Drp1在低氧诱导胶质母细胞瘤细胞迁移侵袭中的作用及其机制

Glioblastoma is the most common and aggressive brain tumor with high mortality. Its response to common therapeutic strategies and prognosis are always poor because of the strong aggression. Recent studies suggest the involvement of mitochondrial dynamics in the aggression of tumor cells. However, its mechanism still remains to be elucidated. In the present study, we will investigate the role of mitochondrial fission protein Drp1 in hypoxia-induced migration and invasion of glioblastoma cells and the underlying mechanism. In preliminary experiments, we found that hypoxia upregulated the transcription and expression of Drp1, and stimulated mitochondrial fission in glioblastoma U251 cells. Inhibition of Drp1 with Mdivi-1 or GFP-Drp1-K38A efficiently attenuated hypoxia-induced migration and ROS production in U251 cells. In addition, GST pull-down experiments demonstrated the interaction between Drp1 and Cortactin. To investigate the mechanism of Drp1 involving in hypoxia-induced migration and invasion, we will further examine the effect of Drp1-mediated mitochondrial fission on ROS production, activation of NF-κB and EMT of glioblastoma cells, and the effect of interaction between Drp1 and Cortactin on remodeling of cytoskeleton and pseudopod formation. Moreover, the role of Drp1 in migration and invasion of glioblastoma cells will also be investigated in vivo. Therefore, this research may provide new therapeutic target to suppress the strong aggression of glioblastoma. Our previous studies were published in J Cell Biol, Neurosci Res and PLoS One, etc.

胶质母细胞瘤是常见的高死亡率恶性脑瘤。其强迁移侵袭能力严重影响临床疗效和预后。最近研究提示线粒体形态与肿瘤迁移侵袭有关，但具体机制尚不清楚。本课题将研究线粒体分裂蛋白Drp1在低氧诱导胶质母细胞瘤迁移侵袭中的作用及其机制。前期实验发现：低氧能上调Drp1转录表达，促进线粒体分裂；抑制Drp1活性能减弱低氧诱导的细胞迁移和ROS产生；GST pull-down证实Drp1能与Cortactin相互结合。为明确Drp1的作用机制，将继续检测Drp1介导的线粒体分裂在ROS产生、NF-κB通路激活和肿瘤细胞EMT中的作用，以及Drp1与Cortactin协同对细胞骨架重构和伪足形成的影响；并在动物水平进一步验证Drp1在细胞迁移侵袭中的作用。故本课题将为抑制胶质母细胞瘤强迁移侵袭能力提供新的治疗靶点。申请者以往相关工作已发表于J Cell Biol,Neurosci Res,PLoS One等。

胶质母细胞瘤是常见的高死亡率恶性脑瘤，其强迁移侵袭能力严重影响临床疗效和预后。低氧微环境是促进肿瘤细胞迁移侵袭的重要因素。最近研究提示线粒体形态与肿瘤迁移侵袭有关，但具体机制尚不清楚。本课题将研究线粒体分裂蛋白Drp1在低氧诱导胶质母细胞瘤迁移侵袭中的作用及其机制。研究中，我们利用低氧刺激神经胶质母细胞瘤U251细胞，western检测发现低氧能上调Drp1的转录表达，棘霉素抑制HIF-1α能有效减弱低氧诱导的Drp1转录表达。低氧条件下Drp1表达增加促进了线粒体显著分裂、细胞内ROS水平增高、NFκB信号通路激活及EMT，从而增强胶质母细胞瘤U251细胞迁移。通过染色质免疫共沉淀方法检测到HIF-1α能与Drp1启动子结合，这提示低氧时可能通过HIF-1α上调Drp1的表达。另外，通过GST pull-down实验和免疫共沉淀实验检测到Drp1与细胞骨架蛋白Cortactin间存在相互作用，且结合部位是SH domain，SH domain缺失的Cortactin与Drp1的结合能力显著下降。Drp1抑制剂Mdivi-1或siRNA敲低Drp1后均能减弱胶质母细胞瘤U251细胞板状伪足的形成（详见报告正文）。这些结果说明低氧可刺激HIF-1α与Drp1启动子结合，上调Drp1表达促进线粒体分裂后ROS/NFκB/EMT和与Cortactin调节细胞骨架两条途径来影响神经胶质瘤细胞的迁移侵袭。我们还成功构建了GFP对照U251细胞系、Lent-GFP-Drp1-K38A细胞系和Lenti-GFP-Drp1细胞系，Drp1与GFP为非融合，故方便在体实验时对肿瘤细胞的荧光示踪，上述三个稳定细胞系的脑瘤动物模型正在建立中，以便检测在体条件下Drp1对胶质母细胞瘤细胞迁移侵袭的影响（在研中）。故本课题将为抑制胶质母细胞瘤强迁移侵袭能力提供新的治疗靶点。本课题基本完成研究计划，已发表SCI论文6篇，仍有核心数据尚待发表1篇高水平SCI论文；并培养了硕士研究生3名。