Department of Epidemiology and Biostatistics, Zhejiang University School of Public Health, 866 Yuhangtang Road, Hangzhou, 310058, China.
Department of Epidemiology and Biostatistics, Zhejiang Chinese Medical University School of Public Health, 548 Binwen Road, Hangzhou, 310053, China.
Clin Epigenetics. 2019 Mar 7;11(1):41. doi: 10.1186/s13148-019-0628-y.
Epigenetic alternation is a common contributing factor to neoplastic transformation. Although previous studies have reported a cluster of aberrant promoter methylation changes associated with silencing of tumor suppressor genes, little is known concerning their sequential DNA methylation changes during the carcinogenetic process. The aim of the present study was to address a genome-wide search for identifying potentially important methylated changes and investigate the onset and pattern of methylation changes during the progression of colorectal neoplasia.
A three-phase design was employed in this study. In the screening phase, DNA methylation profile of 12 pairs of colorectal cancer (CRC) and adjacent normal tissues was analyzed by using the Illumina MethylationEPIC BeadChip. Significant CpG sites were selected based on a cross-validation analysis from The Cancer Genome Atlas (TCGA) database. Methylation levels of candidate CpGs were assessed using pyrosequencing in the training dataset (tumor lesions and adjacent normal tissues from 46 CRCs) and the validation dataset (tumor lesions and paired normal tissues from 13 hyperplastic polyps, 129 adenomas, and 256 CRCs). A linear mixed-effects model was used to examine the incremental changes of DNA methylation during the progression of colorectal neoplasia.
The comparisons between normal and tumor samples in the screening phase revealed an extensive CRC-specific methylomic pattern with 174,006 (21%) methylated CpG sites, of which 22,232 (13%) were hyermethylated and 151,774 (87%) were hypomethylated. Hypermethylation mostly occurred in CpG islands with an overlap of gene promoters, while hypomethylation tended to be mapped far away from functional regions. Further cross validation analysis from TCGA dataset confirmed 265 hypermethylated promoters coupling with downregulated gene expression. Among which, hypermethylated changes in MEEPD2 promoter was successfully replicated in both training and validation phase. Significant hypermethylation appeared since precursor lesions with an extensive modification in CRCs. The linear mixed-effects modeling analysis found that a cumulative pattern of MPPED2 methylation changes from normal mucosa to hyperplastic polyp to adenoma, and to carcinoma (P < 0.001).
Our findings indicate that epigenetic alterations of MPPED2 promoter region appear sequentially during the colorectal neoplastic progression. It might be able to serve as a promising biomarker for early diagnosis and stage surveillance of colorectal tumorigenesis.
表观遗传改变是肿瘤转化的常见致病因素。尽管先前的研究已经报道了一组与肿瘤抑制基因沉默相关的异常启动子甲基化改变，但对于癌发生过程中连续的 DNA 甲基化改变知之甚少。本研究的目的是进行全基因组搜索，以确定潜在的重要甲基化改变，并研究结直肠肿瘤发生过程中甲基化改变的起始和模式。
本研究采用了三阶段设计。在筛选阶段，使用 Illumina MethylationEPIC BeadChip 分析了 12 对结直肠癌 (CRC) 及其相邻正常组织的 DNA 甲基化谱。基于 The Cancer Genome Atlas (TCGA) 数据库的交叉验证分析，选择了具有显著差异的 CpG 位点。使用焦磷酸测序法在训练数据集（来自 46 例 CRC 的肿瘤病变和相邻正常组织）和验证数据集（来自 13 例增生性息肉、129 例腺瘤和 256 例 CRC 的肿瘤病变和配对正常组织）中评估候选 CpG 的甲基化水平。使用线性混合效应模型来研究结直肠肿瘤发生过程中 DNA 甲基化的递增变化。
筛选阶段中正常样本与肿瘤样本的比较显示了广泛的 CRC 特异性甲基组模式，其中 174006 个（21%）CpG 位点发生甲基化，其中 22232 个（13%）呈高甲基化，151774 个（87%）呈低甲基化。高甲基化主要发生在与基因启动子重叠的 CpG 岛，而低甲基化则倾向于远离功能区域。进一步的 TCGA 数据集交叉验证分析证实了 265 个与下调基因表达相关的高甲基化启动子。其中，MEEPD2 启动子的高甲基化改变在训练和验证阶段均得到了成功复制。从癌前病变开始就出现了显著的高甲基化改变，并且在 CRC 中广泛修饰。线性混合效应模型分析发现，从正常黏膜到增生性息肉、腺瘤，再到癌，MPPED2 甲基化变化呈累积模式（P < 0.001）。
我们的研究结果表明，MPPED2 启动子区域的表观遗传改变在结直肠肿瘤发生过程中是依次出现的。它可能成为结直肠肿瘤发生早期诊断和分期监测的有前途的生物标志物。
