AJAP1对MAGEA2的调控机制及其在胶质母细胞瘤恶性进展中的作用

Glioblastoma multiforme (GBM) remains one of the most common and biologically aggressive tumors in adults. Patients with newly diagnosed GBM have a median survival of less than 14 months which mainly resulted from its poor responses to all therapeutic modalities. We for the first time identified that AJAP1 could regulate the expression of MAGEA2 gene on transcription lever by binding the promoter of MAGEA2; AJAP1 could induce the cell apoptosis and inhibit cell proliferation through down-regulating MAGEA2 in GBM; Down-regulation of AJAP1 was related to the decreased survival time of patients with glioma..Based on our previous study, we first aim to investigate if AJAP1 is a special transcription factor or co-factor; to identify other transcription factors synergized with AJAP1. Second, we aim to explore the mechanism of translocation of AJAP1. Next we plan to clarify the relationship between AJAP1 regulating MAGEA2 with malignant biological changes of GBM. .The present study may provide insides for understanding the comprehensive networks of AJAP1 regulating MAGEA2 as well as transcriptional regulation mechanisms include interaction of cis-elements and transcriptional factors. Moreover, it will provide rationals for a new biomarker and treatment strategy in GBM.

前期工作发现AJAP1通过与MAGEA2启动子结合，调控MAGEA2转录水平的表达；AJAP1通过下调MAGEA2的表达，起到了促进胶质母细胞瘤细胞凋亡、抑制细胞增殖的作用；AJAP1的表达下调与胶质瘤患者生存时间缩短具有相关性。.本项目在上述工作基础上，研究AJAP1是否为调控MAGEA2表达的特异性转录因子或与其结合的蛋白，识别和鉴定协同AJAP1调控MAGEA2转录的转录因子；进一步探索AJAP1的核转位机制；明确AJAP1对MAGEA2的调控与胶质母细胞瘤恶性生物学行为改变的关系。.本项目可揭示AJAP1对MAGEA2基因转录调控的复杂机制，在各顺式作用元件与对应转录因子的相互作用调控基因转录方面进行有益的探索,为将 AJAP1应用成为胶质母细胞瘤新的临床标记物和临床干预靶点提供理论基础。

我们通过临床数据分析发现EGFR通路能调控AJAP1的表达：分析TCGA数据库483例胶质瘤基因表达数据，发现EGFR拷贝数与AJAP1没有统计学意义上的相关性，但聚类分析TCGA, CGGA和REMBRANT数据库发现在胶质母细胞瘤的四个亚型（经典型、间叶型、神经型、前神经型）中均有AJAP1表达与EGFR表达的负相关性。研究内容调整如下：EGFR对AJAP1的转录调控机制及其对胶质母细胞瘤恶性生物学行为的影响。通过构建了EGFRvIII的慢病毒载体，转染胶质瘤细胞系和AJAP1的过表达体外模型发现激活EGFR通路能下调AJAP1表达重塑细胞骨架，增强细胞的迁移能力，用LY294002 (PI3K 抑制剂)，MK2206,DZNEP (组蛋白甲基转移酶抑制剂) 处理了U87-EGFRvIII/LN229-EGFRvIII发现，AJAP1呈剂量依赖性增高表达。这些发现表明了EGFRvIII抑制AJAP1的表达是通过PI3K/Akt介导的，EZH2依赖的途径。我们在EGFR阳性的胶质母细胞瘤细胞系中通过染色发现，H3k27me3的表达在U87-EGFRvIII and 和LN229-EGFRvIII组要明显高于对照组，即验证EGFR下游通路的表观遗传学重塑作用：EGFR能通过重塑胶质母细胞瘤的活性增强子促进肿瘤发生。本研究我们发现AJAP1的表达与EGFR表达呈负相关，在胶质母细胞瘤中EGFR通过表观遗传沉默AJAP1表达重塑细胞骨架从而增强了肿瘤细胞的侵袭能力。我们进一步的研究也证明了MK-2206能通过抑制EGFR通路从而逆转AJAP1的表达下调。MK2206有希望成为一个EGFR/EGFRvIII阳性胶质母细胞瘤的有效治疗手段。