Basigin选择性剪切变异体（BSG-2和BSG-3）参与肿瘤细胞侵袭伪足形成及其机制研究

Invadopodia, F-actin-driven membrane protrusions with matrix metalloproteinase (MMPs) activity to degrade the extracellular matrix (ECM), promote tumor metastasis. Invadopodia are enriched in a variety of proteins, including cortactin, cofilin, Arp2/3, and N-WASp. Basigin (BSG), a highly glycosylated type I transmembrane protein, is concerned as a cancer-associated biomarker and serves as a target for cancer therapy. Through extensive pre-mRNA alternative splicing, a large number of BSG isoforms are generated including Basigin alternative splice variant 2 (BSG-2) and variant 3 (BSG-3). Recent study shows that upregulation of CD147 (BSG-2) in non-transformed breast epithelial cells is sufficient for induction of matrix degrading invadopodia-like structures and increases invasion ability of these cells. In this project, we will study the role of BSG alternative splice variants (BSG-2 and BSG-3) in invadopodia formation of epithelial tumor cells, and determine which BSG splicing isoform plays the main role in invadopodia formation. Furthermore, we will demonstrate that BSG, CD44 and Integrin β1 function in one complicated membrane protein complex, which regulates the transition between focal adhesions (FAs) and invadopodia formation. This study is focused on RhoC/ROCK/LIMK/Cofilin and BSG/CD44/Integrin β1/FAK/Src signal pathway. We will block RhoC/ROCK/LIMK/Cofilin signal pathway by using ROCK inhibitor (H-1152 and Y-27632) and LIMK siRNA, to determine whether BSG-2 and BSG-3 regulate actin-barbed end initiation and invadopodia formation through RhoC/ROCK/LIMK/Cofilin signal pathway. We will also knockdown CD44 in BSG re-expression epithelial tumor cells to observe CD44’s function in BSG-dependent invadopodia formation. In short, we will try to reveal the contribution of BSG pre-mRNA alternative splicing and its’ splicing isoforms switch in invadopodia formation and tumor progression, and provide a potential cancer therapeutic target.

侵袭伪足是促进肿瘤细胞向邻近部位侵袭的重要细胞结构。因此了解肿瘤细胞侵袭伪足形成过程，并对此过程进行人为干预，对于抑制恶性肿瘤细胞侵袭及转移过程至关重要。Basigin(BSG)为重要的肿瘤相关分子，参与多种肿瘤细胞侵袭转移过程，其选择性剪切变异体(BSG-2)能使不具有侵袭力的乳腺上皮细胞发生转化并形成侵袭伪足。本项目拟对BSG选择性剪切变异体(BSG-2和BSG-3)在肿瘤细胞侵袭伪足形成中的作用及其作用机制进行研究。以BSG与Annexin II 、Integrin β1和CD44的相互作用为出发点，围绕RhoC/ROCK/LIMK/Cofilin信号通路，及BSG/CD44/Integrin β1/FAK/Src信号通路，分析BSG不同选择性剪切变异体在肿瘤细胞侵袭伪足形成中的交互作用及其作用机制，为将调节BSG前体mRNA选择性剪切过程作为肿瘤转移防治的新策略提供理论依据。

侵袭伪足是肿瘤细胞降解ECM、促进肿瘤细胞向邻近部位侵袭的重要细胞结构。Basigin(BSG)是一个重要的肿瘤相关分子。BSG基因编码产生4种BSG选择性剪切变异体。肿瘤细胞能否通过调节自身BSG前体mRNA的选择性剪切而改变细胞中BSG不同选择性剪切变异体的比例，从而达到调节细胞侵袭伪足形成的目的呢？BSG-3与BSG-2在肿瘤侵袭转移中具有相互拮抗作用，我们推测：BSG-3可能通过拮抗BSG-2的功能改变Annexin II酪氨酸磷酸化，肿瘤细胞通过改变BSG选择性剪切变异体的表达来调节Annexin II酪氨酸磷酸化，进而影响Rho/ROCK信号通路活性和CofilinS3磷酸化，最终影响侵袭伪足的形成。BSG-2为Integrin β1的相互作用分子，参与并调节了FAs的形成过程。在乳腺癌细胞中，同为Integrin β1相互作用用分子的CD44也参与了侵袭伪足的形成过程。因此，我们提出以下假设：分布于细胞膜表面的BSG-2能与CD44和Integrin β1形成复合体，通过调节FAs的形成，及FAs向侵袭伪足的转换而调节肿瘤细胞侵袭伪足过程。.本研究采用基因沉默和基因恢复表达等技术，对BSG选择性剪切变异体(BSG-2和BSG-3)在肿瘤细胞侵袭伪足形成中的作用及其作用机制进行研究。以BSG与Annexin II 、Integrin β1和CD44分子的相互作用为切入点，通过抑制RhoC/ROCK/LIMK/Cofilin信号通路，及检测BSG-2/CD44和Integrin β1复合体在侵袭伪足形成中的功能，揭示BSG不同选择性剪切变异体在肿瘤细胞侵袭伪足形成中的交互作用及作用机制。.本研究证实BSG剪切变异体——BSG2促进肿瘤细胞侵袭伪足的形成，BSG3参与调节BSG2依赖的侵袭伪足形成。细胞通过调节内膜系统BSG选择性剪切变异体的表达来调节Annexin II磷酸化，进而影响RohC/ROCK信号通路活性，调节Cofilin磷酸化及肿瘤细胞侵袭伪足形成；分布于细胞膜表面的BSG分子通过与Integrin β1和CD44分子形成复合体而调节侵袭伪足形成前期黏着斑的形成，及黏着斑向侵袭伪足的转换；从而促进肿瘤细胞侵袭伪足形成及肿瘤细胞侵袭力的增强。.研究结果为将调节BSG选择性剪切过程作为肿瘤转移防治的新策略提供理论支撑。