Department of Gastroenterology, Qingdao Municipal Hospital(East), Medical College of Qingdao University, Qingdao City, Shandong, PR China.
Dis Esophagus. 2011 Aug;24(6):444-9. doi: 10.1111/j.1442-2050.2010.01159.x. Epub 2010 Dec 17.
Cyclooxygenase-2 (COX-2) is overexpressed in various types of human malignancies including esophageal squamous cell carcinoma (ESCC). However, a subset of ESCC either do not express COX-2 or show low level of expression. It is well established that promoter methylation is a major mechanism that mediates transcriptional silencing of COX-2 in gastric and colorectal cancer, but the data on ESCC are very limited. In this study, we attempted to determine whether COX-2 expression was also regulated by promoter methylation in human ESCC cell lines. We examined the methylation status of the COX-2 promoter in five human ESCC cell lines (EC109, EC9706, KYSE 410, KYSE 150, TE-1) using bisulfite sequencing analysis. Western blot analysis was used to determine COX-2 expression. Quantitative real-time polymerase chain reaction was used to determine COX-2 mRNA level. Prostaglandin (PG) E(2) was detected by ELISA. The promoter was densely methylated in TE-1 and KYSE 150, which had a low level of COX-2 expression and less methylated in other three cell lines (EC109, EC9706, KYSE 410), with high level of COX-2 expression. Treatment with 5-aza-deoxycytidine (5-aza-DC), a DNA methyltransferase inhibitor, demethylated the promoter and upregulated COX-2 expression, as well as PGE(2) production in TE-1 and KYSE 150. However, no such effects were observed in EC109. COX-2 protein was negative, but mRNA was positive in TE-1. After treatment with 5-aza-DC, both COX-2 mRNA and protein level had increased. These findings suggest that the promoter methylation may be one of the mechanisms that regulate COX-2 expression in ESCC.
环氧化酶-2（COX-2）在包括食管鳞状细胞癌（ESCC）在内的各种人类恶性肿瘤中过度表达。然而，ESCC 的一部分既不表达 COX-2，也不表现出低水平的表达。众所周知，启动子甲基化是介导胃和结直肠癌中 COX-2转录沉默的主要机制，但 ESCC 的相关数据非常有限。在这项研究中，我们试图确定 COX-2 的表达是否也受到人类 ESCC 细胞系中启动子甲基化的调节。我们使用亚硫酸氢盐测序分析检测了五个 ESCC 细胞系（EC109、EC9706、KYSE410、KYSE150、TE-1）中 COX-2 启动子的甲基化状态。使用 Western blot 分析来确定 COX-2 表达。使用定量实时聚合酶链反应来确定 COX-2 mRNA 水平。使用 ELISA 检测前列腺素（PG）E2。TE-1 和 KYSE150 的启动子高度甲基化，COX-2 表达水平较低，而其他三个细胞系（EC109、EC9706、KYSE410）启动子甲基化程度较低，COX-2 表达水平较高。用 DNA 甲基转移酶抑制剂 5-氮杂-2'-脱氧胞苷（5-aza-DC）处理后，TE-1 和 KYSE150 的启动子去甲基化，上调 COX-2 表达和 PGE2 产生。然而，在 EC109 中没有观察到这种效果。COX-2 蛋白为阴性，但在 TE-1 中 mRNA 为阳性。用 5-aza-DC 处理后，COX-2 mRNA 和蛋白水平均增加。这些发现表明，启动子甲基化可能是调节 ESCC 中 COX-2 表达的机制之一。
