Correlation between the single-site CpG methylation and expression silencing of the XAF1 gene in human gastric and colon cancers
Correlation between the single-site CpG methylation and expression silencing of the XAF1 gene in human gastric and colon cancers
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DOI：
10.1053/j.gastro.2006.09.050
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发表时间：
2006-12-01
期刊：
影响因子：
29.4
通讯作者：
Wong, Benjamin C. Y.
中科院分区：
文献类型：
--
作者：
Zou, Bing;Chim, Chor Sang;Wong, Benjamin C. Y.
背景与目的：X-linked inhibitor of apoptosis protein (XIAP) -associated factor I （XAF1）可拮抗XIAP的抗caspase活性。XAF1信使RNA存在于正常组织中，但在各种癌症中检测不到，因此是一种潜在的肿瘤抑制基因。本研究的目的是研究胃癌和结肠癌中XAF1甲基化的新模式，并定位转录调控和肿瘤进展的重要CpG位点。方法：采用逆转录聚合酶链反应（PCR）和Western blot检测XAF1的表达。克隆了XAF1转录起始位点周围的4个不同片段，并通过荧光素酶报告基因试验检测了推测的启动子活性。利用位点定向诱变技术对F291片段的每个CpG位点进行突变，荧光素酶报告基因法检测该片段启动子活性的变化。通过甲基化特异性PCR （MSP）和亚硫酸氢盐DNA测序PCR分析确定XAF1的甲基化状态。结果：4个胃癌细胞系中的3个和8个结肠癌细胞系中的6个检测到与高甲基化相关的XAF1下调。在4个启动子片段中，F291的启动子活性最高，其启动子活性可因特定CpG位点的突变而明显下调。此外，这些重要CpG位点的异常高甲基化与胃癌和结肠癌的发生密切相关。结论：位于启动子区域的CpG甲基化位点簇而不是CpG岛导致XAF1基因沉默，并且-2、-1和+3位置的CpG在其转录调控中功能更重要。
Background & Aims: X-linked inhibitor of apoptosis protein (XIAP) -associated factor I (XAF1) antagonizes the anti-caspase activity of XIAP. XAF1 messenger RNA is present in normal tissues but undetectable in various cancers and thus poses a potential tumor suppressor gene. The aim of this study was to examine the novel pattern of rnethylation of XAF1 in gastric and colon cancers and locate the important CpG sites for transcriptional regulation and tumor progression. Methods: XAF1 expression was detected by reverse-transcription polymerase chain reaction (PCR) and Western blot analysis. Four different fragments around the transcription start site of XAF1 were cloned and examined putative promoter activities by luciferase reporter assay. Each CpG site in fragment F291 was mutated by site-directed mutagenesis technique, and the change of promoter activity of this fragment was detected by luciferase reporter assay. Methylation status of XAF1 was determined by methylation-specific PCR (MSP) and bisulfite DNA sequencing PCR analysis. Results: Down-regulation of XAF1 in association with hypermethylation was detected in 3 of 4 human gastric cancer cell lines and 6 of 8 colon cancer cell lines. Of the 4 promoter fragments, F291 showed the highest promoter activity, which could be down-regulated obviously by the mutation of particular CpG sites. Moreover, aberrant hypermethylation of these important CpG sites was strongly associated with the development of gastric and colon cancers. Conclusions: A cluster of methylated CpG sites instead of CpG islands located in the promoter area resulted in gene silencing of XAF1, and CpGs at -2nd, -1st, and +3rd positions are functionally more important in its transcriptional regulation.