低氧通过激活TRPM7促进肝癌细胞转移的作用和机制研究

Hypoxic microenvironment is highly correlated with tumor metastasis. Previous studies have focused on adaptive gene expression in hypoxia. Little is known about the electrophysiological properties such as transmembrane transportor of ions in cancer under hypoxia. TRPM7 is a bifunctional membrane protein with both an ion channel pore and a kinase domain. It has been reported that influx of Ca2+ and Mg2+ mediated by TRPM7 is involved in cell growth, adhesion, mobility, and epithelial-mesenchymal transition, etc. Recently，it has also been described that expression of TRPM7 is upregulated in several kinds of cancer tissues and it participates in progression of cancer. However, expression and function of TRPM7 in hepatocellular carcinoma are unclear. Our recent study showed that expression of TRPM7 was upregulated in HCC cells, and downregulation of TRPM7 could inhibit HCC migration. Thus, we propose hypoxic microenvironment may promote HCC metastasis through TRPM7-mediated ion transport. To verify this hypothesis, this project intends to study the regulation of TRPM7 channel characteristics and kinase activity via hypoxia. Furthermore, the effection of hypoxia-activated TRPM7 on HCC metastasis and its possibly molecular mechanisms will be explored. This study will provide new ideas for understanding the role of tumor microenviroment on tumor development and discovering new therapeutic target.

低氧微环境与肿瘤转移高度相关，以往的研究主要关注与低氧相适应的基因表达，而对肿瘤细胞的离子转运等电生理特性认识不足。TRPM7是兼具通道和激酶特性的双功能膜蛋白，其介导的Ca2+、Mg2+参与细胞的生长、黏附、运动和上皮间质转化等病理生理过程。最近研究发现，多种肿瘤组织中TRPM7表达上调并参与肿瘤的恶性进展。然而，TRPM7在肝癌中的表达和功能尚不清楚。我们前期研究发现TRPM7在缺氧的肝癌细胞中表达上调，负调控TRPM7能显著抑制癌细胞的迁移。由此，我们提出肝癌细胞可能通过调控TRPM7介导的离子转运以适应低氧微环境并促进癌细胞的转移。为验证此假说，本项目拟通过膜片钳技术等电生理学方法研究缺氧对肝癌细胞TRPM7通道和激酶活性的调节，以及在细胞和动物水平探讨缺氧通过调节TRPM7促进肝癌细胞转移的作用和分子机制，为进一步理解肿瘤微环境在肝癌发生发展中的作用及新药物靶点研发提供新的思路。

高复发率和高转移率是影响肝癌预后的主要原因，而肿瘤酸性微环境与肝癌的转移密切相关，但机制尚不清楚。TRPM7（Transient receptor potential cation channel, subfamily M, member 7）是兼具激酶特性和通道特性的膜蛋白，能被酸性环境激活，其在胚胎发育、细胞生长、增值、分化和粘附中的重要作用提示其可能参与肿瘤的缺氧耐受。TRPM7是否在肝癌发生过程中发挥重要作用还没有报道。本研究利用肿瘤细胞的低氧模型，检测到TRPM7在多种肝癌细胞系中受低氧影响而上调，TRPM7表达增加与缺氧程度、HIF1a、和HIF2a的表达正相关。通过siRNA干扰HIF1a、HIF2a显著抑制了低氧引起的TRPM7的表达增加。生物信息学分析发现TRPM7的启动子区域有HIF1a的低氧反应元件，进一步我们通过chip和luciferase实验验证了TRPM7受到HIF1a的转录调节。siRNA干扰TRPM7的表达显著抑制了肝癌细胞的增殖和迁移潜能。接下来，我们通过葡萄糖糖代谢相关PCR-array，检测到TRPM7过表达能增加众多的葡萄糖代谢相关基因，这些表达增加的基因与低氧诱导增加的基因很大程度上是一致的，表明TRPM7参与了肿瘤的低氧代谢重塑过程。本研究还比较了21对癌旁和肝癌组织的TRPM7表达水平，结果显示肝癌中TRPM7 mRNA表达与癌旁无显著差异，在后续的研究中需要区分肿瘤的中心缺氧区域和边缘区域，以及扩大样本量进行进一步的验证。本研究为干预肿瘤微环境的治疗策略提供了一定了理论基础。