Genome-wide screen for hepermethylated genes in hepatocellular carcinoma

DNA甲基化异常引起的基因沉默导致抑癌基因失活，在肿瘤发生过程中起重要作用。p16 INK 4a基因的表达在肝细胞癌中由于异常的DNA甲基化而受到抑制，其他新基因的表达也受到同样的机制的抑制。因此，我们利用基因芯片技术分析了6个肝癌细胞系在DNA甲基转移酶抑制剂5-氮杂-2 '脱氧胞苷（5-Aza-dC）处理后基因表达谱的变化。在14个基因中，在多个肝癌细胞系中，5Aza-dC处理后，基于cDNA微阵列分析，包括肝细胞生长因子激活物抑制剂2（HAI 2）、E-钙粘蛋白、1型胶原α 2（COL 1A 2）、胰岛素样生长因子结合蛋白2（IGFBP 2）、结缔组织生长因子（CTGF）和纤连蛋白1（FN 1）在肝癌细胞上表现出CpG岛的高甲基化。在进一步的研究中，原发性肝癌组织中使用甲基化特异性PCR（MS-PCR），HAI 2的高甲基化检测肿瘤组织比相应的非肿瘤组织更频繁。总之，我们可以通过基因芯片的策略，在肝癌中发现6个高甲基化基因。HAI 2基因异常甲基化可能参与了肝细胞生长因子过度表达导致的肝癌发生。

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.