TERT启动子突变区相关G-四链体结构调控端粒酶再活化及在胶质瘤放化疗敏感性中的机制研究

DNA G-quadruplexes are four-stranded globular nucleic acid secondary structures formed in specific G-rich sequences, which play important roles in DNA replication, gene expression and telomere maintenance. However, its biological relevance has not been fully understood. In cancer cells, telomerase reactivation contributes to the limitless proliferation potential and chemoradiotherapy resistance, however, the associated mechanism remains unclear. Mutations within the telomerase promoter region have occurred in many types of cancers, including approximately 80% primary glioblastoma containing these mutations, which combined with IDH mutation and 1p/19q codeletion as novel markers for prognosis and molecular subtyping. However, its biological functions needed to be clarified. We have presented evidence that G-rich sequences within TERT mutation region can adopt a parallel G4 structure; mutation within this region will decrease the stability and inhibit G4-mediated transcriptional silencing effects, resulting increased expression and activation of telomerase, which maybe the important course for telomerase reactivation in cancer cells. We will further use CD, FRET, PAGE, PCR-STOP assay and DMS footprinting to characterize the stability of the formed G4 structure and illustrate the mode of action. Meanwhile, we apply molecular biological techniques, such as ChIP, luciferase assay and EMSA to analyze the effects of G4-induced binding of transcription factors SP1, ETS and p52 to TERT promoter. Finally, we want to use an in vitro and in vivo model to test a specific TERT promoter G4-stabilizing ligand on sensitivity of glioma chemoradiotherapy. This project will help to illustrate the mechanism of G4 structure formed within the promoter mutation region on telomerase reactivation and develop novel therapeutic strategies against glioma through targeting this specific structure.

肿瘤细胞中端粒酶的再激活是其恶性增殖及放化疗抗性的重要因素，但其作用机制尚未阐明。基因组中富G序列形成的G-四链体（G4）结构在基因调控中作用显著，因此其生理意义成为了当前国内外的研究热点。本项目前期研究结果证实端粒酶反转录酶（TERT）启动子在80%的原发性胶质母细胞瘤发生突变，可形成平行G4结构，降低G4结构的稳定性，促进端粒酶表达和激活。申请人由此推测其是肿瘤细胞中端粒酶再活化的重要机制。本项目将进一步借助CD, FRET,NMR及DMS印迹等实验表征突变区形成G4结构的稳定性及作用模式，同时利用ChIP,荧光素酶报告系统，EMSA等手段研究G4形成过程中对转录因子Sp1, ETS以及p52结合的影响。最后利用体内外模型分析选择性稳定启动子G4的配体对胶质瘤放化疗敏感性的影响。本项目的实施将对阐明启动子突变区G4结构在端粒酶再活化中的作用及开发新的胶质瘤靶向治疗策略奠定基础。

肿瘤细胞中端粒酶的再激活是其恶性增殖及放化疗抗性的重要因素，但其作用机制尚未阐明。基因组中富G序列形成的G-四链体（G4）结构在基因调控中作用显著，因此其生理意义成为了当前国内外的研究热点。本项目前期研究结果证实端粒酶反转录酶（TERT）启动子在80%的原发性胶质母细胞瘤发生突变，可形成平行G4结构，降低G4结构的稳定性，促进端粒酶表达和激活。申请人由此推测其是肿瘤细胞中端粒酶再活化的重要机制。.在前期工作的基础上，本项目结合胶质母细胞瘤患者的临床及病理资料，进一步证实了端粒酶的再活化及启动子的突变在胶质瘤患者诊断及预后评估的价值。同时，本项目进一步借助圆二色谱，紫外吸收光谱，非变性-PAGE以及PCR-STOP assay等表征了TERT启动子突变区形成G4结构的稳定因素及其作用模式，证实TERT启动子突变区的确形成平行G4结构，若发生突变，则其G4结构稳定性显著降低。加入特异性稳定的配体，显著下调其的转录，表达以及端粒酶的再活化。荧光素酶报告系统实验证实，加入TERT启动子区突变区G4结构的配体显著降低了启动子的活性。染色质免疫沉淀以及凝胶阻滞实验也证实，TERT启动子区G4结构的形成促进了相关转录因子Sp1以及p52在其启动子区的解离。由于端粒酶活化在胶质瘤放化疗抗性中的重要作用，我们进一步研究证实TERT启动子突变区G4结构的稳定可以显著增强胶质瘤一线化疗药物替莫唑胺的敏感性，同时也增强了其放疗敏感性。联合作用指数表明它们的联合作用具有协同性。同时，进一步研究表明，TERT启动子突变区G4结构的配体联合替莫唑胺以及放疗，可以显著抑制胶质瘤细胞的克隆形成，诱导细胞周期阻滞及凋亡，同时抑制了其的侵袭性。本项目研究也证实，TERT相关G4结构的形成可以显著调节其mRNA的可变剪切，进而影响端粒酶的活性，对胶质瘤细胞也有一定的杀伤作用。在胶质瘤细胞构建的体内移植瘤模型中，选择性稳定TERT启动子G4结构的配体单独或协同替莫唑胺可以显著抑制肿瘤的生长，延长生存期。.本项目研究可以证实，TERT启动子突变区形成一种平行G4结构，这种结构可以抑制转录因子的结合，起到转录沉默的作用。胶质瘤中启动子区的突变破坏了这种转录抑制元件，促进了转录因子的结合，进而促进端粒酶的再活化和端粒结构的维持,因此对于开发新的靶向端粒酶启动子G4结构的治疗策略具有重要的意义。