我们前期研究发现胶质瘤高表达MAGE-D4并与p53相关；鉴于MAGE-D4启动子含多个潜在p53结合位点，推测MAGE-D4功能有赖于p53参与，故拟开展以下研究：①p53对MAGE-D4的转录调控：构建p53上调/下调/突变胶质瘤细胞株，通过MAGE-D4启动子报告基因载体筛选功能性p53结合位点，了解p53对MAGE-D4启动子活性的影响；用qRT-PCR和WB检测p53对MAGE-D4表达的影响；EMSA和ChIP-PCR确认p53与MAGE-D4启动子的结合；②MAGE-D4对胶质瘤细胞增殖和凋亡影响及其p53依赖性：构建MAGE-D4表达上调/下调胶质瘤细胞及其移植瘤动物模型，体内外检测MAGE-D4对不同p53表达状态细胞增殖和凋亡及其相关分子的变化。本项目从p53角度阐明MAGE-D4表达调控机制和部分生物学功能，为将来开发以MAGE-D4为靶标的胶质瘤生物治疗奠定基础。

Our previous study found that the overexpression of MAGE-D4 correlated with p53 in glioma tissues. In view of the fact that MAGE-D4 promoter enriches the consensus binding sites for p53, we speculated that p53 may be involved in MAGE-D4 fuction. Therefore, following contents will be studied: ① Transcriptional regulation of MAGE-D4 by p53. Up- and down- regulation or mutation of p53 will be established in glioma cell lines. Furthermore, the functional p53 binding sites will be screened and promoter activity of MAGE-D4 will be detected by dual-luciferase reporter assay. And MAGE-D4 expression will be observed by qRT-PCR and western blot. Then, EMSA and ChIP-PCR will be used to confirm the binding of p53 protein and the p53 binding sites within MAGE-D4 promoter. ② The influence of MAGE-D4 on glioma cell proliferation and apoptosis and its dependence on p53. up- and down- regulation of MAGE-D4 expression wil be established in different p53-type glioma cell lines. Furthermore, xenograft mouse model will be built. Cell proliferation, cycle,apoptosis and expression of p21, Bax and Bcl-2 gene will be detected in vitro and in vivo. The project will interpret the regulating mechanism and partial biological function of MAGE-D4 from p53 aspect and provide a basis for future glioma biotherapy targeting MAGE-D4.