NEDD4L下调介导的TGF-β/SMAD通路激活及其对胶质瘤细胞线粒体功能和凋亡的调控

TGF-β/SMAD pathway plays critical roles in the development of cancer. Our previous studies have demonstrated that the expression of several members of TGF-β/SMAD pathway is abnormal in glioma. NEDD4L is a potential regulatory molecule for the activation of TGF-β/SMAD pathway. Our previous studies have demonstrated that NEDD4L expression is downregulated in glioma. Moreover, all these dysregulated expression is associated with the progression of glioma and our preliminary study has demonstrated that NEDD4L overexpression promotes the apoptosis of glioma cells. However, the detailed mechanisms by which TGF-β/SMAD pathway is activated and inhibits apoptosis remains unknown. It has been reported that dysregulation of mitochondrial biogenesis exerts an anti-apoptotic effect. Several studies and our preliminary investigation have shown that TGF-β can inhibit mitochondrial biogenesis in cancer cells. Thus, we hypothesize that TGF-β/SMAD pathway may exerts its anti-apoptotic effects through suppression mitochondrial biogenesis in glioma cells, and the downregulation of NEDD4L is one important promoting factor for TGF-β/SMAD pathway activation. In this proposed project, we will examine the alterations in the activation of TGF-β/SMAD pathway, apoptosis, mitochondrial biogenesis, ROS generation and apoptosis-related proteins of glioma cells after changing the expression of SMAD2/3 or NEDD4L or the cell location of SMAD4. We will demonstrate the mechanisms by which mitochondrial biogenesis inhibition mediates the anti-apoptotic effects of TGF-β/SMAD pathway on molecular, cell, animal and human tissue levels in our project. The data generated in our project will provide novel theoretical support for the current understanding on the molecular mechanisms of glioma development.

TGF-β/SMAD通路激活在肿瘤进展中发挥了关键作用，我们前期发现该通路部分成员及其潜在抑制分子NEDD4L在胶质瘤中表达异常，且与其恶性进展密切相关，预实验结果提示NEDD4L过表达可促进胶质瘤细胞凋亡，但TGF-β/SMAD通路在胶质瘤中激活机制及其生物学作用远未阐明。一些研究和我们的预实验结果表明，TGF-β可抑制线粒体生成。进而我们提出如下假设：TGF-β/SMAD通路可通过调控线粒体生成抑制胶质瘤细胞凋亡，而NEDD4L下调则是该通路激活的重要因素。据此，本项目拟通过调控SMAD2/3与NEDD4L表达及SMAD4转位，利用各种生物学技术，检测TGF-β/SMAD通路激活水平、线粒体生成、ROS及细胞凋亡，分别从分子、细胞、动物及组织水平系统探讨线粒体生成异常在TGF-β/SMAD通路介导的胶质瘤细胞凋亡抑制中的作用及分子机制，为深入理解胶质瘤分子病理机制提供新的理论支持。

脑胶质瘤恶性程度高，发病机制亟待阐明。加强胶质瘤分子病因学研究，筛选鉴定新的关键分子并阐明其影响胶质瘤发生发展的病理机制是当前胶质瘤防治研究的重中之重。TGF-β/SMAD 通路的激活在恶性肿瘤进展中发挥极其关键作用。我们的前期研究结果也表明， SMAD4 表达下降与胶质瘤的恶性程度密切相关，且SMAD4表达水平可作为胶质瘤病人生存的预后指标。同时，TGF-β/SMAD通路的重要靶分子基质金属蛋白酶（MMPs）在胶质瘤中表达水平升高，且与较差的胶质瘤预后显著相关。基于上述分析及前期预实验结果，本课题借助我们在线粒体、TGF-β和胶质瘤领域积累的研究基础和丰富经验，应用慢病毒表达系统上调或干扰NEDD4L或SMAD的表达，对TGF-β/SMAD 通路对线粒体功能和胶质瘤凋亡特性的机制进行了深入研究。结果提示：1）SMAD2过表达可抑制脑胶质瘤细胞凋亡，TGF-β通过调控SMAD2抑制脑胶质瘤细胞凋亡。2）SMAD2过表达可抑制细胞色素C向胞浆释放，同时在体载瘤实验表明，SMAD2过表达可促进脑胶质瘤细胞体内生长。3）U251和U87中对SMAD2过表达及干涉的影响，凋亡实验表明SMAD2过表达可抑制脑胶质瘤细胞在体内成瘤组织中的凋亡。4）利用 NEDD4L 稳定表达和干扰细胞系，提取 TGF-β 刺激前后胶质瘤细胞株的线粒体、细胞核和细胞质蛋白，WB检测SMAD4在不同细胞成分的表达水平，并利用免疫荧光技术进一步验证SMAD4在细胞内的定位。最终验证NEDD4L了促进SMAD4线粒体转位的作用。通过上述研究，本课题阐明了TGF-β/SMAD 信号通路可通过调控线粒体生成进而抑制胶质瘤细胞凋亡，而NEDD4L下调则是TGF-β/SMAD信号通路激活的重要促进因素，这为深入理解胶质瘤分子病理机制提供新的理论支持。