TGF-β1及其诱生的AEG-1调控保护性自噬在恶性脑胶质瘤侵袭转移中的机制研究

During the complex biological process in malignant tumor invasion and metastasis, tumor cells can adapt to different survival pressure. Autophagy is a tightly-regulated catabolic process that involves the degradation and utilization of intracellular macromolecules and organelles under metabolic stress. Autophagy not only can be activated to adapt the metabolic stress and micro environmental changes, but also may be involved in the process of epithelial mesenchymal transition (EMT) which is closely related to the mechanism of tumor cells invasion and metastasis. Research shows that astrocyte elevated gene (AEG-1) can induce protective autophagy, however, the regulatory mechanism of autophagy-regulated by AEG-1 during malignant glioma epithelial mesenchymal transition (EMT) and metastasis remain unclear. Our preliminary experimental results show that inhibition of AEG-1 expression by si RNA can inhibit autophagy and EMT, as well as weaken the glioma cell invasion. In this study, we will take cell transfection, siRNA interference, CO-immunoprecipitation, proteomics and mass spectrometry analysis to investigate the molecular mechanism that TGF-β1 and its inducing AEG-1 regulate protective autophagy in malignant glioma EMT-induced by TGF- β1; furtherly, we will establish the malignant glioma animal model with the tumor microenvironment by stable cell lines with high expression and low expression of AEG-1 and clarify the regulation mechanism of protective autophagy by AEG-1 in malignant glioma invasion and metastasis in the level of cell, clinical specimen and in vivo experiment. Therefore, our project aims at an important theoretical basis for the clinical diagnosis and treatment of malignant gliomas.

自噬是代谢压力下对细胞内大分子物质和细胞器降解再利用的过程。自噬不仅可被激活以适应代谢应激和微环境变化，还可能与肿瘤细胞的上皮-间质转化（EMT）等侵袭转移机制密切相关。研究显示，星形胶质细胞升高基因（AEG-1）有诱导保护性自噬的作用，但AEG-1调控的自噬在恶性脑胶质瘤EMT和转移中的作用机制仍不明确。我们的预实验结果显示，阻抑AEG-1的表达可抑制脑胶质瘤细胞发生自噬和EMT，减弱侵袭能力。本课题拟在TGF-β1诱导恶性脑胶质瘤EMT的模型上，通过细胞转染、siRNA干扰、免疫共沉淀、蛋白质组学-质谱分析等技术，重点研究TGF-β1及其诱生的AEG-1调控保护性自噬对恶性脑胶质瘤EMT的分子作用机制；应用稳定AEG-1高表达和低表达的细胞株，建立具有肿瘤微环境的恶性脑胶质瘤动物模型，从细胞、整体和临床样本阐明AEG-1调控保护性自噬在恶性脑胶质瘤侵袭转移中的机制。

脑胶质瘤起源于脑组织的神经胶质细胞，是最常见的一种颅内恶性肿瘤。研究显示，脑胶质瘤约占中枢神经系统肿瘤发生率的70％，居颅内肿瘤发病率的首位，且患者的病情恶化迅速，平均生存期仅为12～15个月。在恶性肿瘤侵袭转移的复杂生物学过程中，肿瘤细胞能够适应不同的生存压力。自噬是代谢压力下对细胞内大分子物质和细胞器降解再利用的过程。自噬不仅可被激活以适应代谢应激和微环境变化，还可能与肿瘤细胞的上皮-间质转化（EMT）等侵袭转移机制密切相关。研究显示，星形胶质细胞升高基因（AEG-1）有诱导保护性自噬的作用，但AEG-1调控的自噬在恶性脑胶质瘤EMT和转移中的作用机制仍不明确。本课题主要研究：（1）TGF-β1信号对恶性脑胶质瘤产生EMT的调节作用和机制；（2）TGF-β1及其诱生的AEG-1对恶性脑胶质瘤产生Autophagy的作用以及对EMT的调节作用；（3）维持AEG-1调控恶性脑胶质瘤细胞保护性自噬的分子机制检测；（4）在恶性脑胶质瘤动物模型上，研究AEG-1诱导细胞自噬的作用和促进肿瘤生长的作用；（5）人的组织样本中AEG-1的高表达与自噬、EMT的指标和恶性脑胶质瘤的分型的相关性研究。研究结果显示：TGF-β1作用不同时间后，恶性脑胶质瘤U251和U87细胞先后发生自噬相关蛋白胞浆型LC3-I向自噬体膜型LC3-II转变和上皮标志基因E-cadherin表达下降，间质标志基因N-cadherin、Vimentin、Snail、Twist1、Slug表达增加，且星形胶质细胞升高基因AEG-1的表达上调，提示在TGF-β1诱导细胞发生EMT的过程中，细胞自噬被激活。另外，应用靶向AEG-1的siRNA阻抑AEG-1表达后，抑制了自噬，并使细胞的增殖和EMT能力减弱；过表达AEG-1可以诱导自噬并促进细胞的EMT能力。我们的研究结果支持了AEG-1有促进自噬和EMT的作用。进一步研究在TGF-β1诱导恶性脑胶质瘤细胞侵袭的模型中，AEG-1通过激活恶性脑胶质瘤发生保护性自噬促进TGF-β1诱导的细胞EMT的进程，从而促进恶性脑胶质瘤细胞的增殖、侵袭和转移的作用和机制。研究为我们通过靶向AEG-1，抑制其诱导的保护性自噬，从而抑制恶性脑胶质瘤细胞侵袭转移提供了理论依据。