Arpin竞争性结合Arp2/3复合体通过TGF-β/EMT通路调控乳腺癌转移的机制研究

Arp2/3 complex-mediated cytoskeletal remodeling involved in cell phenotype transformation is an important mechanism in regulating tumor cell invasion and metastasis. But the regulation mechanism is not clear so far. In the previous study, we found that the newly named Arp2/3 complex inhibitory factor Arpin was closely related to the activity of TGF-β/Smad pathway in breast cancer. After over-expression of Arpin, the morphology of the cancer cells changed and the invasive ability was significantly decreased. WAVE3, an activator of Arp2/3 complex, was associated with TGF-β-mediated epithelial-mesenchymal transition (EMT). Molecular function analysis showed that Arpin and WAVE3 had the same binding region in Arp2/3 complex, and subcellular localization of the three proteins by immunofluorescence assay showed that all of them were detected in restricted segments of the plasma membrane. Based on the above findings, we propose a hypothesis: Arpin and WAVE3 competitive binding Arp2/3 complex regulate the cell phenotype transition and influence the invasion and metastasis of breast cancer through TGF-β / EMT pathway. To verify this hypothesis, this study intends to focus on the role of Arpin in regulating tumor cell invasion and metastasis by gene silence and gene over-expression techniques, clarify the relationship among Arpin, WAVE3 and Arp2/3 complex in breast cancer metastasis. And to further explore the molecular mechanism of cytoskeletal remodeling, epithelial-mesenchymal in breast cancer cells and provide new targets and theoretical basis for the prevention and treatment of breast cancer metastasis.

Arp2/3复合体介导细胞骨架系统重塑参与细胞表型转化是肿瘤细胞侵袭转移的重要机制，但其调控方式尚不明确。我们前期研究发现负性调节Arp2/3复合体新蛋白Arpin在乳腺癌中与TGF-β/Smad通路活性相关；Arpin过表达后癌细胞形态发生改变、迁移能力明显降低。Arp2/3复合体激活因子WAVE3与TGF-β介导的上皮-间质转化（EMT）相关；分子功能分析显示Arpin和WAVE3与Arp2/3复合体具有相同的结合区，且亚细胞定位相似。因此，我们提出Arpin与WAVE3竞争性结合Arp2/3复合体通过TGF-β/EMT通路调控细胞表型从而参与乳腺癌侵袭转移。我们拟采用基因静默和基因过表达等技术，从分子、细胞、动物等多个层面阐明Arpin、WAVE3和Arp2/3复合体的信息交流和相互作用模式，明确细胞骨架和细胞表型转化在乳腺癌转移中的分子机制，为抑制乳腺癌转移提供新的靶点和理论依据。

Arp2/3复合体介导细胞骨架系统重塑，参与细胞表型转化，是肿瘤细胞侵袭转移的重要机制。Arpin是与伪足形成相关的新蛋白，可以负性调节Arp2/3复合体，与细胞的运动和迁移密切相关。本项目深入探究了Arpin与Arp2/3复合体的作用模式以及他们通过TGF-β/EMT通路调控细胞表型转化参与乳腺癌侵袭转移的机制。取得的主要研究结果：1）证实Arpin、WAVE3等在乳腺癌中的表达与乳腺癌临床病理特征相关。检测Arpin、WAVE3及细胞表型特异性标志物分子等在乳腺癌细胞株、乳腺癌组织中的表达，分析证明Arpin低表达和WAVE3高表达与肿瘤的恶性程度及肿瘤细胞表型转化相关。2）明确Arpin和WAVE3对乳腺癌细胞表型转化的调控作用及对肿瘤转移的影响。通过构建Arpin和WAVE3静默或过表达重组慢病毒，观察Arpin和WAVE3的表达对乳腺癌细胞EMT/MET表型转化以及细胞周期、生长曲线、细胞集落形成、侵袭迁移能力等生物学行为的影响。通过体外和体内实验验证了Arpin过表达WAVE3静默的双靶向作用对肿瘤生长及转移具有协同调控作用。3）分析Arpin、WAVE3与Arp2/3复合体之间的作用方式。采用免疫共沉淀和免疫荧光双标记实验证实Arpin和Arp2/3复合体具有相互作用。4）探讨Arpin对TGF-β/EMT信号通路的调节作用及分子机制。在乳腺癌细胞中过表达Arpin，采用基因芯片筛选差异表达的mRNA，经功能富集分析和信号通路富集分析发现Arpin过表达后TGF-β通路抑制，通路的关键分子表达下调。说明Arpin的表达通过TGF-β通路影响了细胞EMT的转化。本项目获得了预期研究结果，达到预期研究目标，研究结果有助于更好地了解TGF-β/EMT信号通路与乳腺癌侵袭转移的关系，为转移性乳腺癌的靶向治疗提供新的靶点和理论依据。