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

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.

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。

本项目证实Prohibitin（PHB）为pig3启动子（TGYCC）n新的结合因子，为阐明PHB与该序列已知的结合因子p53之间相互作用在调控p53相关凋亡通路中的作用提供最直接实验证据。同时我们发现在p53缺陷型细胞中BRCA1丧失了对PIG3的调控作用，这表明BRCA1对PIG3的调控是依赖于p53而非PHB。另外，我们探讨了PHB、p53、PIG3三者之间的调控关系，发现PHB能够在p53存在的情况下通过上调p53的表达，进而调控PIG3的表达。此外，在p53缺失的条件下，PHB亦可以通过直接和PIG3启动子区(TGYCC)n结合，而促进PIG3的表达。此外，我们阐明PHB抑制AR活性的分子机制，通过细胞学实验研究发现，在雌激素受体(ER)阳性乳腺癌细胞中，能够通过促进雄激素受体(AR)的表达，从而抑细胞增殖、促进其凋亡。在ER+乳腺癌细胞株MCF-7和T47D中转染PHB质粒后，发现细胞增殖减慢、G1/S期细胞周期阻滞增加、细胞凋亡增加，证实PHB能够通过诱导AR的表达发挥其抗肿瘤的功能。共在国际学术刊物发表高质量的SCI论文14篇SCI收录论文，其中大于5分的8篇，以上文章被国内外同行在Cell Research，Nature Communication，Oncogene等杂志广泛引用。共培养硕士3名，博士3名。发表的文章和培养研究生的数量均超过计划书要求。

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