RhoA/ROCK信号途径在Sema4D介导的肺癌血管生成拟态形成中的作用及机制研究

Vasculogenic mimicry(VM) is a novel pattern of blood supply in tumor which is associated with the poor response of anti-angiogenesis treatment in lung cancer. The plasticity of malignant tumor cells is an important element of VM. RhoA/ROCK is a key factor of remodeling cell cytoskeleton. However the role and mechanism of RhoA/ROCK in the formation of VM in lung cancer remains unclear. It was reported that RhoA played a critical role in angiogenesis mediated by axon guidance factor semaphorin4D and its receptor plexinB1 through coordinating the cytoskeleton of endothelial cell and promoting endothelial cell motility. Our previous study confirmed that the expression of Sema4D and plexinB1 were higher than that in normal tissue and the high expression of Sema4D was related to the formation of VM in lung cancer. It is postulated that RhoA/ROCK signalling pathway may play an important role in the formation of VM mediated by Sema4D in lung cancer by regulating the cytoskeleton of lung cancer cells. In this study, we will set up the model of VM in lung cancer in vitro by culturing lung cancer cells in 3-dimensional cell cultures and in vivo by transplanting lung cancer cells in nude mice. Then, we will explore the effect and mechanism of RhoA/ROCK in the formation of VM mediated by Sema4D in lung cancer by gene silencing and intervention with Rho kinase inhibitor fasudil. It will help us to find new target blocking the blood supply in lung cancer.

血管生成拟态（VM）为肿瘤中一种全新的特殊血供模式，与目前抗血管生成治疗疗效不佳密切相关。肿瘤细胞自身变形为VM形成的关键步骤，RhoA/ROCK作为细胞骨架重组的关键调节因子，在肺癌细胞自身变形形成VM过程中的作用尚不明确。研究表明，RhoA通过调节细胞骨架促进内皮细胞迁移，在轴突导向因子Sema4D与内皮细胞上受体plexinB1结合促进的内皮依赖性血管新生中起重要作用。我们前期研究表明Sema4D与plexinB1在部分肺癌组织中同时高表达且与肺癌VM形成正相关。鉴于以上因素，我们推测，RhoA/ROCK可能通过调节肺癌细胞骨架促进细胞自身变形在Sema4D介导的肺癌VM形成中起重要作用。本研究拟以三维细胞培养和裸鼠移植瘤技术创建肺癌VM体内外模型，应用药物干预及siRNA技术探索RhoA/ROCK信号途径在Sema4D介导的肺癌VM形成中的作用及机制，为阻断肺癌血供治疗寻求新靶点。

本研究采用小管形成实验、划痕实验、Transwell迁移实验、RT-PCR、Western Blot、免疫组化等方法，研究sema4D在非小细胞肺癌血管生成拟态（VM）中的作用。主要成果表现在以下几个方面:（1）体内外实验证实非小细胞肺癌中存在VM这种特殊的血管生成方式。（2）体内外实验证实sema4D在肺癌VM中发挥重要作用，靶向抑制sema4D能够减少H1299，HCC827细胞肺癌VM的形成及降低VM相关蛋白的表达，同时靶向抑制sema4D可降低肺癌H1299，HCC827细胞的迁移能力。（3）证实sema4D是通过与其受体plexinB1结合激活下游RhoA/ROCK通路影响肺癌H1299，HCC827细胞的VM形成。靶向抑制plexinB1能够减少H1299，HCC827细胞肺癌VM的形成，且给予外源性sema4D后成管能力没有恢复。(4)小鼠恶性黑色素瘤细胞B16构建的动物移植瘤模型中，RhoA/ROCK通路抑制剂fasudil可抑制移植瘤VM形成。上述结果证实了我们提出的假说：sema4D通过激活RhoA/ROCK通路调节肿瘤细胞的迁移能力从而在肺癌VM的形成过程中发挥重要作用，靶向调控非小细胞肺癌中sema4D/plexinB1及下游RhoA/ROCK通路，降低肿瘤细胞的可塑性及迁移能力，有望阻断VM血供模式、遏制肺癌细胞生长。