Beltrami Caroline Moraes, Dos Reis Mariana Bisarro, Barros-Filho Mateus Camargo, Marchi Fabio Albuquerque, Kuasne Hellen, Pinto Clóvis Antônio Lopes, Ambatipudi Srikant, Herceg Zdenko, Kowalski Luiz Paulo, Rogatto Silvia Regina
International Research Center-CIPE-A.C. Camargo Cancer Center and National Institute of Science and Technology in Oncogenomics (INCiTO), São Paulo, Brazil.
Department of Urology, Faculty of Medicine, UNESP, Sao Paulo State University, Botucatu, São Paulo Brazil.
Clin Epigenetics. 2017 May 2;9:45. doi: 10.1186/s13148-017-0346-2. eCollection 2017.
Papillary thyroid carcinoma (PTC) is a common endocrine neoplasm with a recent increase in incidence in many countries. Although PTC has been explored by gene expression and DNA methylation studies, the regulatory mechanisms of the methylation on the gene expression was poorly clarified. In this study, DNA methylation profile (Illumina HumanMethylation 450K) of 41 PTC paired with non-neoplastic adjacent tissues (NT) was carried out to identify and contribute to the elucidation of the role of novel genic and intergenic regions beyond those described in the promoter and CpG islands (CGI). An integrative and cross-validation analysis were performed aiming to identify molecular drivers and pathways that are PTC-related.
The comparisons between PTC and NT revealed 4995 methylated probes (88% hypomethylated in PTC) and 1446 differentially expressed transcripts cross-validated by the The Cancer Genome Atlas data. The majority of these probes was found in non-promoters regions, distant from CGI and enriched by enhancers. The integrative analysis between gene expression and DNA methylation revealed 185 and 38 genes (mainly in the promoter and body regions, respectively) with negative and positive correlation, respectively. Genes showing negative correlation underlined FGF and retinoic acid signaling as critical canonical pathways disrupted by DNA methylation in PTC. mutation was detected in 68% (28 of 41) of the tumors, which presented a higher level of demethylation (95% hypomethylated probes) compared with wild-type tumors. A similar integrative analysis uncovered 40 of 254 differentially expressed genes, which are potentially regulated by DNA methylation in V600E-positive tumors. The methylation and expression pattern of six selected genes (, , , , , and ) were confirmed as altered by pyrosequencing and RT-qPCR.
DNA methylation loss in non-promoter, poor CGI and enhancer-enriched regions was a significant event in PTC, especially in tumors harboring V600E. In addition to the promoter region, gene body and 3'UTR methylation have also the potential to influence the gene expression levels (both, repressing and inducing). The integrative analysis revealed genes potentially regulated by DNA methylation pointing out potential drivers and biomarkers related to PTC development.
甲状腺乳头状癌（PTC）是一种常见的内分泌肿瘤，近年来在许多国家发病率呈上升趋势。尽管已经通过基因表达和DNA甲基化研究对PTC进行了探索，但甲基化对基因表达的调控机制仍未完全阐明。在本研究中，对41例PTC及其配对的非肿瘤性相邻组织（NT）进行了DNA甲基化谱分析（Illumina HumanMethylation 450K），以识别并有助于阐明启动子和CpG岛（CGI）以外的新基因和基因间区域的作用。进行了综合和交叉验证分析，旨在识别与PTC相关的分子驱动因素和途径。
PTC与NT之间的比较揭示了4995个甲基化探针（88%在PTC中低甲基化）和1446个差异表达转录本，这些转录本通过癌症基因组图谱数据进行了交叉验证。这些探针大多位于非启动子区域，远离CGI且富含增强子。基因表达与DNA甲基化之间的综合分析分别揭示了185个和38个基因（主要分别位于启动子和基因体区域）呈负相关和正相关。显示负相关的基因强调了FGF和视黄酸信号通路是PTC中被DNA甲基化破坏的关键经典途径。在68%（41例中的28例）的肿瘤中检测到V600E突变，与野生型肿瘤相比，这些肿瘤呈现出更高水平的去甲基化（95%的低甲基化探针）。类似的综合分析在254个差异表达基因中发现了40个，这些基因可能在V600E阳性肿瘤中受DNA甲基化调控。通过焦磷酸测序和RT-qPCR证实了六个选定基因（、、、、、和）的甲基化和表达模式发生了改变。
在非启动子、CGI较差且富含增强子的区域中DNA甲基化缺失是PTC中的一个重要事件，尤其是在携带V600E的肿瘤中。除了启动子区域外，基因体和3'UTR甲基化也有可能影响基因表达水平（包括抑制和诱导）。综合分析揭示了可能受DNA甲基化调控的基因，指出了与PTC发展相关的潜在驱动因素和生物标志物。
