Microarray analysis of promoter methylation in lung cancers
Microarray analysis of promoter methylation in lung cancers
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DOI：
10.1007/s10038-005-0355-4
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发表时间：
2006-01-01
影响因子：
3.5
通讯作者：
Hatada, I
中科院分区：
文献类型：
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作者：
Fukasawa, M;Kimura, M;Hatada, I
DNA甲基化异常是肿瘤发生过程中的重要事件。在癌症中可以甲基化的基因的各个区域中，启动子对于基因表达的调节是最重要的。在这里，我们描述了一种新开发的启动子相关甲基化DNA扩增DNA芯片(PMAD)用于基因启动子区DNA甲基化的微阵列分析。对每个样本，分别扩增甲基化的HPA II抗性DNA片段和Msp I裂解(未甲基化+甲基化)的DNA片段，分别用Cy3和Cy5标记，然后杂交到包含288个癌症相关基因启动子的微阵列中。来自Hpa II抗性(甲基化)DNA(Cy3)的信号被归一化为来自Msp I裂解(未甲基化+甲基化)DNA片段(Cy5)的信号。将来自肺癌细胞系的归一化信号与来自正常肺细胞的信号进行比较。在肺癌细胞系中，约有10.9%的癌相关基因发生高甲基化。值得注意的是，HIC1、IRF7、ASC、RIPK3、RASSF1A、FABP3、PRKCDBP和PAX3基因在大多数肺癌细胞系中都发生了高甲基化。这些基因的表达谱与基因的甲基化谱相关，表明基因启动子区DNA甲基化的微阵列分析为表观遗传学研究提供了便利。对原发肿瘤的进一步分析表明，ASC(82%)和PAX3(86%)的高甲基化在所有类型的肿瘤中都很高，而RIPK3在小细胞癌中的高甲基化频率(57%)。这表明我们的PMAD方法在发现癌症期间的表观遗传学变化方面是有效的。
Aberrant DNA methylation is an important event in carcinogenesis. Of the various regions of a gene that can be methylated in cancers, the promoter is the most important for the regulation of gene expression. Here, we describe a microarray analysis of DNA methylation in the promoter regions of genes using a newly developed promoter-associated methylated DNA amplification DNA chip (PMAD). For each sample, methylated Hpa II-resistant DNA fragments and Msp I-cleaved (unmethylated + methylated) DNA fragments were amplified and labeled with Cy3 and Cy5 respectively, then hybridized to a microarray containing the promoters of 288 cancer-related genes. Signals from Hpa II-resistant (methylated) DNA (Cy3) were normalized to signals from Msp I-cleaved (unmethylated + methylated) DNA fragments (Cy5). Normalized signals from lung cancer cell lines were compared to signals from normal lung cells. About 10.9% of the cancer-related genes were hypermethylated in lung cancer cell lines. Notably, HIC1, IRF7, ASC, RIPK3, RASSF1A, FABP3, PRKCDBP, and PAX3 genes were hypermethylated in most lung cancer cell lines examined. The expression profiles of these genes correlated to the methylation profiles of the genes, indicating that the microarray analysis of DNA methylation in the promoter region of the genes is convenient for epigenetic study. Further analysis of primary tumors indicated that the frequency of hypermethylation was high for ASC (82%) and PAX3 (86%) in all tumor types, and high for RIPK3 in small cell carcinoma (57%). This demonstrates that our PMAD method is effective at finding epigenetic changes during cancer.