胶质瘤中IDH1突变诱导血管新生相关基因表达促进肿瘤微血管形成的研究

The finding of IDH1 mutations in gliomas is a significant advance in the field of glioma research.Based on the current situation of IDH1 research and our early results that IDH1 mutiations is correlated with high expressions of VEGF,MMP2,MMP9 and high levels of microvessel density in gliomas of the same pathological grade, we propose a hypothesis that glioma-derived IDH1 mutations could induce expressions of angiogenesis-related genes and further upregulate tumor's microvessel density. To confirm this hypothesis, we design the expriments. Firstly, we explore the different exprssions of angiogenesis-related genes with gene micro arrays between IDH1 mutation group and wild-type IDH1 group in the same pathological grade of gliomas. Secondly, we analyze the effect of IDH1 mutations on angiogenesis-related gene expressions in cultured U87(glioma cell line) cells in vitro by RNA interference,and observe the reversive effect of α-KG on angiogenesis-related gene expressions in cultured U87 cells transfected with IDH1 shRNA. Finally, we further verify the results of the experiments above in vivo. To clarify the possible mechanisms of IDH1 mutations contributing to angiogenesis of gliomas and explore new theropeutic targets in glioma treatment offer credible experimental and theoretical gist.

胶质瘤中IDH1突变的发现是近年来胶质瘤研究领域的重大进展。本课题基于当前胶质瘤中IDH1研究现状，结合前期工作中发现：同一病理级别的胶质瘤中IDH1突变与VEGF、MMP2、MMP9等血管新生相关基因表达增加及肿瘤微血管密度上调相关，提出假设即胶质瘤中IDH1突变可通过诱导血管新生相关基因表达上调，进而促进胶质瘤血管新生。本课题拟首先通过基因芯片，在同一病理级别的胶质瘤中，分析IDH1突变组与野生组差异表达的血管新生相关基因；其次采用RNA干扰技术模拟IDH1突变，在体外培养的胶质瘤细胞系U87中，分析IDH1活性下降对前述发现的差异表达的血管新生相关基因表达的影响；最后通过动物实验进一步验证上述实验过程所得到的结论。为阐明胶质瘤中IDH1突变在血管新生过程中的作用，在胶质瘤的治疗中寻找新的靶点提供理论依据及实验基础。

异柠檬酸脱氢酶（IDH）基因突变普遍存在于低级别胶质瘤中，最常见的类型为IDH1突变。重要的是，IDH1突变的胶质瘤患者，其生存时间、预后均显著优于未突变者。目前，已将IDH1作为判断患者预后的重要分子标记物。然而，对于IDH1突变如何影响肿瘤细胞生物学行为乃至患者预后的具体机制，研究尚不清楚。本项目中，首先通过基因芯片技术，对IDH1突变细胞株和野生细胞株的基因表达进行对比和筛选。通过对数据分析后发现，在IDH1突变细胞中，Wnt信号通路相关的Wnt5a及T细胞转录因子(T celltranscription factor, TCF)下调，而抑癌基因APC上调。因此，课题组提出并证实假设即胶质瘤中IDH1突变是通过下调Wnt通路、降低β-catennin蛋白的表达来调控肿瘤细胞的生物学行为、改善患者预后。本项目首先构建IDH1突变组、野生组及空转组的U87、U251细胞株。发现与野生组及空转组相比，IDH1突变组肿瘤细胞中TCF4 、LEF1等Wnt转录因子及效应蛋白β-catennin蛋白明显减少、Wnt/β-catenin通路下调；同时，突变组肿瘤细胞的增殖减少、凋亡增加，其迁徙和侵袭能力降低。通过过表达β-catenin蛋白来干扰此过程，可逆转IDH1突变肿瘤细胞的上述表现，使其出现与野生组及空转组肿瘤细胞相类似的生物学行为。项目组在动物实验中也得到同样的实验结果。 通过一系列研究，本项目初步阐明了IDH1是通过下调Wnt/β-catenin通路而影响胶质瘤细胞生物学行为、改善患者预后；为进一步研究IDH1突变在胶质瘤中的具体机制及寻找的调控手段提供科学依据。