线粒体TRAP1分子介导Ago2蛋白表达在肠癌转移中的作用机制

While previous research has demonstrated that a class of proteins known as molecular chaperones promote tumor cell survival, the specific way in which the proteins achieve this has not been well understood. And although inhibitors of a specific chaperone know as heat shock protein 90 (Hsp90) have been studied for the treatment of cancer, for example, 17-AAG, a small structure of inhibitor of Hsp90, has enter the trial, progress has been questionable. Notably, researcher found a very abundant pool of TRAP1 ( Hsp90-related molecule) in the mitochondrial of tumor cells. This mitochondrial accumulation of TRAP1 as a critical adaptive mechanism that makes cancer cells less susceptible to the unfavorable environment of tumors, and to various anticancer agents. The sub-cellular location of Hsp90 and TRAP1 in the mitochondrial also answers the question as to why the current Hsp90 inhibitors which do not penetrate the mitochondrial are not as effective as hoped in the clinic. In current study, we have collected two types of different metastasis potential colon cell line, SW620/SW480, for the further studying the effect of mitochondrial TRAP1 on the colon cancer metastasis. Based on our preliminary research, combining RNAi technique in colon cancer cell lines and other proteomics techinques were applied for studying the correlation between TRAP1 and key components of the miRNA, Argonaute2(Ago2), in which we try to clarify the possible mechanism of colon cacner metastasis.

目前Hsp90分子抑制剂作为抗肿瘤药物已进入临床试验，但试验发现肿瘤细胞转移无法完全得到抑制。近年来研究表明，肿瘤细胞线粒体中，Hsp90分子相关肿瘤坏死因子受体蛋白TRAP1被认为是肿瘤细胞转移无法完全得到抑制的原因。我们前期研究发现TRAP1分子在转移性肠癌中存在表达差异，TRAP1分子与miRNA结合蛋白Ago2发生免疫共沉淀，并影响肠癌细胞的粘附、迁移能力，提示TRAP1分子可能是影响肿瘤细胞转移的重要因素，但其具体的作用机制尚不清楚。本课题拟选择两种不同转移潜能肠癌细胞，SW480/SW620，通过干预细胞TRAP1表达等方法，观察TRAP1对肠癌细胞转移的生物学行为的影响；探讨TRAP1分子表达及靶基因转录后与Ago2分子的相互作用；并研究TRAP1对EMT转移相关分子的作用，阐明TRAP1分子对肿瘤细胞转移的作用机制及途径，为今后临床有效治疗肠癌寻找相应的治疗靶点提供依据。

构建了HCT116和HCF-8的TRAP1过表达细胞株，构建了SW620和HCT116 的TRAP1干扰细胞株，使用构建的四种细胞株，进行了细胞侵袭，迁移的功能学研究，通过WB、QPCR和免疫荧光技术，对TRAP1与EMT相关基因表达水平的改变进行了研究。使用裸鼠进行了TRAP1基因高表达或干扰细胞株的体内成瘤情况研究，并采用人群肠癌和癌旁组织进行了HE和免疫组化研究，发现在肠癌组织中TRAP1高表达。通过研究完成了 TRAP1 分子对肠癌细胞生物学行为的影响及 EMT 相关基因表达水平的改变， 阐明 TRAP1 对肠癌细胞转移的作用及途径。 对TRAP1 与 Ago2 分子相互作用关系进行了初步的探讨，初步分析 TRAP1 分子影响肠癌细胞转移作用机制。通过体内外实验研究，我们发现在肠癌中TRAP1高表达是与EMT密切相关的重要基因，为今后肠癌临床治疗寻找相应治疗靶点提供了充分的科学依据。