Regulation of hMSH2 and hMLH1 expression in the human colon cancer cell line SW1116 by DNA methyltransferase 1
Regulation of hMSH2 and hMLH1 expression in the human colon cancer cell line SW1116 by DNA methyltransferase 1
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DOI：
10.1016/j.canlet.2005.03.005
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发表时间：
2006-02-20
期刊：
影响因子：
9.7
通讯作者：
Chen, YX
中科院分区：
文献类型：
--
作者：
Fang, JY;Lu, R;Chen, YX
异常 DNA 甲基化现在被认为是人类癌症早期发生的重要表观遗传改变。为了直接研究 DNA 甲基转移酶 1 (DNMT1) 在人结肠癌细胞肿瘤相关基因表达调节中的作用，我们将含有正义或反义 DNMT1 的表达构建体稳定转染到人结肠癌细胞系 SW1116 中。通过实时 RT-PCR 监测错配修复基因 (MMR)、人 mut-L 同源物 1 (hMLH1) 和人 Mut S 同源物 2 (hMSH2) 的表达水平。通过亚硫酸氢盐修饰和甲基化特异性 PCR (MSP) 测定 hMLH1 和 hMSH2 启动子的甲基化状态。通过 Western 分析测定 DNMT1、hMSH2 和 hMLH1 的蛋白水平。结果表明，在含有有义或反义 DNMT1 构建体的转染细胞系中，DNMT1 蛋白表达分别增加或减少。在表达有义 DNMT1 构建体的细胞中，hMLH1 和 hMSH2 的表达通过其各自启动子的高甲基化而下调。此外，反义 DNMT1 表达诱导 SW 1116 细胞中 hMSH2 (P < 0.05) 和 hMLH1 (P=0.064) 启动子去甲基化和转录上调。 (c) 2005 Elsevier Ireland Ltd. 保留所有权利。
Aberrant DNA methylation is now recognized as an important epigenetic alteration occurring early in human cancer. To directly study the role of DNA methltransferase 1 (DNMT1) in the regulation of expression of tumor-related genes in human colon cancer cells, we stably transfected expression constructs containing sense or antisense DNMT1 into the human colon cancer cell line, SW1116. The expression level of mismatch repair genes (MMR), human mut-L homologue 1 (hMLH1) and human Mut S homologue 2 (hMSH2), was monitored by real-time RT-PCR. The methylation status of hMLH1 and hMSH2 promoters was determined by bisulfite modification and methylation-specific PCR (MSP). The protein levels of DNMT1 hMSH2 and hMLH1 were determined by Western analysis. The results show that DNMT1 protein expression was increased or decreased in transfected cell lines containing sense or antisense DNMT1 constructs, respectively. In cells expressing the sense DNMT1 construct, the expression of hMLH1 and hMSH2 was down-regulated through hypermethylation of their respective promoters. Furthermore, antisense DNMT1 expression induced promoter demethylation and up-regulated transcription of hMSH2 (P < 0.05) and hMLH1 (P=0.064) in SW 1116 cells. (c) 2005 Elsevier Ireland Ltd. All rights reserved.