PinX1基因通过c-Myc抑制胶质瘤增殖的分子机制研究

In our previous study, we identified that PinX1 as a tumor suppressor gene involved in glioma development process. However, the underlying molecular mechanism is not clear. In our preliminary experiment, we found that silencing PinX1 gene can increase c-Myc and Cyclin D1 expression in glioma cells, and the cells proliferation ability is also increasing. The biological information software predicted that there were three potential PinX1 binding sites in the c-Myc promoter. Consequently, we suppose that PinX1 regulate glioma proliferation through inhibiting c-Myc transcriptional expression and Cyclin D1 expression by binging with c-Myc promoter..To confirm this hypothesis, we will investigate the effect of PinX1 on c-Myc transcriptional expression, and verify the binding sites in the c-Myc promoter by EMSA, ChIP and site mutation. Then, we will verify if PinX1 can suppress glioma grow through c-Myc/Cyclin D1 signal pathway by in vivo nude moues model. Lastly, we will analysis the correlation between the PinX1, c-Myc, Cyclin D1 and the pathology, and prognosis of glioma patients by tissue microarray.Our study will provide evidence for new therapeutic target for glioma.

我们既往研究证实PinX1作为一种抑癌基因参与了胶质瘤的发生与发展过程。但如何实现这一作用的具体机制不详。申请者前期研究发现，沉默胶质瘤细胞PinX1基因，显著增加c-Myc和Cyclin D1表达，促进胶质瘤细胞增殖。生物信息分析发现c-Myc核心启动子区域内存在3个潜在的PinX1结合位点。由此，我们提出：PinX1通过与c-Myc启动子结合抑制c-Myc转录，进而影响Cyclin D1表达，抑制胶质瘤增殖。.本研究拟在分子水平，通过双荧光素酶实验、ChIP实验、启动子点突变以及EMSA实验，确认PinX1与c-Myc启动子结合位点，以及对其转录的影响；然后在动物模型上研究PinX1通过c-Myc/Cyclin D1对胶质瘤细胞生长的影响；最后通过胶质瘤组织芯片分析PinX1和c-Myc、Cyclin D1表达之间的关系，以及与临床病理和预后的关系。为寻找胶质瘤新的治疗靶点提供理论依据

胶质瘤的发病机制尚未阐明，发现影响其发生、发展和预后的关键基因，并从中寻找到治疗标靶成为研究热点。PinX1是近年发现的与端粒及端粒酶关系密切的核仁蛋白，PinX1在肿瘤中作用，特别是在胶质瘤中作用，报道甚少。本项目通过对具有完整临床资料和随访信息的胶质瘤患者的肿瘤组织芯片，分析PinX1表达与胶质瘤病理、分级和预后的关系；通过构建PinX1高表达和低表达的胶质瘤细胞株，用细胞模型研究PinX1在胶质瘤增殖、迁移和侵袭中的作用及c-Myc/CyclinD1参与的分子机制；用裸鼠模型验证PinX1在体内的抑癌作用，及其对c-Myc/CyclinD1的影响。本研究为胶质瘤的分子诊断和基因治疗提供新的靶点，PinX1有望成为判断胶质瘤患者术后生存的分子标志物以及治疗胶质瘤的靶基因。我们发现，PinX1在胶质瘤组织以及胶质瘤细胞系中表达显著下降，并且PinX1与胶质瘤分级有关；Kaplan-Meier 生存分析提示PinX1的低表达与胶质瘤患者的5年总生存率和疾病特异性生存率均呈负相关。PinX1可以通过c-Myc/CyclinD1显著抑制胶质瘤细胞增殖能力和裸鼠皮下成瘤。PinX1可能成为胶质瘤的预后标志物或治疗靶点。