神经胶质瘤细胞中GDNF基因高转录的表观遗传学机制研究

The mRNA expression of glial cell-derived neurotrophic factor (GDNF) is increased significantly in glioma cells. However, little is known about its transcriptional regulation mechanisms. Our preliminary data showed that the methylation of GDNF gene promoter regionⅡ is altered in glioma cells and this alteration of methylation is highly related to the grade and classification of glioma cells. We also found that the capacity of specific transcription factors binds to the regions with altered methylation has been changed. We predict that the changes in the binding capacity to specific transcription factors induced by altering methylation level of GDNF gene promoter region Ⅱ contribute to the enhancement of GDNF gene transcriptional level in glioma cells. The experiments proposed will 1), test whether change the methylation level at GDNF gene promoter region Ⅱ can affect it's binding activity to specific transcription factors by assessing the binding capacity of transcription factors and GDNF gene promoter region Ⅱ with altered methylation in glioma cells; 2) investigate the relationship between the binding capacity of transcription factors and the GDNF gene transcriptional level by changing transcription factors activity specifically; 3), explore the effects of altering methylation level at GDNF gene promoter region Ⅱ on mRNA expression of GDNF by target silencing the sequence of GDNF gene promoter regionⅡwith altered methylation in glioma cells. This study can reveal the occurrence and development mechanism of glioma.

神经胶质瘤细胞中胶质细胞系源性神经营养因子（GDNF）mRNA表达水平显著升高，但其转录调控机制尚不明确。预实验发现：胶质瘤细胞中GDNF基因启动子Ⅱ区存在甲基化修饰改变，其变化与胶质瘤细胞分级分型存在密切关联，并且其甲基化修饰变化区域与特异性转录因子的结合能力也发生了改变。由此我们推测：胶质瘤细胞中GDNF基因启动子Ⅱ区甲基化修饰水平的变化，改变了其与特异性转录因子的结合能力，从而导致GDNF基因转录水平的升高。本项目拟通过检测胶质瘤细胞中发生甲基化修饰改变的GDNF基因启动子Ⅱ区与特异性转录因子的结合能力，探索此种甲基化修饰水平的变化是否影响其与特异性转录因子的结合；特异性改变转录因子活性，探讨转录因子结合能力与GDNF转录水平的关系；导向沉默胶质瘤细胞中GDNF基因启动子Ⅱ区发生甲基化修饰改变的序列，探索其对GDNFmRNA水平的影响。本项目有助于揭示胶质瘤的发生发展机制。

胶质细胞系源性神经营养因子（glial cell—line derived neurotrophic factor， GDNF）属于转化生长因子（TGF-β）超家族成员，在胶质瘤发生发展过程中发挥着重要的作用。有研究发现，在胶质瘤细胞中GDNF mRNA的表达量异常升高，但其转录机制尚不明确。本研究的结果显示：胶质瘤细胞中GDNF启动子区的碱基序列并未变化，但启动子区甲基化修饰程度发生了显著性改变：启动子Ⅰ区甲基化修饰水平显著升高（P﹤0.05），且高低级别胶质瘤组织之间无显著差异（P>0.05）；启动子Ⅱ区在低级别胶质瘤组织中甲基化修饰水平显著降低（P﹤0.05），且降低的位点主要位于增强子区，而高级别胶质瘤组织中甲基化修饰水平显著升高（P﹤0.05），且升高的位点主要位于沉默子区。与这些甲基化修饰水平改变的区域特异性结合的转录因子AP-2、SP1、ERG-2和CREB，随着胶质瘤病理级别的增高，其结合量呈现下降的趋势，且高级别组与正常脑组织组间差异显著（P<0.05），表明GDNF基因启动子区甲基化改变引起的转录因子结合量的改变可能是GDNF基因在胶质瘤中异常高表达的原因。