EMX2在结直肠癌中的甲基化状态、临床意义及作用机理研究

Epigenetic study of gene has become a new era of developing early diagnositic tumor marker, prediction of prognosis and target therapy development in colorectal cancer. Wnt signaling pathway has been confirmed as the key pathway in tumorgeneisis. Recently, it has been shown that empty spiracles homeobox 2 (EMX2), a human homolog of Drosophila empty spiracles gene (ems),may regulate neural stem cell proliferation,endometrial cell proliferation and cell differentiation through the Wnt signaling pathway. Our research group found that the methylation of EMX2 promoter was significantly different (p<0.001) between colorectal cancer tissue (78.9%) and normal surgical margin tissue(42.1%).These pre-study results suggest that EMX2 may be a new tumor suppressor which is epigenetically silenced in colorectal cancer. Based on the previous results, this project is going to detect the expression level of EMX2 in colorectal cancer specimens and understand the relation between EMX2 expression level and prognosis. To study the relation between EMX2 promoter methylation and EMX2 expression level, EMX2 promoter methylation will be detected in colorectal cancer specimen and colorectal cell lines by methylation-specific PCR. Moreover, to provide the experimental data of EMX2 as a new tumor suppressor gene in colorectal cancer, the mechanism of EMX2 regulating cell proliferation and migration as a tumor suppressor will be investigated in colorectal cell lines.

基因的表观遗传学研究目前已成为结直肠癌早期诊断、预后预测及靶向治疗研究的新方向。已证明Wnt信号通路是多种肿瘤发生的关键通路，EMX2基因是果蝇空泡基因的人同源物，其可通过Wnt信号通路抑制神经细胞、子宫内膜细胞的增殖。本课题组前期研究发现EMX2基因启动子区甲基化率在19例结直肠癌组织（78.9%）和切缘正常组织（42.1%）中存在显著差异（p<0.001），提示EMX2可能是结直肠癌中新的受表观遗传调控的抑癌基因。本项目拟在前期工作基础上，检测结直肠癌组织中的EMX2表达量，分析EMX2表达量与病理、预后的关系；通过甲基化特异性PCR检测结直肠癌组织和细胞系中EMX2基因启动子甲基化状态，研究EMX2启动子甲基化和表达量的关系，并在细胞水平研究EMX2对细胞增殖、迁移的影响，深入探讨EMX2是否通过Wnt通路发挥抑癌作用，为验证EMX2作为一个新的结直肠癌抑癌基因提供重要试验依据。

基因的表观遗传学研究目前已成为结直肠癌早期诊断、预后预测及靶向治疗研究的新方向。已证明Wnt信号通路是多种肿瘤发生的关键通路，EMX2基因是果蝇空泡基因的人同源物，其可通过Wnt信号通路抑制神经细胞、子宫内膜细胞的增殖。本课题组发现EMX2基因启动子区甲基化率在结直肠癌组织和切缘正常组织中存在显著差异（p<0.001），提示EMX2可能是结直肠癌中新的受表观遗传调控的抑癌基因。在研究过程中我们发现，EMX2在正常结肠上皮细胞和LOVO细胞系中高表达，在HCT116和HT29细胞系中低表达。而经过去甲基化试剂DAC处理后EMX2在HCT116，HT29中表达量明显上调。并且，高表达EMX2可以抑制细胞的生长。在稳定表达EMX2shRNA细胞系中，EMX2表达明显下调，其迁移速度显著增高。在转染了EMX2的HCT116细胞中，细胞质β-Catenin和Cyclin D1蛋白表达受到抑制。因此，我们得出结论，EMX2在结直肠癌中发挥抑癌基因的作用，其发挥抑癌基因作用的重要途径是Wnt信号通路，并且EMX2基因启动子区的高甲基化会下调EMX2的表达量。在后续实验中将进一步验证EMX2甲基化与预后的关系。