Bcl2L13基因介导线粒体分裂对胶质瘤侵袭的影响及机制研究

Highly invasive nature is the main cause of glioma recurrence. Bcl2L13 expression is abnormally up-regulated in many solid and blood cancers, including glioma, and closely related with tumor development. Whether Bcl2L13 contributes to cell invasion has not been well studied. We have found that knock-down Bcl2L13 expression in U251 cells decreased the wound closure rate and the migrated cell numbers, which is the first report on Bcl2L13 and glioma cell migration. It was also showed that Bcl2L13 enhanced mitochondrial fission in glioma cells. What is the effect on glioma invasion by mitochondrial fission mediated by Bcl2L13? In the present study, we intend to confirm that Bcl2L13 could enhance glioma invasion in clinical glioma specimens, cell lines and animal models. Firstly, we try to clarify mitochondrial fission is essential for Bcl2L13 enhancing glioma invasion. Then we will study whether Bcl2L13 enhancing mitochondrial fission depends on Drp1 or Mfn1/Mfn2. Moreover, the modulation of cell bioenergetics and subcellular trafficking of mitochondria fragments by mitochondrial fission mediated by Bcl2L13 will be analyzed. In addition, clinical glioma specimens will be collected for detecting the expression of Bcl2L13, and we will study the correlation between Bcl2L13 expression level and post-operative outcome. This study should contribute to evaluating the role of Bcl2L13 in gliomagenesis and development, which may broaden our understanding of the function of mitochondrial dynamics in tumor cell invasion and migration, and provide theoretical evidence and therapeutic target for the treatment of glioma.

高度侵袭性是胶质瘤复发的主要原因。Bcl2L13在胶质瘤中高表达，我们前期实验首次发现干扰Bcl2L13基因表达能够降低胶质瘤细胞迁移能力，提示Bcl2L13可能增强胶质瘤侵袭性。我们还初步证实Bcl2L13促进胶质瘤细胞中线粒体分裂。那么Bcl2L13介导线粒体分裂是否以及如何参与促进胶质瘤侵袭呢？本课题将在临床肿瘤标本、细胞和动物水平进一步研究Bcl2L13对胶质瘤侵袭性的促进作用。首先验证Bcl2L13介导的线粒体分裂是否是其促进胶质瘤侵袭所必需的，然后探讨Bcl2L13促进线粒体分裂是否通过作用于Drp1或Mfn1/Mfn2蛋白实现，进一步明确Bcl2L13介导线粒体分裂对细胞能量代谢和线粒体向细胞皮层区域转运的影响，最后结合胶质瘤临床组织标本中Bcl2L13的表达研究其潜在临床应用价值。课题实施将有助于评价Bcl2L13在胶质瘤发生发展中的作用，为抑制胶质瘤侵袭迁移提供新靶标。

背景: BCL2 like 13（BCL2L13）是BCL2家族的成员，它参与细胞生长和凋亡的调节。 但是，其在胶质瘤中的临床意义、生物学作用和潜在机制仍有待进一步探索。.方法: 通过癌症基因组图谱（TCGA）数据库，REMBRANDT数据库，Oncomine数据库，Proteinatlas数据库，癌细胞系百科全书（CCLE）数据库以及免疫组化（IHC）和蛋白质印迹来获得表达水平数据和临床信息进行分析。通路分析用于预测BCL2L13的潜在功能。用慢病毒干扰和过表达BCL2L13蛋白的合成，构建稳定的神经胶质瘤细胞系，分别使用划痕实验，侵袭实验，透射电子显微镜，细胞免疫荧光测定和小鼠体内原位肿瘤种植来评估迁移，侵袭，自噬和线粒体动力学中的生物学功能。 Mdivi-1是一种选择性的线粒体裂变抑制剂，用于检查线粒体裂变在BCL2L13诱导的线粒体自噬中的作用。.结果: BCL2L13的高表达与胶质瘤等级升高（P <0.01）和间充质分子胶质瘤亚型恶性程度（P <0.01）密切相关。通过BCL2L13抗体对独立队列的患者（n = 30）进行的免疫组织化学证实了这些结果。BCL2L13基因敲减显著降低了U251，GBM＃P3和GBM＃BG5细胞的迁移和侵袭能力。RNA SEQ.相关分析表明，BCL2L13与胶质母细胞瘤中的自噬/线粒体自噬密切相关。透射电子显微镜，免疫荧光和蛋白质印迹分析进一步证实BCL2L13促进了胶质母细胞的保护性线粒体自噬。 此外，BCL2L13通过促进DNM1L在Ser616位点的磷酸化诱导胶质母细胞线粒体裂变，从而促进线粒体自噬。最后，在荧光素酶转导的GBM＃P3细胞原位种植到小鼠头颅中，BCL2L13的敲低显著抑制了肿瘤的生长（P <0.01）。.结论: 我们的研究结果表明，BCL2L13通过DNM1L介导的胶质母细胞线粒体裂变，在促进胶质母细胞保护性线粒体自噬中起着重要的作用。因此，BCL2L13蛋白在胶质母细胞瘤中的重要调控和生物学功能，使其成为胶质母细胞瘤治疗的候选分子靶标。