Quantitative assessment of RUNX3 methylation in neoplastic and non‐neoplastic gastric epithelia using a DNA microarray
Quantitative assessment of RUNX3 methylation in neoplastic and non‐neoplastic gastric epithelia using a DNA microarray
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DOI：
10.1111/j.1440-1827.2006.02010.x
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发表时间：
2006-10
影响因子：
2.2
通讯作者：
K. So;G. Tamura;T. Honda;N. Homma;M. Endoh;N. Togawa;S. Nishizuka;T. Motoyama
中科院分区：
文献类型：
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作者：
K. So;G. Tamura;T. Honda;N. Homma;M. Endoh;N. Togawa;S. Nishizuka;T. Motoyama
RUNX3 基因启动子 CpG 岛超甲基化导致的沉默在胃癌发生中起重要作用。为了定量评估 RUNX3 甲基化，使用了纤维型 DNA 微阵列，其上安装了甲基化和非甲基化序列探针。经过亚硫酸氢盐修饰后，使用 Cy5 末端标记的引物通过聚合酶链反应扩增了 RUNX3 启动子 CpG 岛的一部分（甲基化对于基因沉默至关重要）。甲基化率（MR）计算为甲基化序列探针的荧光强度与甲基化和非甲基化探针的总荧光强度的比率。分析了 5 种胃癌细胞系以及 26 种原发性胃癌及其相应的非肿瘤性胃上皮。四种丢失 RUNX3 mRNA 的癌细胞系中的 MR 范围为 99.0% 至 99.7%（平均 99.4%），而表达 RUNX3 mRNA 的其余细胞系中的 MR 为 0.6%。在原发性胃癌及其相应的非肿瘤性胃上皮中，MR 范围为 0.2% 至 76.5%（平均 22.7%）和 0.7% 至 25.1%（平均 5.5%）。 26 例胃癌中有 10 例 (38.5%) 及其相应的非肿瘤性胃上皮细胞的 MR > 30%。大多数 MR > 10% 的样品通过传统的甲基化特异性聚合酶链反应 (MSP) 检测出甲基化阳性。这种基于微阵列的甲基化测定是定量评估基因甲基化的一种有前途的方法。
Silencing of the RUNX3 gene by hypermethylation of its promoter CpG island plays a major role in gastric carcinogenesis. To quantitatively evaluate RUNX3 methylation, a fiber‐type DNA microarray was used on which methylated and unmethylated sequence probes were mounted. After bisulfite modification, a part of the RUNX3 promoter CpG island, at which methylation is critical for gene silencing, was amplified by polymerase chain reaction using a Cy5 end‐labeled primer. Methylation rates (MR) were calculated as the ratio of the fluorescence intensity of a methylated sequence probe to the total fluorescence intensity of methylated and unmethylated probes. Five gastric cancer cell lines were analyzed, as well as 26 primary gastric cancers and their corresponding non‐neoplastic gastric epithelia. MR in four of the cancer cell lines that lost RUNX3 mRNA ranged from 99.0% to 99.7% (mean, 99.4%), whereas MR in the remaining cell line that expressed RUNX3 mRNA was 0.6%. In primary gastric cancers and their corresponding non‐neoplastic gastric epithelia, MR ranged from 0.2% to 76.5% (mean, 22.7%) and from 0.7% to 25.1% (mean, 5.5%). Ten (38.5%) of the 26 gastric cancers and none of their corresponding non‐neoplastic gastric epithelia had MR >30%. Most of the samples with MR >10% tested methylation‐positive by conventional methylation‐specific polymerase chain reaction (MSP). This microarray‐based methylation assay is a promising method for the quantitative assessment of gene methylation.