Arg激酶通过αvβ3整合素信号通路促进黑色素瘤细胞迁移的机制研究

Iintegrin is critical for cell migration because integrin can integrate intracellular and extracellular signals to regulate cell migration. αvβ3 integrin in many tumor cells have high expression, including breast cancer, prostate cancer cells, melanoma cells and glioma cells. αvβ3 integrin express in tumor cells and it closely relate to tumor occurrence, especially in tumor cell invasion and metastasis.. Integrin mediates adhesion and migration of tumor cells mainly through contacting with the cytoskeleton to form focal adhesion, and through regulating the dynamic temporal and spatial variation of focal adhesion assembly and disassembly. Integrin itself has no activity, its function generally regulated by a number of related kinases. Arg kinase as an intracellular non-receptor tyrosine kinases can phosphorylate many proteins, activate the corresponding target molecules, regulate cytoskeletal reorganization or gene expression, consequently, participate in cell apoptosis, differentiation, aging, etc. variety of physiological processes, there were studies also reported that it participates in cell migration.. In this study, we will establish the experimental model to study tumor cell invasion and migration and then determine the role of Arg kinase in tumor cell cytoskeleton rearrangement, pseudopodia formation, and cell migration, which were mediated by the integrin αvβ3. we will analysis the signal transduction events and the interaction between the proteins to clear the function and related mechanisms of Arg kinases in affecting tumor cell adhesion and migration. The understanding of related issues is an important aspect to clarify the mechanism of tumor cell invasion and migration molecules.

整合素在肿瘤细胞的黏附和迁移中起到非常重要的作用，研究发现，αvβ3整合素在黑色素瘤细胞中的高表达与肿瘤的发生发展及恶性程度密切相关。整合素本身没有酶活性，其作用一般由一些相关的激酶来调节。Arg激酶是一种非受体酪氨酸激酶，参与了细胞信号转导、细胞骨架重排等生命活动。最近，我们发现Arg激酶参与了αvβ3整合素介导的黑色素瘤细胞的迁移，且二者存在于同一免疫复合体内，这提示Arg激酶在αvβ3整合素介导的细胞迁移信号通路中可能是一个关键性调控分子。本研究拟探索Arg激酶对αvβ3整合素介导的肿瘤细胞的骨架的重排、伪足形成、以及迁移的影响；通过免疫共沉淀、FRET等技术研究信号转导事件中的蛋白质相互作用，揭示Arg激酶影响肿瘤细胞黏附和迁移的作用环节以及相关的作用机制，相关问题的阐明是了解肿瘤细胞浸润和迁移分子机制的重要方面。

整合素在肿瘤细胞的黏附和迁移中起到非常重要的作用。αvβ3整合素在黑色素瘤细胞中的高表达与肿瘤的发生发展及恶性程度密切相关。整合素介导肿瘤细胞的黏附和迁移主要是通过与细胞骨架联系形成黏着斑，并通过黏着斑的组装与去组装的动态时空变化来实现的。整合素本身没有酶活性，其作用一般由一些相关的激酶来调节。Arg激酶是一种非受体酪氨酸激酶，参与了细胞信号转导、细胞骨架重排等生命活动。. 在本项目中，我们通过transwell、单层细胞划痕和琼脂糖3D培养的细胞迁移实验模型发现Arg激酶参与了PDGF诱导的αvβ3整合素介导的黑色素瘤A375细胞的黏附、迁移，并且是呈正相关的影响。我们确定了Arg激酶与αvβ3整合素之间能够相互作用，但二者之间是间接结合。免疫荧光标记的结果显示细胞在PDGF刺激以后Arg激酶与αvβ3整合素在胞内有共定位的现象。通过体外激酶反应等实验我们发现Arg激酶与αvβ3整合素的相互作用依赖于Arg激酶的活性。同时Arg激酶对αvβ3整合素的调控不受F-肌动蛋白的影响，Arg激酶也没有参与αvβ3整合素与胞外配体结合引发的整合素outside-in的信号通路。通过pull down实验证明了Arg激酶是通过它的SH3结构域与αvβ3整合素形成复合体的。我们还进一步证实Arg激酶是通过与Talin蛋白直接连接来调控αvβ3整合素在细胞迁移中的功能的。小鼠体内肺转移实验证实Arg激酶抑制剂STI571能够明显抑制小鼠肺部转移灶的形成。平板克隆形成实验结果显示Arg激酶抑制剂STI571能够明显抑制细胞克隆的形成。Arg激酶能够调节几种增殖相关蛋白P27、AKT、PTEN、PARP的蛋白活性及表达。免疫共沉淀实验结果显示PDGF刺激后Arg与PI3K的调节亚基P85在同一免疫复合体中，说明Arg通过PI3K/AKT信号通路影响细胞的增殖。这些研究为揭示黑色素瘤细胞迁移和增殖的机制提供了可靠的数据，并且对筛选以阻断细胞信号通路为靶点的抗癌症药物具有重要的意义。同时为进一步的深入研究奠定了基础。