DNA methylation, induced by beta-carotene and arachidonic acid, plays a regulatory role in the pro-angiogenic VEGF-receptor (KDR) gene expression in endothelial cells.
DNA methylation, induced by beta-carotene and arachidonic acid, plays a regulatory role in the pro-angiogenic VEGF-receptor (KDR) gene expression in endothelial cells.
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发表时间：
2009-12
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通讯作者：
B. Kieć-Wilk;U. Raźny;J. Mathers;A. Dembińska-kieć
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作者：
B. Kieć-Wilk;U. Raźny;J. Mathers;A. Dembińska-kieć
未标记DNA甲基化是基因表达的有效调节因子。本文报道了β-胡萝卜素（BC）和花生四烯酸（AA）对新生血管形成的影响。血管内皮生长因子激活酪氨酸激酶VEGFR-2受体（KDR）是主要的血管生成机制之一。本研究的目的是探讨CpG岛甲基化对人内皮细胞与BC和/或AA孵育后促血管生成KDR基因表达的调节作用。方法将人脐静脉内皮细胞（HUVEC）与1-10 μ M的BC和/或3 μ M的AA共同孵育24小时。使用COBRA方法和限制酶消化（NewEngland BioLabs）定量CpG岛甲基化。使用特异性抗体（圣克鲁斯）通过蛋白质印迹分析测定细胞内蛋白质浓度。结果BC和AA共同孵育后，KDR基因启动子区甲基化水平降低。这些结果与通过qRT-PCR检测到的BC（p=0.035）以及AA对KDR基因表达的上调密切相关。与BC（p=0.02）和AA（p=0.0014）孵育增加了HUVEC中的KDR蛋白水平。结论促血管生成基因KDR启动子区CpG岛甲基化的改变是BC和AA调控血管生成反应的机制之一。
UNLABELLED DNA methylation is a potent regulator of gene expression. The influence of beta-carotene (BC) and arachidonic acid (AA) on angiogenesis--a new blood vessel formation, was reported. The tyrosine kinase VEGFR-2 receptor (KDR) activation by vascular endothelial growth factor is one of the main angiogenic mechanisms. This study was aimed to investigate a possible role of CpG island methylation on regulation of the pro-angiogenic KDR gene expression after incubation of human endothelial cells with BC and/or AA. METHODS Human umbilical vein endothelial cells (HUVEC) were incubated with BC (1-10 microM) and/or 3 microM AA for 24 hours. The CpG island methylation was quantified using the COBRA method and restriction enzymes' digestion (NewEngland BioLabs). Intracellular protein concentrations were determined by Western blot analysis using the specific antibodies (Santa Cruz). RESULTS Incubation with BC and AA, decreased methylation of the KDR promoter region. These results well-correlated with the detected, by qRT-PCR, up-regulation of KDR gene expression by BC (p=0.035) as well as by AA. Incubation with BC (p=0.02) and AA (p=0.0014) increased the KDR protein levels in HUVECs. CONCLUSION The changes in CpG island methylation of the KDR the pro-angiogenic gene promoter, represents one of the mechanisms involved in regulation of angiogenic response by BC and AA.