MC3-Ag/Txl-2与小G蛋白家族分子Ran相互作用介导结肠癌转移的机制研究

MC3 is a monoclonal antibody previously prepared in our laboratory, which could detect colon cancer with high sensitivity and specificity. Our previous work identified thioredoxin-like protein 2 (Txl-2) as the target of MC3 and indicated that overexpression of Txl-2 in colon cancer cells significantly promoted cancer cell motility, and the invasive and metastatic abilities. Furthermore, isoform-specific interaction was identified between Txl-2b and Ran, a Ras-related protein, by yeast two-hybrid assay and co-immunoprecipitation. Recently, emerging evidence has been reported on Ran associating with various cancer malignant phenotypes. However, the underlying mechanism is still poorly understood. In the present study, confocal study, paired immunostaining,deleting mutation and co-IP will be employed to further confirm the interaction between Txl-2b and Ran and detect the detailed binding sequences. Lentiviral co-transfection of Txl-2 and Ran will be done, followed by the observation of changes in cell invasive and metastatic potential in vitro and in vivo. Ran GTP level will be monitored before or after Txl-2b and Ran interaction. Mutant variants will be generated to determine whether the enzyme active sites of Txl-2b may influence the interaction between Txl-2b and Ran. Gene arrays, pathway inhibitors, qPCR and western blot analysis will be used to further explore the involved downstream events and pathways of Txl-2b-Ran mediated colon cancer metastasis. The current study may provide a deep insight into how protein interaction functions in cancer metastasis and open an avenue to a novel therapeutic strategy against colon cancer.

MC3是我们自主建立的一株结肠癌特异的单抗，前期研究利用蛋白质组学技术鉴定出其识别的抗原为Txl-2,并发现Txl-2能够显著促进结肠癌转移。酵母双杂和免疫共沉淀发现Txl-2的第2个亚型Txl-2b与小G蛋白家族成员Ran相互作用，而Txl-2其它2个亚型与Ran没有相互作用；Ran近年来被报道在多种肿瘤中高表达且促进肿瘤进展，但机制不清。本课题拟采用激光共聚焦、配对免疫组化和截短突变免疫共沉淀等进一步证实Ran与Txl-2b亚型特异的相互作用；慢病毒共感染、体内体外功能实验观察两者相互作用对结肠癌细胞转移能力的影响;Pull Down实验检测二者相互作用后Ran GTP活化形式的表达水平；位点突变明确Txl-2b酶学催化位点对其与Ran相互作用的影响；芯片筛选、抑制剂干预进一步寻找下游信号通路；本项目将深入阐明蛋白相互作用对肿瘤恶性表型影响的机制，为开发肿瘤新的治疗策略提供新思路。

本项目进展顺利，达到预期目标。结直肠癌是世界范围内发病率和死亡率均高的恶性肿瘤。传统的肿瘤标志物CEA、CA199等敏感性较差，在早期患者中阳性率低，不利于结直肠癌的早期筛查。寻找和鉴定新的分子标志物并经临床研究得到验证和转化，将有助于结直肠癌的早期诊断、预后判断和治疗监测。Txl-2是本实验室具有自主知识产权建立的结肠癌特异性单克隆抗体MC3所识别的抗原。本研究通过免疫组织化学研究MC3-Ag/Txl-2在243例结肠癌组织中的表达，发现其表达水平与患者病理分期和患者预后密切相关。接着，我们重点针对Txl-2促进结肠癌细胞侵袭转移进行了深入研究， 特别阐明了Txl-2的三个选择性剪接体发挥的不同作用。下调Txl-2表达能够明显抑制结肠癌细胞的迁移转移能力，其中，选择性剪接体Txl-2b的功能最为明显。酵母双杂交、免疫沉淀和免疫荧光显示Txl-2和Ran蛋白共定位并相互作用，PI3K/Akt通路介导了Txl-2/Ran-MMP介导的结肠癌细胞侵袭转移。此外，我们还研究了Txl-2b介导结肠癌细胞增殖和促进凋亡方面的作用，阐明NF-κB通路介导了Txl-2参与的细胞增殖。以上工作深入研究了结肠癌新抗原Txl-2在结肠癌恶性表型中发挥的作用，为结肠癌提供了新的潜在治疗靶点。本课题在Antioxid Redox Signal.（封面文章, IF=7.6），Cancer Lett (IF=5.6) 等杂志发表论文4篇，培养2名硕士研究生, 2次参加国内会议交流。