Prrx1动态调控胃癌细胞EMT-MET转换的机制及意义

EMT-MET conversion theory has becoming the focus in researching the mechanism of tumor metastasis in recent years , but the role in metastasis of gastric cancer has not yet been confirmed. Our previous studies have found Prrx1, an important regulator in the process of EMT-MET, discrepantly expressed in primary tumor and the metastases, thus suggesting EMT-MET is a possible mechanism in the process of gastric cancer cell metastasis. Our study is designed to detect the expression of Prrx1 and some epithelial mesenchymal phenotypic molecular markers in human gastric primary tumor, metastases and other organizations, then analyzing the relationship between Prrx1 expression and EMT-MET status, Prrx1 expression and clinicopathological factors. Screening the gastric cancer cells lines, in which Prrx1 low expression or high expression ; utilizing RNAi or recombinant DNA technology，and by means of 3D substrate cultivation、Transwell、Migration Assay、FACS and orther methods to observe the effect on epithelial and mesenchymal phenotypic molecular markers , the biological behavior of gastric cancer cells, chemotherapy sensitivity. Models of human gastric carcinoma would be constructed, utilizing fluorescent tracer technique to observe gastric cancer cells shedding process dynamically; via Cell Search screening and separation of CTCs to assess the status and the degree of EMT-MET. Understanding the relationship between epithelial and mesenchymal phenotypic markers expression and chemotherapy sensitivity, discussing the relationship between EMT status and the tolerability of chemotherapy，the mechanism of EMT process promoting tumor metastasis; building simulation gastric lymph, liver and peritoneal metastasis model to investigate the mechanism of MET process promoting CTCs colonization, to clarify the mechanism of EMT-MET in the process of gastric cancer cell metastasis and colonization, to get the theory of against gastric cancer metastasis in stages. Try to provide new ideas for overcoming tumor metastasis and chemotherapeutic resistance.

EMT-MET转换理论是近年肿瘤转移研究的热点，但其在胃癌转移过程中的作用尚未被证实。我们前期研究发现EMT-MET转换的重要调节因子Prrx1在肿瘤原发灶及转移灶中差异性表达，提示EMT-MET转换是胃癌细胞动态转移的可能机制。本实验检测人胃癌组织Prrx1及上皮、间质表型分子标志物表达，分析Prrx1表达与EMT、MET状态及临床病理因素的关系；利用RNAi或重组DNA技术干预，通过Transwell、 FACS等手段观察其对胃癌细胞生物学行为、化疗药物敏感性等方面的作用；建立胃癌原位移植瘤及体内转移模型，荧光细胞示踪动态观测胃癌细胞脱落、转移、定殖过程，CellSearch分离CTCs，评估EMT及MET状态及程度，探讨EMT状态与化疗耐药性的关系及EMT-MET过程促进肿瘤转移、定殖的机制，探索分阶段抗胃癌转移的可能性。为克服肿瘤转移及化疗药物耐受提供新的思路。

主要研究内容：检测人胃癌组织Prrx1及上皮、间质表型分子标志物表达，分析Prrx1表达与EMT、MET状态、CTCs及临床病理因素的关系；利用RNAi或重组DNA技术干预Prrx1表达，通过Transwell、wound healing等手段观察其对胃癌细胞生物学行为、化疗药物敏感性等方面的作用；同时对EMT相关LncRNA进行筛选。重要结果：胃癌组织Prrx1表达与EMT相关蛋白（E-cadherin、N-cadherin、Vimentin）表达密切相关,可以增加CTCs数量，参与胃癌淋巴结转移；Prrx1表达与胃癌细胞迁移、侵袭、增殖能力相关，高表达可增强细胞株微血管生成拟态VM能力，参与细胞株耐药；Prrx1可能在TGFβ Smad2 通路介导的上皮间质转化起作用，并与p53通路相关；发现LncRNA MT1JP低表达水平可能会促进胃癌细胞EMT过程；初步探讨了LncRNA MT1JP甲基化对胃癌细胞的影响。科学意义： 阐述了胃癌演进的新机制，为抑制肿瘤转移及化疗药物耐受提供参考。