高脂饮食通过增加肠道胆酸激活VDR-FOXK2通路促进结直肠癌发病的机制研究

It has been recently reported that high-fat diet promotes carcinogenesis of colorectal cancer, but the underlying mechanisms is not fully elucidated. We have also observed increased tumor incidence of high-fat diet-fed mice in an AOM-induced intestinal tumor model. Since most studies suggested the intestinal stem cells located at the base of intestinal crypt was the origin of colorectal cancer, we performed RNA-seq of isolated crypts and found that FOXK2 expression was elevated in high-fat diet-fed mice. FOXK2 has been reported to enhance nuclear translocation and transactivity of β-catenin, which is critical for the intestinal stem cell homeostasis maintenance. Bioinformatics analysis showed the existence of vitamin D receptor (VDR) binding site at the promoter region of FOXK2. VDR, in addition to response to active vitamin D, is also a natural receptor for the secondary bile acid lithocholic acid (LCA). It has been reported that high-fat diet increase intestinal LCA concentration and excessive LCA causes DNA damage. Taking all the evidence together, we hypothesize that increased intestinal LCA concentration resulting from high-fat diet promotes colorectal cancer carcinogenesis by directly causing DNA damage and at the same time promoting intestinal stem cell proliferation which accumulates the DNA damage. To test the hypothesis we will employed the AOM-induced intestinal tumor animal model and in vitro Organoid culture system. The accomplishment of this project helps understand why and how high-fat diet contributes to increased colorectal cancer risk and develop novel intervention methods.

近年来陆续有研究证实高脂饮食增加结直肠癌发病，但其机制尚未完全阐明。我们的预实验结果也显示高脂饮食增加了AOM诱导的小鼠肠道肿瘤。由于主流观点认为肠癌起源于肠道隐窝干细胞，我们分离高脂饮食小鼠隐窝细胞进行RNA测序发现既往报道可以通过增强β-catenin活性调控肠道干细胞增殖的FOXK2表达升高。生物信息学预测FOXK2启动子区存在转录因子VDR的结合序列。VDR是石胆酸受体，预实验显示石胆酸处理可以促进体外培养Organoid表达FOXK2。文献报道高脂饮食可以升高肠道石胆酸，而石胆酸可以损伤细胞DNA。据此，我们提出高脂饮食导致肠道石胆酸升高造成肠道干细胞DNA损伤，并通过VDR诱导FOXK2表达促进肠道干细胞增殖累积DNA损伤从而促进结直肠癌发病的假说。我们将综合采用动物、Organoid等模型对这一假说进行验证。该项目有利于深入理解高脂饮食促进结直肠癌的机制并提供相应干预靶点。

高脂饮食和肥胖是结肠癌发生的高危因素，但是其致癌的分子机制尚未完全阐明。根据文献回顾和本课题组前期工作基础，我们提出高脂饮食导致肠道石胆酸升高造成肠道干细胞DNA损伤，并通过VDR诱导FOXK2表达促进肠道干细胞增殖累积DNA损伤从而促进结直肠癌发病的假说。针对这一假说，我们采用AOM诱导的结肠癌小鼠动物模型、肠道类器官体外培养模型以及荧光素酶报告基因等一系列分子生物学实验方法开展了以下研究：1.对比AOM结肠癌造模小鼠普通饮食±石胆酸以及高脂饮食±胆酸螯合剂条件下结肠癌的发生情况，结果表明高脂饮食和石胆酸可以促进小鼠结肠肿瘤的发生，胆酸螯合可以逆转上述效应；2.石胆酸短期处理可以抑制肠道类器增殖并促进其死亡，但是部分类器官可以克服石胆酸的毒性作用得以长期快速增殖，机制研究显示p53表达缺失与石胆酸的协同作用在这一过程中发挥关键作用；3.逃逸石胆酸毒性作用的肠道类器官形成次级类器官的能力增强且在分化培养基中分化不良，表明其干细胞特性增强，进一步研究显示石胆酸通过其天然受体VDR促进FOXK2表达是其关键分子机制。通过上述研究我们阐明了石胆酸通过VDR-FOXK2信号通路调控肠道干细胞干性并与p53功能缺失协同促进结肠癌发生的分子机制。除了上述理论成果，我们还改良了本项目所涉及的类器官基因操作的关键技术，建立的2D-3D续贯基因操作策略将传统方法的1-2周缩短至10小时，同时极大的提高了基因投递操作的效率。同时，我们还建立了用于肠道类器官培养的一系列工具细胞。上述研究为后续开展以类器官为模型的研究奠定了丰富的技术和资源基础。