Identification of differentially methylated sequences in colorectal cancer by methylated CpG island amplification.
Identification of differentially methylated sequences in colorectal cancer by methylated CpG island amplification.
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发表时间：
1999-05
期刊：
影响因子：
11.2
通讯作者：
M. Toyota;Coty Ho;N. Ahuja;K. Jair;Qing Li;Mutsumi Ohe-Toyota;S. Baylin;J. Issa
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文献类型：
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作者：
M. Toyota;Coty Ho;N. Ahuja;K. Jair;Qing Li;Mutsumi Ohe-Toyota;S. Baylin;J. Issa
CpG 岛甲基化与肿瘤中的抑癌基因失活有关，并且可能作为克隆新的癌症相关基因的有用标记。我们开发了一种基于 PCR 的新型方法，即甲基化 CpG 岛扩增 (MCA)，该方法可用于甲基化分析和克隆差异甲基化基因。使用对 5-甲基胞嘧啶具有不同敏感性的限制性内切酶，然后进行接头连接和 PCR 扩增，可以优先扩增富含甲基化 CpG 的序列。在使用 p16 基因外显子 1 探针的模型实验中，从结直肠癌细胞系的 MCA 产物中检测到信号，但在正常结肠粘膜中未检测到信号。为了识别结直肠癌中差异甲基化的新型 CpG 岛，我们将 MCA 与代表性差异分析结合起来，以结肠癌细胞系 Caco2 作为测试者，以正常结肠粘膜作为驱动者。使用这种策略，我们分离了 33 个差异甲基化 DNA 序列，包括与几个已知基因（PAX6、Versacan、α-微管蛋白、CSX、OPT 和 rRNA 基因）相同的片段。通过检查 Versican 基因，证实了获得的克隆的高甲基化与结直肠癌转录抑制之间的关联，我们发现该基因在甲基化细胞系中被沉默，并被甲基化抑制剂 5-aza-2'-deoxycytidine 重新激活。因此，我们认为 MCA 是研究甲基化和分离癌症中差异甲基化的 CpG 岛的有用技术。
CpG island methylation has been linked to tumor suppressor gene inactivation in neoplasia and may serve as a useful marker to clone novel cancer-related genes. We have developed a novel PCR-based method, methylated CpG island amplification (MCA), which is useful for both methylation analysis and cloning differentially methylated genes. Using restriction enzymes that have differential sensitivity to 5-methyl-cytosine, followed by adaptor ligation and PCR amplification, methylated CpG rich sequences can be preferentially amplified. In a model experiment using a probe from exon 1 of the p16 gene, signal was detected from MCA products of a colorectal cancer cell line but not in normal colon mucosa. To identify novel CpG islands differentially methylated in colorectal cancer, we have applied MCA coupled with representational difference analysis to the colon cancer cell line Caco2 as a tester and normal colon mucosa as a driver. Using this strategy, we isolated 33 differentially methylated DNA sequences, including fragments identical to several known genes (PAX6, Versican, alpha-tubulin, CSX, OPT, and rRNA gene). The association of hypermethylation of the clones obtained and transcriptional suppression in colorectal cancer was confirmed by examining the Versican gene, which we found to be silenced in methylated cell lines and reactivated by the methylation inhibitor 5-aza-2'-deoxycytidine. We therefore propose that MCA is a useful technique to study methylation and to isolate CpG islands differentially methylated in cancer.