细胞骨架调节蛋白Coronin3促进胃癌细胞侵袭转移的分子机制研究

Multiple steps are involved in the metastasis of cancer cells from primary sites to distant organs. Invadopodia formation initiated by the nucleation of F-actin plays a crutial role in tumor metastasis, which enables the tumor cells to migrate through the extracellular matrix. Coronins are a conserved family of actin cytoskeleton regulators that participate in the nucleation of F-actin and modulate other actin-dependent processes together with other molecules including N-WASP and Cortactin. Coronin 3 is the only member of coronin family that has been related to human tumor. Our previous study detected an increasing level of Coronin3 expression in gastric cancer cells with high metastatic potential and found that down-regulation of Coronin 3 expression significantly suppressed the invasive and metastatic abilities of gastric cancer cells. Based on the preliminary results, in the present study, we will first detect coronin 3 expression in a larger group of patients by immunostaining, qPCR and Western blot analysis. Then, by gain of function and lose of function studies, the morphological changes of invadopodia will be detected by confocal study while the alterations in the distribution, localization and expression levels of those molecules that participate in F-actin nucleation will be observed by immunofluorescence,Western blot analysis and qPCR analysis respectively. Furthermore, potential interactions between Coronin 3 and other F-actin nucleation related molecules as well as other unknown molecules will be detected by immunoprecipitation,multiple reaction monitoring(MRM) and liquid chromatography/ tandem mass spectrometry(LC-MS/MS). Together, the current project will deepen the understanding of how cytoskeleton regulators participate in the process of tumor metastasis and provide a novel target molecule and therapeutic strategy for the treatment of gastric cancer metastasis.

肿瘤转移是多步骤参与的复杂过程，其中肿瘤细胞F-actin核化启动侵袭性伪足形成，进而穿越基底膜是重要环节。Coronin家族与N-WASP、Cortactin等分子在F-actin核化过程中发挥重要作用。Coronin3是其家族中唯一被报道与肿瘤相关的分子，但机制不清。我们的前期研究发现Coronin3在胃癌高转移潜能细胞系中高表达，下调其表达能够抑制胃癌细胞的侵袭转移能力。本研究拟进一步检测coronin3在胃癌组织中的表达及其临床意义；通过功能获得和缺失研究，共聚焦观察Coronin3对F-actin核化及侵袭性伪足的影响，Western、qPCR等检测F-actin核化相关分子表达；IP、MRM、LC-MS/MS测序探索Coronin3与F-actin核化相关分子及其它未知分子间的相互作用。本项目有助于深入阐明细胞骨架调节蛋白参与肿瘤转移的机制，为有效干预胃癌转移提供新的治疗策略。

肿瘤转移是多步骤参与的复杂过程，其中肿瘤细胞F-actin核化启动侵袭性伪足形成，进而穿越基底膜是重要环节。Coronin家族在F-actin核化过程中发挥重要作用。Coronin3是其家族中唯一被报道与肿瘤相关的分子，但机制不清。本研究针对Coronin3在胃癌中的表达及其功能进行了深入研究，具体研究发现如下：①检测coronin3在胃癌组织中的表达及其临床意义， 发现Coronin3在胃癌患者淋巴结转移灶中表达明显高于原发灶，且与胃癌患者分期和淋巴结转移密切相关，是影响胃癌患者预后的危险因素。②体外功能实验和体内裸鼠转移瘤实验表明，在胃癌细胞系中上调Coronin3能够明显增强肿瘤细胞的迁移和侵袭能力；反之下调Coronin3表达能够明显抑制肿瘤细胞的迁移和转移能力。③通过转移相关PCR芯片，发现Coronin3能够通过调控MMP-9 和Cathepsin K表达调控肿瘤细胞侵袭转移。④通过蛋白质组学技术发现并验证Arp2是与Coronin3相互作用的F-actin核化分子。Arp2在胃癌高转移细胞系中表达高于亲本细胞及低转移细胞系。进一步证实下调Arp2可抑制Coronin3介导的胃癌细胞的迁移和侵袭能力，上调Arp2可促进细胞的迁移和侵袭。⑤Arp2表达与胃癌的分化和TNM分期相关，在淋巴结转移灶中的阳性率高于原发灶。联合检测Coronin3和Arp2表达与胃癌患者的生存期密切相关，能够准确预测胃癌患者预后，两者表达越高，生存期越短。. 本项目深入研究了Coronin3在胃癌中的表达、作用及其机制，鉴定了Coronin3与F-actin核化相关分子Arp2相互作用，并初步研究了Arp2在胃癌中的表达及作用。本项目有助于深入阐明细胞骨架调节蛋白参与肿瘤转移的机制，为有效干预胃癌转移提供新的治疗策略。

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