Supervillin(SVIL)调控细胞迁移与侵袭的分子机制研究

Cell migration is a fundamental process in the development and the maintenance for multi-cellular organisms, including embryonic development, wound healing, immune response and tumor metastasis. The remodeling of membrane-actin cytoskeleton plays a central role during this process. Supervillin(SVIL) is a very important membrane-cytoskeleton binding protein, it directly interacts with F-actin and actin-binding proteins such as Myosin II, Cortactin to regulate cell migration and cell invasion. We recently found SVIL is specially over-expressed in tumor tissues and tumor cell lines. However, it is still very limited to the understanding of the function of SVIL in its interacting networks, signaling transduction and biochemical pathway. We propose to combine bio-photonics, cell biology, biophysics and experimental animal model to dissect the properties of SVIL in the actin-based membrane-cytoskeletal dynamics. The aim is to demonstrate molecular mechanism of SVIL-mediated remodeling of membrane - actin cytoskeleton during cell migration and cell invasion. We also want to identify more SVIL interaction partners and reveal the regulatory effect by the interaction network, the signaling pathway and post-modification of phosphorylation. Furthermore, we try to explore the pathological mechanism of the disease resulted from the abnormal SVIL function, which will provide theoretical basis for intervention of related diseases and lay out a solid foundation for future clinical research.

细胞迁移是器官形成与发育的基础，参与胚胎发生、伤口愈合、免疫反应以及肿瘤转移等过程，细胞膜-微丝骨架动态重排在这过程中起着关键性调节作用。Supervillin(SVIL)是一个重要的膜-微丝骨架结合蛋白，它与微丝，以及MyosinII、Cortactin等微丝调节蛋白相互作用，调控细胞迁移与侵袭；我们最近发现其在肿瘤组织与细胞中特异高表达，但涉及的相互作用网络、信号转导分子机理、生化途径不甚明了。我们拟采用先进的生物光子学、细胞生物学、生物物理学及实验动物学等手段，对SVIL在微丝介导的膜-细胞微丝骨架动力学调控中的功能特性进行精确解析，揭示SVIL相互作用网络、信号传导、磷酸化修饰对其功能调节效应，通过体内外系统阐明SVIL介导的微丝细胞骨架重排在细胞迁移与侵袭过程中的作用机制。进而探索SVIL功能异常引起的病理机制，为相关疾病临床治疗与研究提供理论基础。

肿瘤细胞的侵袭与迁移是受多因素调控的复杂过程，细胞膜－微丝骨架的动态调控细胞迁移与侵袭的重要特征。SVIL是一个膜-微丝骨架结合蛋白，阐明其参与肿瘤细胞的侵袭与迁移的调控网络及作用机制是本项目的主要目标。综合应用细胞生物学、生物化学、分子生物学、动物模型等技术，利用TAP（串联亲和纯化）结合质谱鉴定SVIL体内互作网络，主要包括微丝骨架蛋白、膜结合蛋白、信号通路蛋白、细胞粘附蛋白等。通过分子与细胞水平探索，我们鉴定了一个新的SVIL剪切异构体SV5，在组织与细胞水平都表达，与其它异构体之间存在功能冗余性，促进细胞的增殖与迁移。深入探究SVIL促细胞迁移与侵袭的病理功能，发现在肝癌组织中SVIL表达上调，且与肿瘤缺氧微环境相关，SVIL增强肝癌细胞的体内转移。具体作用机制表现为SVIL与小分子GTPase包括RhoA、Cdc42、Rac1存在互作，主要通过与RhoA直接相互作用，促进其激活，并通过RhoA-ROCK信号通路激活下游的ERK/p38信号传递，影响EMT相关基因表达变化，在小鼠体内成瘤模型中增强肝癌细胞的转移。此外，细胞迁移能力与个体发育进程密切相关，以斑马鱼与小鼠为模型，抑制svila表达影响斑马鱼胚胎的集中延伸运动。通过CRISPR技术获得基因敲除成年小鼠存在发育迟缓现象，但生理功能影响仍需进一步试验确认。.通过本项目的研究，丰富了SVIL相互作用蛋白网络，阐明SVIL介导的肿瘤细胞侵袭与迁移调控机制，加深了对肿瘤浸润与迁移的本质和分子调控机制的认识，为临床潜在应用提供了理论依据。也初步探索SVIL参与发育过程，为下一步研究奠定了基础。