缺氧微环境下TRPC1/3通道介导的钙信号对胶质瘤细胞迁移与侵袭影响的分子机制研究

Hypoxia, a common phenomenon both in vitro and vivo, occurred in lots of physiological and pathological processes. However the definite molecular mechanism in hypoxic micro-environmental condition is unclear. Our previous researches found that the calcium influx induced by hypoxia was attenuated by downregulation of TRPC3; the migration of glioma cells was suppressed when TRPC channels were inhibited or silenced under hypoxia. The expression of MMP2 was increased in glioma cells exposed to hypoxia. And it was generally known that the migration of cancer cells could by promoted by STAT3/MMPs pathway. Therefore, we speculated that, under hypoxia, the migration and invasion of glioma cells mediated by STAT3/MMPs activation could be enhanced by calcium influx through TRPC1/3 channels. This project aims to validate this hypothesis as follows. Firstly, a hypoxic model is established by treating the cells with 3% oxygen or 150μM cobalt chloride. Secondly, the influences and molecular mechanism of calcium reflux through TRPC1/3 channels on the migration and invasion of glioma cells under hypoxia are authenticated and investigated. Finally, the roles of TRPC1/3 channels in cell invasion in vivo are confirmed by tumor formations in nude mice. Based on the above study, the problem on whether the TRPC1/3 channels could promote the invasive growth of glioma cells under hypoxia through STAT3/MMPs pathway will be elucidated. Therefore, this project is helpful to appreciate the signaling pathway in glioma cells under hypoxic microenvironment and provide important clue for the molecular therapy on glioblastoma multiforme.

缺氧存在于多种病理生理过程并且可通过钙信号调控肿瘤细胞的恶性生物学行为，然机制不明。我们前期研究发现在胶质瘤细胞中缺氧诱导TRPC通道开放进而介导钙内流，调节MMPs表达上调，促进细胞迁移。又文献提示缺氧及钙信号均可激活STAT3/MMPs通路，而该通路对细胞迁移侵袭具有显著的调控功能。因此推测：缺氧微环境下TRPC1/3调控的钙信号激活STAT3/MMPs通路促进胶质瘤细胞迁移与侵袭。本项目拟在缺氧模型中，以药理学及分子生物方法调控TRPC1/3活化与表达，明确TRPC1/3介导的钙信号促进迁移侵袭，研究TRPC1/3对STAT3/MMPs表达与活化的影响，探讨STAT3/MMPs信号在TRPC1/3调控细胞迁移侵袭中的作用；并通过裸鼠荷瘤体内验证TRPC1/3生理功能。以揭示缺氧时TRPC1/3促进细胞迁移侵袭的分子机制，为缺氧微环境中细胞信号调控研究及恶性胶质瘤防治提供新思路新靶点。

以3%O2物理缺氧与150μM CoCl2化学模拟缺氧建立缺氧模型，在此基础上探讨TRPC1/3通道对胶质瘤细胞增殖与迁移的影响：① 缺氧条件下TRPC通道阻滞剂(2APB，SKF96365)及TRPC通道表达下调均可抑制胶质瘤细胞增殖，并引起G1期周期阻滞，提示缺氧微环境下TRPC通道参与胶质瘤细胞增殖的调控。②缺氧条件下TRPC1表达增加，TRPC通道阻滞剂(2APB，SKF96365)及TRPC1/3通道表达下调均可抑制细胞迁移，证实TRPC1/3通道在缺氧微环境下调控胶质瘤细胞的迁移； TRPC1/3通道表达下明显降低缺氧诱导的细胞钙内流；同时发现TRPC1/3通道表达下调可抑制胶质瘤细胞中STAT3活化，并降低了MMP2表达，提示缺氧微环境中TRPC1/3通道可能通过STAT3/MMP2信号促进胶质瘤细胞迁移。. 另外研究中发现胶质瘤细胞中基础IL‐37表达水平极低，将IL‐37真核表达载体转染入U87MG及U251胶质瘤细胞，转染后细胞中IL‐37的mRNA及蛋白表达均显著上调。IL‐37在胶质瘤细胞中的过表达显著抑制了的细胞增殖并诱导其凋亡， IL‐37的过表达同时抑制了AKT的磷酸化；研究还发现PI3K抑制剂处理和IL-37有相近生物学效应，提示PI3K/AKT信号可能介导了IL-37对胶质瘤细胞增殖与凋亡的调控。. 通过以上研究，明确缺氧微环境下TRPC通道参与了胶质瘤细胞的增殖的调控；证实缺氧微环境下TRPC1/3通道介导的钙信号通过STAT3/MMP2通路促进胶质瘤细胞的迁移。发现IL-37可能通过PI3K/AKT信号抑制胶质瘤细胞的增殖并诱导其凋亡。更深入认识了胶质瘤在实际生理条件下侵袭性生长的分子机制，项目的拟解决的关键问题得到证实。本研究不仅为胶质瘤的防治提供理论基础和实验依据，也为其分子治疗提供新的靶点。部分数据已发表，一篇SCI论文在投稿过程中，另外仍有数据资料分析撰写过程中，期待更多的研究成果呈现。目前为止发表标注本研究资助的论文3篇，并且在此期间培养副高职称人员一名（申请人由讲师晋升副教授），协助培养研究生2名，单独指导培养研究生1名（在读）。