Tissue factor pathway inhibitor-2 was repressed by CpG hypermethylation through inhibition of KLF6 binding in highly invasive breast cancer cells
Tissue factor pathway inhibitor-2 was repressed by CpG hypermethylation through inhibition of KLF6 binding in highly invasive breast cancer cells
复制标题
在高侵袭性乳腺癌细胞中，CpG 高甲基化通过抑制 KLF6 结合来抑制组织因子途径抑制剂-2
DOI：
10.1186/1471-2199-8-110
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发表时间：
2007-12-03
影响因子：
--
通讯作者：
Ma, Duan
中科院分区：
文献类型：
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作者：
Guo, Hongshen;Lin, Yifeng;Ma, Duan
背景资料：组织因子途径抑制物-2（TFPI-2）是基质相关Kunitz抑制剂，其抑制纤溶酶和胰蛋白酶介导的酶原基质金属蛋白酶的活化，所述酶原基质金属蛋白酶参与肿瘤进展、侵袭和转移。结果：在高侵袭性乳腺癌细胞株MDA-MB-435中，TFPI-2的蛋白和mRNA均未检测到。为了进一步研究乳腺癌细胞中TFPI-2的阻遏机制，克隆了1.5Kb TFPI-2启动子，发现启动子区的点突变不影响荧光素酶活性，缺失突变进一步证实了这一现象。扫描突变和信息学分析确定了TFPI-2启动子中一个潜在的KLF 6结合位点。通过亚硫酸氢盐修饰的序列显示，在MDA-MB-435中，TFPI-2启动子区的CpG岛被高甲基化。最后，通过EMSA和ChIP实验，我们证明KLF-6结合位点的CpG甲基化降低了KLF-6与TFPI-2启动子的结合。在本研究中，我们发现TFPI-2启动子中的CpG岛在高侵袭性乳腺癌细胞系中高甲基化，整个启动子区域的DNA甲基化通过诱导非活性染色质结构和减少KLF 6与其DNA结合序列的结合而引起TFPI-2抑制。
Background: Tissue factor pathway inhibitor-2 (TFPI-2) is a matrix-associated Kunitz inhibitor that inhibits plasmin and trypsin-mediated activation of zymogen matrix metalloproteinases involved in tumor progression, invasion and metastasis. Here, we have investigated the mechanism of DNA methylation on the repression of TFPI-2 in breast cancer cell lines.Results: We found that both protein and mRNA of TFPI-2 could not be detected in highly invasive breast cancer cell line MDA-MB-435. To further investigate the mechanism of TFPI-2 repression in breast cancer cells, 1.5 Kb TFPI-2 promoter was cloned, and several genetic variations were detected, but the promoter luciferase activities were not affected by the point mutation in the promoter region and the phenomena was further supported by deleted mutation. Scan mutation and informatics analysis identified a potential KLF6 binding site in TFPI-2 promoter. It was revealed, by bisulfite modified sequence, that the CpG island in TFPI-2 promoter region was hypermethylated in MDA-MB-435. Finally, using EMSA and ChIP assay, we demonstrated that the CpG methylation in the binding site of KLF-6 diminished the binding of KLF6 to TFPI-2 promoter.Conclusion: In this study, we found that the CpG islands in TFPI-2 promoter was hypermethylated in highly invasive breast cancer cell line, and DNA methylation in the entire promoter region caused TFPI-2 repression by inducing inactive chromatin structure and decreasing KLF6 binding to its DNA binding sequence.