泛素化蛋白酶A20抑制caspase-8活化在脑肿瘤干细胞发生TRAIL抵抗中的作用机制

Brain tumor stem cells（BTSCs）determined the tumor's growth and response to recurrence and therapies,tumor necrosis factor-related apoptosis inducing ligand (TRAIL) is normally expressed in the human immune system and plays a critical role in antitumor immunity. However,the most of malignant glioma cells indicated the tumor resistance to TRAIL. A20 has been reported as a deubiquitin-editing enzyme and protected against the abnormal programmed cell death. Our prelimiary studies have shown that the TRAIL-induced caspase-8-initiated apoptosis is the main reason of TRAIL resistance in BTSCs, A20 may be the regulatory genes of caspase-8 cleavage. In this study, we have established BTSCs from surgically removed malignant glioma, untransfected and transfected with A20 shRNA through lentivirus, and immunoprecipitated from the death-inducing signaling complex (DISC) and examined the ubiquitination after BTSCs treated with TRAIL, also generated BTSCs xenografts in nude mice, etc. In this project, we will discuss the relationship between A20 and TRAIL resistance in BTSCs, study the role of A20 in BTSCs resistance to TRAIL, and reveal the mechanisms of A20 mediated TRAIL resistance in BTSCs. This propasal has a sufficient theoretical basis, innovative ideas, solid preliminary studies and reasonable experimental design and methods, this study will lay a theoretical foundation for the development of new drug and establishment of cancer therapeutics, also provide a new target for cancer researsh.

脑肿瘤干细胞（brain tumor stem cells，BTSCs）是脑肿瘤发生、复发及治疗抵抗的根源，TRAIL凋亡传导途径是人体内重要的、天然的抗肿瘤途径，但大多数恶性胶质瘤细胞都存在TRAIL抵抗现象。泛素化蛋白酶A20具有去泛素化蛋白酶活性，可以保护细胞免于非正常细胞程序性死亡。我们前期研究发现，在BTSCs中caspase-8活化受抑制是TRAIL抵抗的根源，A20可能是caspase-8活化的调控基因。因此，本课题采用BTSCs培养、shRNA、免疫共沉淀等技术及BTSCs动物模型方法，探讨A20与TRAIL抵抗的关系，研究A20在BTSCs发生TRAIL抵抗中的作用，揭示A20介导BTSCs发生TRAIL抵抗的机制。本项目的提出具有充分的理论依据、新颖的设计思路、扎实的前期工作及合理的研究方案，将为TRAIL作为新药开发及多种肿瘤的治疗奠定理论基础，为肿瘤研究提供新靶点。

脑肿瘤以胶质瘤最为常见，恶性居多，即使结合替莫唑胺在内的多种疗法效果亦不显著。最近肿瘤生物学研究表明，脑肿瘤干细胞（BTSCs）是脑肿瘤发生、复发及治疗抵抗的根源，众多凋亡因素决定了胶质瘤的发生发展。肿瘤坏死因子相关凋亡诱导配体（TRAIL）凋亡传导途径是人体内重要的、天然的抗肿瘤途径，但大多数恶性胶质瘤细胞都存在TRAIL抵抗现象。本项目研究发现，基因沉默A20表达可以增加TRAIL敏感性。因此，我们详细地研究了A20通过下调caspase-8表达使BTSCs发生TRAIL抵抗的机制及相关因素。本项目通过研究最终建立和完善了BTSCs的培养方法，使其制备工艺化。并通过大量实验发现，在BTSCs中存在着TRAIL抵抗，caspase-8的表达水平及功能状态是BTSCs发生TRAIL抵抗的关键，而A20和RIP的表达水平与caspase-8功能状态也密切相关。深入研究发现，这是因为A20引起RIP泛素化而使caspase-8处于失活状态，不能引发肿瘤细胞凋亡，BCL-2也参与了这一过程。小泛素化（SUMO）相关蛋白和DNA修复基因也影响了胶质瘤的发生发展，Sch B可以诱导胶质瘤细胞凋亡。本项目的提出具有充分的理论依据、新颖的设计思路、扎实的前期工作及合理的研究方案，将为TRAIL作为新药开发及多种肿瘤的治疗奠定理论基础，为胶质瘤的治疗提供新靶点和新药。