Yoshida Satoshi, Yamashita Satoshi, Niwa Tohru, Mori Akiko, Ito Seiji, Ichinose Masao, Ushijima Toshikazu
Division of Epigenomics, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan.
Second Department of Internal Medicine, Wakayama Medical University, Wakayama, Japan.
Gastric Cancer. 2017 Jan;20(1):136-145. doi: 10.1007/s10120-016-0593-5. Epub 2016 Jan 20.
Gastric cancer (GC) is highly influenced by aberrant methylation, and accumulation of aberrant methylation in gastric mucosae produces an epigenetic field for cancerization. Nevertheless, the individual driver genes involved in such field cancerization are still unclear. Here, we aimed to demonstrate that FAT4, a novel tumor suppressor identified by exome sequencing of GC, is methylation-silenced and that such methylation is involved in epigenetic field cancerization for GC.
A transcription start site was determined by the 5' rapid amplification of complementary DNA ends method. DNA methylation was analyzed by bisulfite sequencing with use of a next-generation sequencer or quantitative methylation-specific PCR. Gene expression was analyzed by quantitative reverse transcription PCR.
A single transcription start site was identified for FAT4 in gastric epithelial cells, and a CpG island was located in the FAT4 promoter region. FAT4 was highly methylated in two of 13 GC cell lines and was not expressed in them. Removal of FAT4 methylation by a DNA demethylating agent (5-aza-2'-deoxycytidine) restored its expression in the two cell lines. In primary GC samples, FAT4 was methylated in 12 of 82 GCs (14.6 %). FAT4 methylation was associated with the presence of the CpG island methylator phenotype but not with prognosis, tumor invasion, lymph node metastasis, or histological types. In noncancerous gastric mucosae, high FAT4 methylation levels were associated with the presence of GC and Helicobacter pylori infection.
FAT4 was methylation-silenced in GCs. Its methylation in gastric mucosae was associated with H. pylori infection and likely contributed to epigenetic field cancerization.
胃癌（GC）受异常甲基化的影响很大，胃黏膜中异常甲基化的积累产生了一个致癌的表观遗传区域。然而，参与这种区域致癌作用的个体驱动基因仍不清楚。在此，我们旨在证明，通过胃癌外显子测序鉴定出的一种新型肿瘤抑制因子FAT4被甲基化沉默，且这种甲基化参与了胃癌的表观遗传区域致癌作用。
通过5' 互补DNA末端快速扩增法确定转录起始位点。使用二代测序仪通过亚硫酸氢盐测序或定量甲基化特异性PCR分析DNA甲基化。通过定量逆转录PCR分析基因表达。
在胃上皮细胞中鉴定出FAT4的单个转录起始位点，且一个CpG岛位于FAT4启动子区域。FAT4在13个胃癌细胞系中的2个中高度甲基化，且在这些细胞系中不表达。用DNA去甲基化剂（5-氮杂-2'-脱氧胞苷）去除FAT4甲基化可恢复其在这两个细胞系中的表达。在原发性胃癌样本中，82例胃癌中有12例（14.6%）FAT4被甲基化。FAT4甲基化与CpG岛甲基化表型的存在相关，但与预后、肿瘤侵袭、淋巴结转移或组织学类型无关。在非癌性胃黏膜中，FAT4高甲基化水平与胃癌的存在及幽门螺杆菌感染相关。
FAT4在胃癌中被甲基化沉默。其在胃黏膜中的甲基化与幽门螺杆菌感染相关，并可能促成了表观遗传区域致癌作用。
