cell lines by HBV-Alu-PCR[J ] . Chin J Exp Clin Infect Dis Electron Ed, 2015, 9(5): 134-138. DOI: 10.3877/cma.j.issn.1674-1358.2015.05.027.

阮鹏, 何春萍, 黄超, 等. 改良Alu-PCR技术检测HepG2.2.15细胞HBV整合的研究[J]. 肝脏, 2022, 27(7): 785-788. DOI:10.14000/j.cnki.issn.1008-1704.2022.07.012.

RUAN P, HE C P, HUANG C, et al . Detection of hepatitis B viral DNA integration in HepG2.2. 15 cells by a modified Alu-PCR method[J ] . Chin Hepatol, 2022, 27(7): 785-788. DOI:10.14000/j.cnki.issn.1008-1704.2022.07.012.

NO J S, KIM W K, CHO S, et al . Comparison of targeted next-generation sequencing for whole-genome sequencing of Hantaan orthohantavirus in Apodemus agrarius lung tissues[J ] . Sci Rep, 2019, 9(1): 1-9. DOI: 10.1038/s41598-019-53043-2..

MINAMI M, POUSSIN K, BRÉCHOT C, et al . A novel PCR technique using Alu -specific primers to identify unknown flanking sequences from the human genome[J ] . Genomics, 1995, 29(2): 403-408. DOI: 10.1006/geno.1995.9004..

ZHENG B, LIU X L, FAN R, et al . The landscape of cell-free HBV integrations and mutations in cirrhosis and hepato cellular carcinoma patients[J ] . Clin Cancer Res, 2021, 27 (13): 3772-3783. DOI: 10.1158/1078-0432.CCR-21-0002.

JANG J W, KIM J S, KIM H S, et al . Persistence of intrahepatic hepatitis B virus DNA integration in patients developing hepatocellular carcinoma after hepatitis B surface antigen seroclearance[J ] . Clin Mol Hepatol, 2021, 27(1): 207-218. DOI: 10.3350/cmh.2020.0115.

TU T, BUDZINSKA M, VONDRAN F, et al . Hepatitis B virus DNA integration occurs early in the viral life cycle in an in vitro infection model via sodium taurocholate cotransporting polypeptide-dependent uptake of enveloped virus particles[J ] . J Virol, 2018, 92(11): e02007-17. DOI:10.1128/JVI.02007-17..

MEIER M A, CALABRESE D, SUSLOV A, et al . Ubiquitous expression of HBsAg from integrated HBV DNA in patients with low viral load[J ] . J Hepatol, 2021, 75(4): 840-847. DOI: 10.1016/j.jhep.2021.04.051.

SELLS M A, CHEN M L, ACS G. Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA[J]. Proc Natl Acad Sci USA, 1987, 84(4): 1005-1009. DOI: 10.1073/pnas.84.4.1005.

LADNER S K, OTTO M J, BARKER C S, et al . Inducible expression of human hepatitis B virus (HBV) in stably transfected hepatoblastoma cells: a novel system for screening potential inhibitors of HBV replication[J ] . Antimicrob Agents Chemother, 1997, 41(8): 1715-1720. DOI: 10.1128/AAC.41.8.1715.

HU X G, JIANG J H, NI C, et al . HBV integration-mediated cell apoptosis in HepG2.2.15[J ] . J Cancer, 2019, 10(17): 4142-4150. DOI: 10.7150/jca.30493.

TIAN X, DONG H J, LAI X Y, et al . TRIM56 impairs HBV infection and replication by inhibiting HBV core promoter activity[J ] . Antivir Res, 2022, 207: 105406. DOI: 10.1016/j.antiviral.2022.105406.

JIANG D L, ZHANG Y S, WANG Y N, et al . Diagnostic accuracy and prognostic significance of Glypican-3 in hepatocellular carcinoma: a systematic review and meta-analysis[J ] . Front Oncol, 2022, 12: 1012418. DOI:10.3389/fonc.2022.1012418.

Views

下载量

CSCD

Alert me when the article has been cited

提交

Tools

Publicity Resources

SRSF1 inhibits HBV replication by enhancing the stability of P53 in cell models

Dynamic analysis of serum markers of HBV in 302 infants of pregnant women with HBV infection

Comprehensive bioinformatics analysis of vitiligo melanocytes based on high-throughput sequencing

Therapeutic effect of compound Lactobacillus acidophilus tablets on constipation and its impact on intestinal flora in mice

Effect of interferons on expression of adenosine deaminase acting on RNA 1 and hepatitis B virus replication in human hepatocellular carcinoma cells in vitro

Related Author

LIU Jiajun

LONG Shaoyuan

HU Jieli

CUI Jing

Jing WANG

Yan ZHU

Zhongwei LIU

Huimin LIU

Related Institution

First Affiliated Hospital， Air Force Medical University

Key Laboratory of Molecular Biology for Infectious Diseases of Ministry of Education， Chongqing Medical University

Institute of Infectious Diseases, Chongqing Key Laboratory of Infectious Diseases Research, First Affiliated Hospital, Army Medical University (Third Military Medical University)

Department of Dermatology, Xijing Hospital, Air Force Medical University

Department of Gastroenterology, the First Affiliated Hospital of Chongqing Medical University

⁰