Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Beijing Cancer Hospital and Institute, Peking University School of Oncology, Haidian District, Beijing, 100142, China.
BMC Cancer. 2010 Feb 17;10:44. doi: 10.1186/1471-2407-10-44.
Alu methylation is correlated with the overall level of DNA methylation and recombination activity of the genome. However, the maintenance and methylation status of each CpG site within Alu elements (Alu) and its methylation status have not well characterized. This information is useful for understanding natural status of Alu in the genome and helpful for developing an optimal assay to quantify Alu hypomethylation.
Bisulfite clone sequencing was carried out in 14 human gastric samples initially. A Cac8I COBRA-DHPLC assay was developed to detect methylated-Alu proportion in cell lines and 48 paired gastric carcinomas and 55 gastritis samples. DHPLC data were statistically interpreted using SPSS version 16.0.
From the results of 427 Alu bisulfite clone sequences, we found that only 27.2% of CpG sites within Alu elements were preserved (4.6 of 17 analyzed CpGs, A approximately Q) and that 86.6% of remaining-CpGs were methylated. Deamination was the main reason for low preservation of methylation targets. A high correlation coefficient of methylation was observed between Alu clones and CpG site J (0.963), A (0.950), H (0.946), D (0.945). Comethylation of the sites H and J were used as an indicator of the proportion of methylated-Alu in a Cac8I COBRA-DHPLC assay. Validation studies showed that hypermethylation or hypomethylation of Alu elements in human cell lines could be detected sensitively by the assay after treatment with 5-aza-dC and M.SssI, respectively. The proportion of methylated-Alu copies in gastric carcinomas (3.01%) was significantly lower than that in the corresponding normal samples (3.19%) and gastritis biopsies (3.23%).
Most Alu CpG sites are deaminated in the genome. 27% of Alu CpG sites represented in our amplification products. 87% of the remaining CpG sites are methylated. Alu hypomethylation in primary gastric carcinomas could be detected with the Cac8I COBRA-DHPLC assay quantitatively.
Alu 甲基化与 DNA 甲基化的总体水平和基因组的重组活性相关。然而，Alu 元件（Alu）内每个 CpG 位点的维持和甲基化状态及其甲基化状态尚未得到很好的描述。这些信息有助于了解基因组中 Alu 的自然状态，并有助于开发一种最佳的检测方法来量化 Alu 低甲基化。
最初在 14 个人胃样本中进行了亚硫酸氢盐克隆测序。开发了 Cac8I COBRA-DHPLC 测定法来检测细胞系和 48 对胃癌和 55 例胃炎样本中甲基化-Alu 的比例。使用 SPSS 版本 16.0 对 DHPLC 数据进行统计学解释。
从 427 个 Alu 亚硫酸氢盐克隆序列的结果中，我们发现只有 27.2%的 Alu 元件内的 CpG 位点得到保留（在 17 个分析的 CpG 中，有 4.6 个，A 大约为 Q），而其余-CpGs 均被甲基化。脱氨基是导致甲基化靶标保留率低的主要原因。Alu 克隆与 CpG 位点 J（0.963）、A（0.950）、H（0.946）、D（0.945）之间观察到高度相关的甲基化系数。H 和 J 位点的共甲基化被用作 Cac8I COBRA-DHPLC 测定中甲基化-Alu 比例的指标。验证研究表明，在分别用 5-aza-dC 和 M.SssI 处理后，该测定法可以敏感地检测出人细胞系中 Alu 元件的过度甲基化或低甲基化。胃癌（3.01%）中甲基化-Alu 拷贝的比例明显低于相应的正常样本（3.19%）和胃炎活检（3.23%）。
基因组中大多数 Alu CpG 位点脱氨。我们扩增产物中代表的 Alu CpG 位点为 27%。其余 CpG 位点的 87%被甲基化。原发性胃癌中 Alu 低甲基化可以通过 Cac8I COBRA-DHPLC 测定法进行定量检测。
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