Institute of Biochemistry, Stuttgart University, Stuttgart, Germany.
Max-Planck-Genomzentrum Köln, Köln, Germany.
PLoS One. 2014 Jan 29;9(1):e87703. doi: 10.1371/journal.pone.0087703. eCollection 2014.
The Epithelial Cell Adhesion Molecule (EpCAM) is overexpressed in many cancers including ovarian cancer and EpCAM overexpression correlates with decreased survival of patients. It was the aim of this study to achieve a targeted methylation of the EpCAM promoter and silence EpCAM gene expression using an engineered zinc finger protein that specifically binds the EpCAM promoter fused to the catalytic domain of the Dnmt3a DNA methyltransferase. We show that transient transfection of this construct increased the methylation of the EpCAM promoter in SKOV3 cells from 4-8% in untreated cells to 30%. Up to 48% methylation was observed in stable cell lines which express the chimeric methyltransferase. Control experiments confirmed that the methylation was dependent on the fusion of the Zinc finger and the methyltransferase domains and specific for the target region. The stable cell lines with methylated EpCAM promoter showed a 60-80% reduction of EpCAM expression as determined at mRNA and protein level and exhibited a significantly reduced cell proliferation. Our data indicate that targeted methylation of the EpCAM promoter could be an approach in the therapy of EpCAM overexpressing cancers.
上皮细胞黏附分子（EpCAM）在许多癌症中过度表达，包括卵巢癌，并且 EpCAM 过表达与患者生存率降低相关。本研究旨在利用一种工程化的锌指蛋白实现 EpCAM 启动子的靶向甲基化，该蛋白特异性结合 EpCAM 启动子，并融合了 Dnmt3a DNA 甲基转移酶的催化结构域，从而沉默 EpCAM 基因表达。我们发现，该构建体的瞬时转染可将 SKOV3 细胞中 EpCAM 启动子的甲基化水平从未经处理的细胞中的 4-8%提高到 30%。在表达嵌合甲基转移酶的稳定细胞系中，观察到高达 48%的甲基化。对照实验证实，甲基化依赖于锌指和甲基转移酶结构域的融合，并且是针对靶区域特异性的。具有 EpCAM 启动子甲基化的稳定细胞系在 mRNA 和蛋白质水平上显示 EpCAM 表达降低了 60-80%，并且细胞增殖明显减少。我们的数据表明，靶向 EpCAM 启动子的甲基化可能是治疗 EpCAM 过表达癌症的一种方法。
