恶性胶质瘤细胞中GDNF基因启动子上特定顺式作用元件甲基化水平升高的分子机制研究

Research shows that Glial cell line derived neurotrophic factor (GDNF) is highly expressed in glioblastoma (GBM) and blocking its expression can inhibit the occurrence and development of GBM. The silencer II hypermethylation in the gdnf promoter II may underlie high gdnf gene transcription in GBM cells. According to existing work, we hypothesize that in normal astrocytes (NA), the silencer II in the gdnf promoter II may combine with a certain binding factor, which play a role in blocking methyl transferase. However, this binding factor changed in GBM cells, resulting in the removal of the methyltransferase inhibition. This leads to high methylation which enhances RNA polymerase II activity, and consequently the GDNF transcription level increases. With existing knowledge that the binding factor is located in the silencer II of the gdnf promoter II, we intend to figure out the similarities and differences between the binding factor of the NA and GBM cells by Reverse ChIP combined with mass spectrometry, and to investigate the effects of the Modified or unmodified binding factor on the methylation, RNA polymerase Ⅱ activity and gdnf transcription level when combined with or without silencerⅡ, then we go deeper into the cause of changes in these binding factors.

研究发现，胶质母细胞瘤（GBM）细胞显著高表达胶质细胞系源性神经营养因子（GDNF)，阻断其高表达能抑制GBM的发生发展；GDNF基因启动子Ⅱ区之沉默子Ⅱ区的甲基化水平升高是该基因高转录的重要原因，然而机制不清。依据已有的工作，我们推测：在正常星形胶质细胞（NA），GDNF基因启动子Ⅱ区沉默子Ⅱ区结合着某种因子，其对甲基转移酶起阻碍作用；但是，在GBM细胞，该因子发生变化，其结果是该处甲基转移酶的障碍被解除，于是，这里甲基化水平升高。基于此，我们拟以反向CHIP联合质谱分析技术，了解NA和GBM细胞的GDNF基因启动子Ⅱ区沉默子Ⅱ区结合因子之异同；研究有无结合因子、或不同结合因子、或修饰与否的结合因子结合或不结合该沉默子Ⅱ区时，对沉默子Ⅱ区甲基化、RNA聚合酶Ⅱ的活性及GDNF基因转录水平的影响，并深入研究在GBM细胞导致该处结合因子变化的原因。

胶质细胞系源性神经营养因子（GDNF）在胶质母细胞瘤（GBM）中显著性高表达，与GBM的发生发展密切相关。研究表明，不同表观修饰异常参与了GBM细胞中GDNF基因的高转录调控，且该基因启动子Ⅱ区内沉默子Ⅱ的沉默效应异常是导致其高转录的先决条件之一，但具体机制不清。本研究结果显示：GDNF基因沉默子II区经高DNA甲基化和组蛋白H3K9三甲基（H3K9me3）修饰形成了沉默型染色质，其迫使高表达的CREB选择性与增强子II结合进而促进GBM细胞中该基因的高转录，且低水平的CUE结构域包含蛋白2（CUEDC2）通过减少CREB泛素化降解的途径进一步维持了该机制的运行。组蛋白H3K9甲基转移酶SETDB1能够结合于GDNF沉默子Ⅱ区潜在的结合位点；敲减或过表达SETDB1能够显著下调或上调SETDB1与GDNF沉默子Ⅱ区的结合量、该区域组蛋白H3K9me3水平和GDNF基因的转录水平；U251细胞中转录因子STAT1能够与SETDB1结合，且能结合至GDNF沉默子II区的潜在结合位点，该位点与SETDB1的结合位点部分重合，由此推测SETDB1经STAT1募集至GDNF基因沉默子II区升高该区域组蛋白H3K9me3水平，抑制沉默子II的沉默效应，进而促进该基因的高转录。此外，我们发现GDNF通过激活MARK信号通路促进双皮质素（DCX）入核进而促进GBM的侵袭性生长；lncRNA TCONS_00020456在GBM中显著性低表达，且其能够经阻抑Smad2/PKCα通路降低EMT转化的方式抑制GBM的进展；鉴定了GBM中存在新型的融合转录本Bcl2l2-Pabpn1，且发现其参与了GBM的恶性进展。本项目进一步揭示了GBM中GDNF高转录的表观遗传机制，并阐明了GDNF等分子促进GBM进展的新机制，为GBM的防治提供了新思路。