PTBP1介导的survivinΔEx3过表达调控胶质母细胞瘤微血管增生的机制研究

Glioblastoma is the most frequent malignant tumor of the central nervous system. Microvascular proliferation is a histopathological hallmark of glioblastoma. Inhibiting angiogenesis would be an important approach for the treatment of glioblastoma. In our previous study, we found the level of survivinΔEx3 was increased in glioblastoma tissues and cell lines. However, the mechanisms underlying the alternative splicing of survivin remain unknown. A number of studies have revealed that survivinΔEx3 not only plays an important role in inhibiting apoptosis, but also takes part in regulating tumor angiogenesis. After downregulated splicing factor PTBP1 expression via specific siRNA in glioblastoma cell line, we found that the level of survivinΔEx3 mRNA was dramatically decreased. Based on bioinformatics analysis of the genome sequence of survivin, we put forward the hypothesis that PTBP1 involves in the regulation of survivinΔEx3 alternative splicing, and PTBP1-mediated survivinΔEx3 over-expression induces microvascular proliferation in glioblastomas. This project aims to confirme that PTBP1 regulates survivinΔEx3 alternative splicing through specific recognition and combination the binding sites, by using specific RNA splicing pathway models cell culture, EMSA and ChIP experiments. This project also intends to clarify the significance of PTBP1- mediated survivinΔEx3 over-expression in microvascular proliferation of glioblastomas through matrigel tubule formation assay in vitro and in vivo experiments. The results of this project would provide a novel anti-angiogenesis target for the treatment of glioblastoma.

胶质母细胞瘤是发病率最高的脑部恶性肿瘤，微血管丛状增生是其典型特征，抑制血管生成是胶质母细胞瘤治疗的重要方面。本课题组前期发现胶质母细胞瘤组织及细胞中过表达survivinΔEx3。survivinΔEx3不但具有抗细胞凋亡功能，还参与调控肿瘤血管生成。在胶质母细胞瘤细胞中特异性下调剪接因子PTBP1的表达，导致survivinΔEx3表达量明显降低；结合生物信息学分析结果，我们提出PTBP1参与调控survivinΔEx3的剪接，导致胶质母细胞瘤微血管增生的假说。本课题拟通过RNA剪接模型细胞培养及EMSA和ChIP等实验证实PTBP1通过结合在特定剪接识别位点，参与调控survivinΔEx3的选择性剪接；并拟通过小管形成实验及移植瘤动物模型实验阐明PTBP1介导的survivinΔEx3过表达在胶质母细胞瘤微血管增生方面的意义，旨在为胶质母细胞瘤靶向抑制血管生成提供一个新的靶点。

目的：探讨剪接因子PTBP1在胶质瘤中参与调控survivinΔEx3选择性剪接的可能性。.方法: 对survivin基因组序列进行生物信息学分析，找出PTBP1可能结合并参与调控survivinΔEx3剪接的序列；采用分子克隆技术，构建小基因剪接报告质粒，转染U251细胞及对照细胞中培养，检测各报告质粒3号外显子及3’-UTR区的纳入或排除；运用染色质免疫沉淀实验及凝胶电泳迁移实验，检测PTBP1与survivin 3号内含子及3’-UTR区识别序列的特异性结合。.结果: 小基因剪接报告质粒模型实验，明确survivin基因内含子3区域以及3’-UTR区的 PTBP1识别原件的关键结合位点。CHIP和EMSA实验结果分别在细胞内外水平证实PTBP1与survivin 3号内含子及3’-UTR区识别结合元件的特异性结合。.结论: PTBP1通过结合在特定的剪接识别位点，参与调控survivinΔEx3选择性剪接，导致survivinΔEx3在胶质瘤中高表达。