Title应用HBV-Alu-PCR研究肝癌细胞株中乙型肝炎病毒整合
Other TitlesIdentiifcation of hepatitis B virus integration in human hepatocellular carcinoma cell lines by HBV-Alu-PCR
Authors赵杨静
李国力
张恒辉
谢兴旺
贺改霞
闫琳琳
李明珍
魏来
曾辉
Affiliation212013镇江市,江苏大学医学院
100044北京,北京大学人民医院北京大学肝病研究所,丙型肝炎及肝病免疫治疗北京市重点实验室
100015北京,首都医科大学附属北京地坛医院,新发突发传染病研究北京市重点实验室
100044北京,北京大学人民医院北京大学肝病研究所,丙型肝炎及肝病免疫治疗北京市重点实验室
北京市理化分析测试中心,北京,100089
Keywords肝炎病毒,乙型
肝癌细胞株
病毒整合
Hepatitis B virus
Hepatocellular carcinoma cell
Virus integration
Issue Date2015
Publisher中华实验和临床感染病杂志(电子版)
Citation中华实验和临床感染病杂志(电子版).2015,9,(5),708-713.
Abstract目的 探讨常见肝癌细胞株中整合的乙型肝炎病毒(HBV)序列和整合位点.方法 分别将来源于HBV感染者的肝癌细胞株(MHCC97H、MHCC97L、MHCCLM3和PLC/PRF/5),稳定转染HBV的肝癌细胞株(HepG2.2.15、HepAD38和DE19),和无HBV感染者来源的肝癌细胞株(HepG2、HuH-7)培养72 h,检测培养上清中乙型肝炎病毒表面抗原(HBsAg)、乙型肝炎病毒E抗原(HBeAg)及HBV DNA滴度.采用HBV-Alu-PCR方法,扩增肝癌细胞株中整合的HBV基因X/C/S片段及其侧翼的人基因组DNA片段,对扩增片段进行测序确定HBV整合在人类染色体的精确位置,并用生物信息学分析确定其上下游基因.结果 MHCC97H、MHCC97L和MHCCLM3未检出HBsAg和HBeAg,但HBV DNA滴度为2.00 × 105~4.00 × 105IU/ml;HepG2.2.15、HepAD38、DE19和PLC/PRF/5 HBsAg阳性,且HBV DNA滴度> 5.00 ×105IU/ml,其中HepG2.2.15、HepAD38HBeAg阳性;HepG2、HuH-7 HBsAg、HBeAg和HBV DNA均低于检测下限.除HepG2和HuH-7未检测到整合外,其余细胞株中可检测到1个或多个整合的不同HBV亚基因组片段:HepG2.2.15检测到5个HBx和HBc整合片段,HepAD38和PLC/PRF/5各检测到2个HBx整合片段,DE19检测到一个HBc整合片段.3个MHCC97的衍生细胞株:MHCC97L(低转移潜能肝癌细胞)、MHCC97H(高转移潜能肝癌细胞)和MHCCLM3(MHCC97H肺转移肝癌细胞)检测到完全相同的HBc片段整合在16q13上IRX3和IRX5基因之间的非编码区.此外,各细胞株HBV整合位点上下游的基因主要包括肿瘤相关基因,核糖体蛋白编码基因和钙信号相关基因.结论 HBV感染者来源和稳定转染HBV的肝癌细胞株存在HBV整合;HBV基因X/C片段比S片段有更高的整合频率;同一个克隆来源的肝癌细胞株的HBV整合位点稳定,提示HBV整合分析可能对肝细胞癌原发灶和转移灶癌细胞克隆来源的鉴定提供参考.
Objective To investigate the integration of hepatitis B virus (HBV) in human hepatocellular carcinoma (HCC) cell lines.Methods HCC cell lines, which established from patients with HBV infection (MHCC97H, MHCC97L, MHCCLM3 and PLC/PRF/5), stably transfected with HBV (HepG2.2.15, HepAD38 and DE19) and without HBV infection (HepG2 and HuH-7), were cultured for 72 h and cell supernatants were harvested for HBsAg, HBeAg and HBV DNA quantiifcation. HBV-Alu-PCR was used to amplify the integrated HBV X/C/S genes and their lfanking sequences in human genomic DNA of the HCC cell lines. The integrated HBV fragments and integration sites were determined by sequencing the PCR products. The upstream and downstream human genes adjacent to the integration sites were identiifed by bioinformatics analysis.Results MHCC97H, MHCC97L and MHCCLM3 were negative for HBsAg andHBeAg, but positive for HBV DNA with a titer ranged between 2.00× 105to 4.00× 105IU/ml. HepG2.2.15, HepAD38, DE19 and PLC/PRF/5 were positive for HBsAg with a HBV DNA titer higher than 5.00× 105 IU/ml. HepG2.2.15 and HepAD38 were positive for HBeAg. HepG2 and HuH-7 were negative for HBsAg,HBeAg and HBV DNA. One or more integrated HBV sub-genome fragments were detected in cell lines except for HepG2 and HuH-7. Among them, up to five integration sites were detected in HepG2.2.15 (HBx/HBc), two in HepAD38 and PLC/PRF/5 (HBx), one in DE19 (HBc). Interestingly, three HCC cells (MHCC97H, MHCC97L and MHCCLM3), which had the same clone origin from MHCC97, contained exactly the same HBC fragment integrated into the same site. Furthermore, HBV integration mainly targeted genes belonging to distinct pathways: cancer related genes, calcium signaling related genes, ribosomal protein encoding genes. Conclusions HBV integration occurs in HCC cell lines from patients with HBV infection and HCC cell lines stably transfected with HBV virus genome. HBx and HBc are the main HBV fragments integrated into the genomic DNA of HCC cells. HCC cells from the same clone origin contain the same HBV integration site, which may be used to trace the clone origin of primary or metastatic HCC cells.
URIhttp://hdl.handle.net/20.500.11897/433046
ISSN1674-1358
DOI10.3877/cma.j.issn.1674-1358.2015.05.027
Indexed中国科技核心期刊(ISTIC)
Appears in Collections:人民医院

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