Department of General Surgery, First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, China.
Hepatobiliary Pancreat Dis Int. 2011 Feb;10(1):95-100. doi: 10.1016/s1499-3872(11)60014-3.
A growing body of evidence suggests that many tumors are initiated by both epigenetic abnormalities and gene mutations, which promote tumor progression. Epigenetic abnormalities include changes in DNA methylation and in the modification of histones. This study aimed to assess the status of methylation in the CpG island (CGI) of the tumor necrosis factor receptor superfamily member 10c (TNFRSF10C) with combined bisulfite restriction analysis (COBRA) and to evaluate its role in the progression of pancreatic cancer (PC).
The methylation status of four PC cell lines was assessed using COBRA and/or bisulfite genomic sequencing (BGS). Changes in methylation and TNFRSF10C expression in PC cell lines before and after treatment with 5-aza-2'-deoxycytidine (5-aza-dC) and/or trichostatin A (TSA) were assessed by BGS and real-time RT-PCR. Apoptosis in the four cell lines was tested by flow cytometry (FCM) and TUNEL assay.
The methylation status of the TNFRSF10C promoter was assessed in PC cells (BxPC-3: 68.84+/-8.71%; CFPAC-1: 0; PANC-1: 96.77+/-4.57%; SW1990: 54.97+/-7.33%) with the COBRA assay, which was confirmed by the results of BGS. After treatment with 5-aza-dC and/or TSA, apoptosis was induced in PC cells to different degrees, and the levels of TNFRSF10C transcriptional expression in the PC cell lines (except CFPAC-1) increased markedly after 5-aza-dC treatment.
A high frequency of CGI methylation in the TNFRSF10C promoter results in inactivation of the gene and enhancement of tumor growth in most PC cell lines (except CFPAC-1). Inactivation of TNFRSF10C by CGI hypermethylation can play an important role in PC progression and be potentially useful as a diagnostic marker and a new therapeutic approach for PC.
越来越多的证据表明，许多肿瘤是由表观遗传异常和基因突变共同引发的，这些异常和突变促进了肿瘤的进展。表观遗传异常包括 DNA 甲基化和组蛋白修饰的改变。本研究旨在通过联合亚硫酸氢盐限制分析（COBRA）评估肿瘤坏死因子受体超家族成员 10c（TNFRSF10C）的 CpG 岛（CGI）甲基化状态，并评估其在胰腺癌（PC）进展中的作用。
使用 COBRA 和/或亚硫酸氢盐基因组测序（BGS）评估 4 种 PC 细胞系的甲基化状态。用 BGS 和实时 RT-PCR 检测 5-氮杂-2'-脱氧胞苷（5-aza-dC）和/或曲古抑菌素 A（TSA）处理前后 PC 细胞系中 TNFRSF10C 甲基化和表达的变化。用流式细胞术（FCM）和 TUNEL 检测 4 种细胞系的凋亡。
用 COBRA 检测法评估了 PC 细胞（BxPC-3：68.84+/-8.71%；CFPAC-1：0；PANC-1：96.77+/-4.57%；SW1990：54.97+/-7.33%）中 TNFRSF10C 启动子的甲基化状态，并用 BGS 结果进行了验证。用 5-aza-dC 和/或 TSA 处理后，PC 细胞发生不同程度的凋亡，除 CFPAC-1 外，PC 细胞系中 TNFRSF10C 转录表达水平在 5-aza-dC 处理后显著升高。
TNFRSF10C 启动子 CGI 高甲基化导致大多数 PC 细胞系（除 CFPAC-1 外）中基因失活，促进肿瘤生长。CGI 过度甲基化导致 TNFRSF10C 失活可能在 PC 进展中起重要作用，并可能作为 PC 的诊断标志物和新的治疗方法。
