Park In Young, Sohn Bo Hwa, Yu Eunsil, Suh Dong Jin, Chung Young-Hwa, Lee Je-Ho, Surzycki Stefan J, Lee Young Ik
Liver Cell Signal Transduction Laboratory, Molecular Cancer Research Center, KRIBB, Daejeon, Korea.
Gastroenterology. 2007 Apr;132(4):1476-94. doi: 10.1053/j.gastro.2007.01.034. Epub 2007 Jan 25.
BACKGROUND & AIMS: The involvement of the hepatitis B virus X (HBx) protein in epigenetic modifications during hepatocarcinogenesis has not been previously characterized. The aim of the present study was to identify the involvement of HBx in regional hypermethylation and global hypomethylation during the formation of hepatocellular carcinoma (HCC).
Liver cell lines were transiently or stably transfected with an HBx-expressing vector. DNA methyltransferase (DNMT) promoter activity changes were examined by luciferase assay and chromatin immunoprecipitation. The methylation status of insulin-like growth factor binding protein-3 was examined by methyl-specific polymerase chain reaction and bisulfite sequencing. Global DNA methylation levels were examined using 5-methylcytosine dot blot and methylation-sensitive Southern blot analysis. HBx-mediated DNA methylation abnormalities were confirmed in patient HCC samples using methyl-specific polymerase chain reaction and 5-methylcytosine dot blot analysis.
HBx expression increased total DNMT activities by up-regulation of DNMT1, DNMT3A1, and DNMT3A2 and selectively promoted regional hypermethylation of specific tumor suppressor genes. HBx specifically repressed insulin-like growth factor-3 expression through de novo methylation via DNMT3A1 and DNMT3A2 and by inhibiting SP1 binding via recruiting methyl CpG binding protein 2 to the newly methylated SP1 binding element. HBx also induced global hypomethylation of satellite 2 repeat sequences by down-regulating DNMT3B. The prevalence of these specific methylation abnormalities by HBx was significantly correlated with HBx expression in HBV-infected HCC patients.
Targeted deregulation of DNMTs by HBx promotes both specific regional hypermethylation and global hypomethylation. These epigenetic modulations by HBx may suggest a mechanism for epigenetic tumorigenesis during HBV-mediated hepatocarcinogenesis.
此前尚未明确乙型肝炎病毒X（HBx）蛋白在肝癌发生过程中表观遗传修饰方面的作用。本研究旨在确定HBx在肝细胞癌（HCC）形成过程中区域高甲基化和整体低甲基化方面的作用。
用表达HBx的载体对肝细胞系进行瞬时或稳定转染。通过荧光素酶测定和染色质免疫沉淀检测DNA甲基转移酶（DNMT）启动子活性变化。用甲基化特异性聚合酶链反应和亚硫酸氢盐测序检测胰岛素样生长因子结合蛋白-3的甲基化状态。使用5-甲基胞嘧啶斑点印迹和甲基化敏感的Southern印迹分析检测整体DNA甲基化水平。通过甲基化特异性聚合酶链反应和5-甲基胞嘧啶斑点印迹分析在患者HCC样本中证实HBx介导的DNA甲基化异常。
HBx表达通过上调DNMT1、DNMT3A1和DNMT3A2增加总DNMT活性，并选择性促进特定肿瘤抑制基因的区域高甲基化。HBx通过DNMT3A1和DNMT3A2从头甲基化并通过招募甲基化CpG结合蛋白2到新甲基化的SP1结合元件来抑制SP1结合，从而特异性抑制胰岛素样生长因子-3的表达。HBx还通过下调DNMT3B诱导卫星2重复序列的整体低甲基化。HBx导致的这些特定甲基化异常的发生率与HBV感染的HCC患者中HBx的表达显著相关。
HBx对DNMTs的靶向失调促进了特定区域的高甲基化和整体低甲基化。HBx的这些表观遗传调控可能提示了HBV介导的肝癌发生过程中表观遗传肿瘤发生的机制。
