神经钙粘素前体介导胶质瘤细胞迁移的机制研究

Glial cell-line Derived Neurotrophic Factor (GDNF) can promote glioma cells to metastasize. Recent publications suggest increased levels of the non-adhesive transmembrane pre-protein, proN-cadherin, is the main reason why glioma cells are prone to metastasis. Further research has found that the increase of pro-N-cadherin in glioma cells is due to a decreased expression of furin. In our previous research we showed that GDNF and proN-cadherin interact. We believe there is a positive feedback loop in glioma cells that promotes metastasis by overproduction of proN-cadherin. The signaling pathway begins with GDNF and proN-cadherin's interaction. This results in decreased expression of furin and therefore increases production of proN-cadherin in glioma cells. In order to prove and elucidate this positive feedback loop, we plan to demonstrate that β-catenin is released from the pro-N-cad/β-catenin membrane complex when the concentration of GDNF is increased. Once free, cytoplasmic β-catenin can enter the nucleus and form a β-catenin/CDX2/TCF complex. CDX2's sequestration in this complex will keep it from interacting with the promoter of furin gene, which in turn will downregulate its expression. Our research project would provide the mechanistic foundation of metastasis in glioma cells, which will bring new ideas for the clinical treatment of glioma.

胶质细胞系源性神经营养因子（GDNF）能促进胶质瘤细胞迁移。研究发现，胶质瘤细胞膜上存在大量无粘附能力的神经钙粘素前体（pro-N-cad）,这是胶质瘤细胞迁移的主要原因，而pro-N-cad增加是成对碱性氨基酸蛋白酶(furin)减少所致。我们前期研究显示，GDNF能与pro-N-cad相互作用，故此，我们认为，GDNF促胶质瘤细胞迁移是经pro-N-cad介导、激活一条细胞内信号通路、下调furin表达，进而产生更多pro-N-cad而实现的。为验证该正反馈环路的存在，本项目拟研究在GDNF作用下，pro-N-cad/β-catenin复合体上β-catenin的脱落；胞核内β-catenin/CDX2/TCF复合体的形成；CDX2与furin基因启动子结合量下降等系列因素致furin基因表达下调。该项目的完成能为深入了解胶质瘤细胞迁移提供理论依据，并为防治胶质瘤转移提供新思路。

胶质细胞系源性神经营养因子（glial cell—line derived neurotrophic factor， GDNF）属于转化生长因子（TGF-β）超家族成员，在胶质瘤发生发展过程中发挥着重要作用，但机制尚未明确。本项目旨在揭示细胞外可溶性因子GDNF是如何经由细胞膜上的神经型钙粘附分子前体（pro-N-cad）介导，激活细胞内β-catenin相关信号通路，进而发挥促进胶质瘤细胞侵袭性生长的机制。结果显示：GDNF与pro-N-cad之间存在着氢键连接的相互作用，该作用会促进pro-N-cad胞内段Tyr142、Tyr498和Tyr654位点磷酸化，进而促使pro-N-cad/β-catenin复合体中的β-catenin脱落，并移入细胞核发挥转录调控作用。另外：GDNF作用后，细胞外基质中的四种粘附蛋白（I型胶原；IV型胶原；纤连蛋白；层粘连蛋白）表达会显著升高，细胞与细胞外基质的粘附能力增加，细胞之间的粘附相对减弱。细胞膜上pro-N-cadherin的表达增加也会促进胶质瘤细胞分泌MMP9，这就帮助降解细胞外基质而进一步促进的肿瘤细胞的迁移和侵袭。.基于生物信息学和分子生物学实验，我们观察到：GDNF可以招募神经纤毛蛋白1（neuropilin-1，NRP1）富集于细胞膜上，并由其介导信号转导，同样是激活了胞内β-catenin，并促进β-catenin发生核内转移；在核内，β-catenin与Rac1结合作为共转录因子调节与增殖相关的基因CXCL1表达，进而发挥促C6细胞增殖作用。GDNF作用于胶质瘤细胞后，发现可显著增加七种miRNA的表达，分析发现这七种miRNA所调节的靶基因多参与细胞粘附和细胞周期的调控，进而影响胶质瘤细胞增殖、迁移和侵袭。同时，观察到GDNF能提高大鼠原代星形胶质细胞的生长活性，且呈浓度和时间依赖性，通过二代测序发现GDNF促进星形胶质细胞增殖的基因表达上调，抑制凋亡相关的基因表达。所激活的增殖相关信号通路与胶质瘤细胞的促增殖通路相同。基于此，我们推测，异常高剂量GDNF可能与星形胶质瘤的发生有关。这部分工作正在补充和整理之中，预计将有较好文章发表。.综上可知GDNF在胶质瘤发生方面有着重要作用，其分子基础是从细胞膜上粘附分子的结构和性质变化开始的。我们下一步工作将继续围绕全面揭示GDNF在胶质瘤发生发展中的影响及作用。