Analysis of genes upregulated by the demethylating agent 5-aza-2′-deoxycytidine in gastric cancer cell lines
Analysis of genes upregulated by the demethylating agent 5-aza-2′-deoxycytidine in gastric cancer cell lines
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DOI：
10.1002/ijc.21968
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发表时间：
2006-10-01
影响因子：
6.4
通讯作者：
Saisho, Hiromitsu
中科院分区：
文献类型：
--
作者：
Mikata, Rintaro;Yokosuka, Osamu;Saisho, Hiromitsu
据报道，在胃癌中，越来越多的基因被异常甲基化沉默。然而，很少对癌细胞中的表观遗传失活进行全面分析。为了筛选被去甲基化剂5-氮杂-2 ′-脱氧胞苷（DAC）上调的基因，使用用DAC处理的胃癌细胞系（AGS、MKN 74、MKN 1、MKN 45和Kato 3）进行cDNA微阵列分析（AceGene（R），含有30，000个基因）。通过实时荧光定量PCR（real-time PCR）确定上调的候选基因，并通过亚硫酸氢盐DNA测序或甲基化特异性PCR确定5 'CpG岛的甲基化状态。在被认为在其启动子区具有CpG岛的上调基因中，我们选择了在至少2个细胞系中表现出大于3倍增加的5个基因（BCL 2L 10、DKK 1、DNAJD 1、GAGED 2和NMU）。其中，我们可以确定BCL 2L 10、DKK 1和DNAJD 1的5 'CpG岛的甲基化状态。BCL 2L 10和DNAJD 1的5 'CpG在5个胃癌细胞系中有4个高甲基化，而DKK 1的5' CpG仅在1个细胞系中高甲基化。BCL 2L 10的MSP分析显示，DAC处理后CpG岛被去甲基化。此外，我们观察到BCL 2L 10的过表达可以促进胃癌细胞株的凋亡和生长抑制作用。总之，DAC治疗上调的一些基因可能是转录抑制启动子超甲基化。这些基因可能与胃癌的发生有关。尤其是BCL 2L 10的抑制作用，它可以诱导癌细胞凋亡并抑制癌细胞的增殖，这可能是胃癌发生的潜在机制之一。(c)2006 Wiley-Liss，Inc.
In gastric cancer, increasing numbers of genes have been reported to be silenced by aberrant methylation. However, global analysis of epigenetic inactivation in cancer cells has rarely been performed. For screening the genes upregulated by the demethylating agent 5-aza-2'-deoxycytidine (DAC), cDNA microarray analysis (AceGene (R), containing 30,000 genes) was performed using gastric cancer cell lines (AGS, MKN74, MKN1, MKN45 and Kato3) treated with DAC. The candidate upregulated genes were confirmed by real-time PCR, and the methylation status of 5'CpG islands was determined by bisulfite DNA sequencing or methylation-specific PCR. Among the upregulated genes considered to have CpG island in their promoter regions, we selected 5 genes (BCL2L10, DKK1, DNAJD1, GAGED2 and NMU) that exhibited a greater than 3-fold increase in at least 2 cell lines. Of these, we could determine the methylation status of 5'CpG islands of BCL2L10, DKK1 and DNAJD1. 5'CpG of BCL2L10 and DNAJD1 was hypermethylated in 4 of 5 gastric cancer cell lines, whereas 5'CpG of DKK1 was hypermethylated in only 1 cell line. MSP analysis for BCL2L10 revealed that the CpG island was demethylated after DAC treatment. In addition, we observed that overexpression of BCL2L10 could promote apoptosis and growth-inhibitory effect in gastric cancer cell lines. In conclusion, some of the genes upregulated by DAC treatment may be transcriptionally repressed by promoter hypermethylation. These genes might be related to gastric carcinogenesis. In particular, the suppression of BCL2L10, which could induce apoptosis and inhibit proliferation of cancer cells, might be one of the underlying mechanisms for gastric carcinogenesis. (c) 2006 Wiley-Liss, Inc.