β-catenin/Ets1复合体在胶质母细胞瘤中对hTERT表达调控机制的研究

Mutations in the core promoter region of human telomerase reverse transcriptase (hTERT) gene are implicated in cancer pathological processes. In previous studies, we screened for TERT promoter mutations by nested PCR and Sanger sequencing in a population-based collection of glioblastomas(GBMs) and found the promoter mutation was enriched in GBMs and significantly associated with prognosis. Bioinformatics analysis found that hTERT promoter mutation (C228T and C250T) can obtain a new site for transcription factors Ets1 loci. The Co-IP showed that Ets1 and β-catenin can be combined with each other. Real-time PCR indicated that expression level of hTERT and β-catenin in mutation group increased significantly, and is closely related to the prognosis, but had nothing to do with the wild type group. Here, we propose a hypothesis that β-catenin/Ets1 complex binding with hTERT promoters mutations may enhance the hTERT gene transcription, and promote glioblastoma malignant phenotype. To verify the above hypothesis, we will explore the way of β-catenin/Ets1 binding with mutated site and molecular mechanism of β-catenin/Ets1 in regulation of hTERT activity in glioblastoma malignant process. Taken together, we will provide a novel targets and develop optimization strategies for glioblastomas biological therapy dependent on the studies.

人端粒酶逆转录酶（hTERT）启动子突变与多种肿瘤发生发展关系密切。我们前期基于巢式PCR联合Sanger测序证实hTERT启动子突变在胶质母细胞瘤中高频存在并与患者预后显著相关。生物信息学分析发现C228T及C250T突变可获得新的Ets结合位点。免疫共沉淀结果示Ets1和β-catenin蛋白可相互结合；RT-PCR结果表明hTERT和β-catenin表达水平在突变组中显著提高,且与患者预后显著相关。据此我们提出假说：β-catenin/Ets1复合体与hTERT启动子突变位点结合后增强hTERT转录，进而促进肿瘤恶性表型。本项目拟研究β-catenin/Ets1复合体在胶质母细胞瘤中对hTERT表达调控的机制，系统阐述β-catenin/Ets1对hTERT表达的调控机制在胶质母细胞瘤分子诊断、预后判断及靶向治疗中的作用，为恶性胶质瘤个体化治疗方案及基因治疗靶点的寻找奠定基础。

人端粒酶逆转录酶（hTERT）启动子突变在胶质母细胞瘤中高频存在并与患者预后显著相关。我们利用全转录组测序数据对已收集到的样本进行了TCGA分子亚型的注释，从而在不同的分子亚型中分析TERT突变对患者预后的影响。结果显示：在Proneural亚型中，携带TERT启动子突变的患者，其预后生存期明显好于TERT野生组的患者。此外通过生物信息学分析发现C228T及C250T突变可获得新的Ets结合位点，免疫共沉淀结果示Ets1和β-catenin蛋白可相互结合；RT-PCR结果表明hTERT和β-catenin表达水平在突变组中显著提高，且与患者预后显著相关。TERT作为胶质瘤的分子标志物，在WHO II级和胶质母细胞瘤中具有一定的预后评估价值，当联合IDH突变和EFGR扩增的时候则更为显著。此外，WHO II级胶质瘤中，TERT突变与IDH1突变存在共生关系，但在高级别胶质瘤中则存在着互斥关系。C228T和C250T虽然同为TERT启动子区域上具有相同作用的突变位点，但它们之间却存在着明显的预后分层，结果显示含有C250位点突变的患者预后要好于C228T位点突变和野生型的的患者。本研究系统阐述了hTERT启动子突变在胶质瘤分子诊断、预后判断及靶向治疗中的作用，为恶性胶质瘤个体化治疗方案及基因治疗靶点的寻找奠定基础。