prohibitin与PIG3基因启动子区（TGYCC）n序列结合并调控其转录的分子机制

项目名称： prohibitin与PIG3基因启动子区（TGYCC）n序列结合并调控其转录的分子机制

项目编号： No.81470357

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 医药、卫生

项目作者： 管晓翔

作者单位： 中国人民解放军东部战区总医院

项目金额： 80万元

中文摘要： p53是目前唯一被证实与PIG3启动子区微卫星序列（TGYCC）n相互结合的元件，其介导PIG3参与的细胞凋亡通路在血液系统肿瘤发生发展过程中起着至关重要的作用。本课题组运用高效液相色谱分析法及多肽测序发现prohibitin(PHB)为PIG3启动子（TGYCC）n的新结合因子。为阐明PHB与PIG3启动子区结合并调控其转录的分子机制及其在p53/PIG3介导细胞凋亡通路中的作用，本项目拟采用荧光共振能量转移(FRET)方法，实时观察p53 、PHB以及PIG3分子之间相互作用动态过程，并观察凋亡诱导药物喜树碱（CPT）刺激对以上动态过程的影响。位点突变及RNA干涉等实验方法阐明阻断和抑制PHB功能后对p53/PIG3信号通路的调控机制。以上研究为阐明PHB在介导p53/PIG3细胞凋亡通路的作用机制提供实验依据，并为逆转肿瘤细胞生物学特性提供潜在的干预靶点。

中文关键词： PIG3；prohibitin；转录调控

英文摘要： The promoter of p53-induced gene 3 (PIG3) contains a variable number of tandem repeats (VNTRs) of pentanucleotides (TGYCC)n that is known as a p53 binding site. In our previous study,using ligand-chromatography combined with liquid chromatography-tandem mass spectrometry analyses, we indicated that direct interactions of prohibitin and/or prohibiton with the (TGYCC)15 motif, which was confirmed by electrophoretic mobility shift assay and super-gel shift analysis with anti-prohibitin(PHB) antibodies. Using the chromatin immunoprecipitation assay, we further demonstrated that prohibitin and prohibiton associated with the (TGYCC)15 motif in vivo regardless of the p53 status and apoptotic stress. We also found that prohibitin up-regulated PIG3 transcription independent of p53, although p53 obviously enhanced this process, and that the knock-down of prohibitin and prohibiton inhibited camptothecin-induced apoptosis. Taken together, our findings suggest that prohibitin and prohibiton contribute to PIG3-mediated apoptosis by binding to the PIG3 promoter (TGYCC)15 motif. However,much less is known about the role of PHB in the transcriptional regulation of PIG3 gene. To find the molecular mechanism of PHB /PIG3 mediated apoptosis process,FRET and immunoprecipitation were examined. The differential protein-spots were identified by peptide mass fingerprint based on mass spectrometry and database searching. Our research maybe contribute to elucidate the different signaling pathways that cause deregulation of PIG3 expression and localization in human cancer, demonstrating the molecular mechanisms of PIG3 down-expression and mislocalization in tumor cells and provide additional insight into control of the cell cycle. Besides, Our observation can offer a potential target for the development of drugs ,which, if successful, may prevent or retard tumor cell formation.

英文关键词： PIG3;prohibitin;transcriptional regulation