神经胶质瘤中同源盒基因six3启动子甲基化修饰的分子机制及其功能初步研究

A lot of homeobox genes invovle in tumor development.In our previous study, MeDIP combination with CpG island and promoter microarrays were applied to screen aberrant methylation regions in glioma tissues. We found that the methylation level of homeobox gene Six3 promoter in glioma tissues was significant higher compared with the normal control. Furthermore, the real-time PCR results showed that the Six3 expression were inversely associated with the level of its promoter methylation in some degree. Treatment with demethylating agent 5-aza-2'-deoxycytidine could significantly up-regulat the Six3 expression in glioma cell lines in which Six3 promoter were hypermethylated. Thus, we speculated that Six3 might invovle in initiation and development of glioma. It has been reported that Six3 played an important role in nervous system development and it could inhibit Wnt1 expression and interact with nuclear orphan receptor Nor1 in order to ensure forebrain normal development. Nor1 is normally invovled in the cell differentiation. Six3 being directly regulated by metastasis-Associated gene MTA1 has also been reported. Therefore, we put forward a scientific hypothesis: In glioma, homeobox gene Six3 expression was down-regulated due to its promoter being methylated and other epigentic modifications. The down-regulation of Six3 increased the activity of Wnt1 pathway and reduced the interaction with Nor1. All the changes enhanced the glioma cells invasion and metastasis and blocked the glioma cells differentiation. If the hypothesis could be verfied, the regulatory molecular mechanism of Six3 promoter methylation and the preliminary role of Six3 in glioma would be clarified. In addition, it would also provide new clues for glioma diagnosis,therapy or prognosis.

同源盒基因异常参与了肿瘤的发生发展。前期我们采用甲基化芯片对胶质瘤组织中异常甲基化位点进行了初步筛选，其中发现同源盒基因Six3启动子在胶质瘤中甲基化水平显著高于正常对照；实时定量PCR结果显示Six3表达与启动子甲基化水平呈一定程度的负相关；甲基化酶抑制剂处理胶质瘤细胞后，Six3的表达显著升高。文献报道，Six3是神经系统发育调节基因，Six3能抑制Wnt1，并与细胞分化调节因子核孤儿受体Nor1相互作用，以确保脑的正常发育；同时Six3也受转移相关基因MAT1直接调控。由此，我们提出科学假说：胶质瘤中Six3因甲基化等表观遗传学修饰导致其表达下调，从而导致Wnt1通路活性增加，与Nor1交互作用减少，使胶质瘤细胞侵袭转移增强，分化受阻。验证该假说，不仅能明确胶质瘤中Six3启动子甲基化修饰的调控机制，初步阐明其在胶质瘤发生发展中的作用，也将为胶质瘤的诊断、治疗或预后等提供新的线索。

Six3蛋白为同源盒转录因子SIX家族成员之一，有研究表明其不仅在前脑和眼睛发育中起重要的调节作用，而且参与某些肿瘤如肺癌、乳腺癌的发生发展。我们前期结果表明，与正常脑组织相比，Six3在神经胶质瘤组织及细胞中呈高水平的甲基化及低水平的表达。Six3是否参与了神经胶质瘤的发生发展，不是十分清楚。通过本项目的研究，我们明确了Six3基因启动子甲基化对其表达的调控；证实了Six3通过抑制Wnt1/beta-catenin通路活性，进而抑制了胶质瘤细胞的侵袭转移；同时也证实了Six3通过调节细胞周期（G1/S过渡）而抑制了胶质瘤细胞的增殖及体外成瘤的能力。以上结果不仅表明Six3在胶质瘤中潜在的抑瘤作用，也为胶质瘤的诊断、治疗等提供新的线索。