Genome-wide screen for hepermethylated genes in hepatocellular carcinoma

DNA甲基化异常导致的基因沉默导致肿瘤抑制基因失活，而肿瘤抑制基因在癌变过程中起着重要作用。p16INK4a基因在肝细胞癌中由于DNA甲基化异常而被抑制，其他新的基因也可能通过相同的机制被抑制。因此，我们使用cDNA芯片分析了DNA甲基转移酶抑制剂5-aza-2'脱氧胞苷（5Aza-dC）处理后6个肝组细胞系的基因表达谱变化。在多种肝癌细胞系中，5Aza-dC处理后14个显著诱导（超过5倍变化）的基因中，基于cDNA芯片分析，肝细胞生长因子激活因子抑制剂2 （HAI2）、E-cadherin、1型胶原α 2 （COL1A2）、胰岛素样生长因子结合蛋白2 （IGFBP2）、结缔组织生长因子（CTGF）和纤维连接蛋白1 （FN1）等6个基因在肝癌细胞上表现出CpG岛的高甲基化。在使用甲基化特异性PCR （MS-PCR）对原发性肝癌组织的进一步研究中，在肿瘤组织中检测到HAI2的高甲基化比在相应的非肿瘤组织中更频繁。总之，我们可以利用微阵列技术鉴定出肝癌中6个高甲基化基因。HAI2基因的异常甲基化可能与肝细胞生长因子的过度表达有关。

Gene silencing due to aberrant DNA methylation causes inactivation of tumor suppressor genes, which plays an important role during carcinogenesis. Expression of p16INK4a gene was suppressed due to aberrant DNA methylation in hepatocellular carcinoma and other novel genes could be suppressed by the same mechanism. Therefore, we analyzed gene expression profile altered after DNA methyltransferase inhibitor, 5-aza-2' deoxycytidine (5Aza-dC) treatment using cDNA microarray in 6 hepatome cell lines. Among 14 genes exhibiting the substantial induction (more than 5-fold changes) after 5Aza-dC treatment in multiple hepatoma cell lines, based on cDNA microarray analysis, 6 genes including hepatocyte growth factor activator inhibitor 2 (HAI2), E-cadherin, collagen type 1 alpha 2 (COL1A2), insulin-like growth factor binding protein 2 (IGFBP2), connective tissue growth factor (CTGF) and fibronectin 1 (FN1) exhibited hypermethylation of CpG islands on hepatoma cells. In further studies of primary hepatoma tissues using methylation-specific PCR (MS-PCR), hypermethylation of HAI2 was detected in tumorous tissues more often than in corresponding nontumorous tissues. In conclusion, we could identify 6 hypermethylated genes in hepatoma by microarray based strategy. Aberrant methylation of HAI2 gene might be involved in hepatocarcinogenesis due to overexpression of hepatocyte growth factor.