Table_1_Deep Sequencing Revealed a CpG Methylation Pattern Associated With ALDH1L1 Suppression in Breast Cancer.DOCX

Hypermethylation of promoter CpG islands is generally recognized epigenetic mechanism responsible for gene silencing in cancer. However, molecular details on how this epigenetic mark triggers the process of gene downregulation are still elusive. Here, we used deep bisulfite sequencing and qPCR analysis to investigate the pattern of CpG methylation of ALDH1L1 promoter region and its association with the gene expression level in 16 paired breast cancer (BC) samples of different clinical stages. Expression of ALDH1L1 gene was suppressed in all examined BC samples up to 200-fold, and average hypermethylation level of the promoter region correlated positively with ALDH1L1 downregulation. We determined the role of every individual CpG site within the ALDH1L1 promoter, including upstream untranscribed region, first untranslated exon, and the start of the first intron, in aberrant gene expression by correlation analysis. The search revealed CpG sites which methylation has the highest impact on intensity of gene transcription. The majority of such CpG sites are located in a compact region in the first intron of the ALDH1L1 gene. These results assist in unraveling of dynamic nature of CpG promoter hypermethylation as well as demonstrate the efficiency of deep bisulfite sequencing in search for novel epigenetic markers in cancer.

启动子 CpG 岛的过度甲基化通常被认为是导致癌症中基因沉默的表观遗传机制。然而，关于这一表观遗传标记如何触发基因下调过程的分子细节仍不明确。在本研究中，我们利用深度亚硫酸氢盐测序和定量聚合酶链反应分析，探讨了 ALDH1L1 启动子区域 CpG 甲基化模式及其与 16 对不同临床阶段的乳腺癌（BC）样本中基因表达水平的相关性。在所有检测到的 BC 样本中，ALDH1L1 基因的表达被抑制，高达 200 倍，且启动子区域的平均高甲基化水平与 ALDH1L1 下调呈正相关。通过相关性分析，我们确定了 ALDH1L1 启动子内每个单独的 CpG 位点的作用，包括上游未转录区、第一个未翻译外显子和第一个内含子的起始部位，在异常基因表达中的作用。研究揭示了那些对基因转录强度影响最大的甲基化 CpG 位点，其中大多数位于 ALDH1L1 基因第一个内含子中的一个紧密区域。这些结果有助于揭示 CpG 启动子高甲基化的动态特性，并展示了深度亚硫酸氢盐测序在寻找癌症中新型表观遗传标记方面的有效性。