利用肠道特异性FBX8条件敲除小鼠探讨FBX8在结直肠癌侵袭和转移中的作用机制

In our previous study, we found that FBX8 was a key functional target of metastasis promoting miR-223. FBX8 was down-regulated in colorectal cancer and suppressed cell proliferation and invasion of colorectal cancer. Based on the bioinformatic analyses and results of preliminary experiment, we proposed a novel regulatory mechanism of FBX8 in tumor metastasis: FBX8 is transcriptionally regulated by SOX17 and inhibits invasion and metastasis of colorectal cancer by targeting mTOR for ubiquitin degradation. This study will validate this mechanism by using cell lines,nude mouse, transgenic mouse and tissue sample (1) To reveal the role of FBX8 in proliferation, invasion and metastasis (including intravasation, extravasation and colonization at the metastatic sites) of colorectal cancer; (2) To verify that SOX17 transcriptionally regulates FBX8 and FBX8 targets mTOR for ubiquitin degradation and inhibits the expressions of downstream target genes; (3) To detect the effect of ubiquitin degradation of mTOR in FBX8-induced invasion and metastasis of colorectal cancer; (4) To establish the gene knockout model of tumorigenesis and spontaneous metastasis that is consistant with colorectal cancer progression and validate the novel mechanism of FBX8. Our study aims to give new clues for molecular mechanism of tumor metastasis, and provide novel target and animal model for clinical anti-metastasis.

前期工作中我们发现FBX8是转移促进因子miR-223的一个重要功能靶基因，FBX8在结直肠癌组织中表达下调，抑制结直肠癌细胞的增殖和侵袭能力。根据生物信息学分析及预实验结果，我们提出FBX8可能参与肿瘤转移调控的假设：即FBX8可能在SOX17转录调控下，通过泛素化降解mTOR抑制结直肠癌侵袭和转移。为证实这一假设，本研究拟利用细胞株、临床标本、裸鼠及转基因小鼠模型等技术；（1）明确FBX8在结直肠癌增殖、侵袭、各阶段转移（侵入血管、侵出血管及转移部位增殖）中的作用；（2）验证SOX17对FBX8的转录调控及FBX8对mTOR的泛素化降解并抑制下游靶基因；（3）检测mTOR泛素化降解对FBX8诱导结直肠癌侵袭和转移中的作用；（4）建立与结直肠癌演进规律相一致的肿瘤发生和（或）自发转移基因敲除小鼠模型并进行验证。为肿瘤转移机制研究提供新思路，为肿瘤转移的诊治提供新的靶点和理想的动物模型。

结直肠癌是当今世界对人类生存和健康构成极大威胁的恶性肿瘤之一，在各种恶性肿瘤中发病率高居第三位。在我国，结直肠癌的发病率和死亡率有逐年上升的趋势，治疗失败的主要原因是术后的局部复发和远处转移。阐明结直肠癌转移分子机制，鉴定出其特异性分子标志物是结直肠癌转移研究的关键科学问题。我们通过荧光素酶报告系统证实FBX8是miR-223的下游靶基因；通过免疫荧光及CO-IP验证了FBX8与mTOR存在直接相互作用，可通过泛素化降解mTOR调控mTOR信号通路；通过体内外功能试验实验证实FBX8调控结直肠癌的增殖、侵袭及转移；通过免疫组化证实结直肠癌组织FBX8表达水平与与肿瘤分化程度和病人预后存在相关性；利用基因敲除技术成功构建了肠道特异性FBX8 f/f，FBX8 f/+小鼠模型。本项目提出了一个FBX8参与结直肠癌侵袭及转移中的新机制，即FBX8 通过泛素化降解 mTOR抑制结直肠癌侵袭和转移，为全面理解 FBX8 在肿瘤侵袭和转移中的功能提供科学依据，并为结直肠癌演进的诊治、预后评估、药物筛选等提供有效的分子靶点和理想的动物模型。