BEX2通过Mcl-1调控Hedgehog通路维持大肠癌干细胞干性特征的分子机制研究

Studies have shown that colorectal cancer is closely related to colorectal cancer stem cells (CCSC). CCSC has the features of self-renewal, differentiation potential and strong tumorigenesis, and resistance to chemo-therapy drugs. Therefore, to clarify the mechanism involved in maintaining the characteristics of CCSC is important to target CCSC selectively. Our previous study had firstly found that silencing BEX2 can promote the stemness in CCSC , including forming colo-sphere, promoting metastasis, enhancing drug resistance and higher tumorigenesis, which indicates that BEX2 may be one of the important molecular promoting the stemness of CCSC. However, the molecular mechanisms are unknown. Based on our previous study that BEX2 maintains the CCSC stemness by regulating Hedgehog pathway, and the results that Mcl-1, a Hedgehog inhibiting protein, interacts with BEX2, we hypothesize that BEX2 regulates the Hedgehog pathway maintaining the CCSC stemness by Mcl-1. This project aims to further explore the biological functions of BEX2 in CCSC, and clarify the key binding site between BEX2 and Mcl-1 by multiple biological techniques such as molecular docking, gene mutation and fluorescence reporter gene system. By completing this project, we will able to provide a new target for CCSC target therapy.

研究表明大肠癌发生发展与大肠癌干细胞(CCSC)密切相关。CCSC具有自我更新能力、多向分化潜能、治疗抵抗及强致瘤性等干性特征，因此阐明维持CCSC干性特征分子机制是靶向清除CCSC的基础。我们前期研究首次发现BEX2沉默后可增强CCSC克隆球形成、转移侵袭、化疗耐药和致瘤性等干性特征，提示BEX2是维持CCSC干性特征的重要分子，但机制尚不明了。进一步研究发现BEX2通过调控Hedgehog通路维持CCSC干性特征，结合前期研究发现BEX2与Hedgehog通路抑制蛋白Mcl-1相互结合，提示BEX2通过Mcl-1调控Hedgehog通路维持CCSC干性特征。本课题拟在前期研究基础上，进一步完善BEX2在CCSC中的功能，利用分子对接、基因突变和荧光报告基因技术明确BEX2与Mcl-1的关键结合位点和调控方式，由此解析BEX2维持CCSC干性特征的分子机制，为CCSC靶向治疗提供新靶点。

前期研究发现BEX2沉默后可增强大肠癌干细胞克隆球形成、转移侵袭、化疗耐药和致瘤性等干性特征，提示BEX2是维持大肠癌干细胞干性特征的重要分子，在此基础上，我们进一步构建BEX2敲除及过表达肠癌细胞系，同时检测BEX2不同水平的肠癌细胞的干性性能，发现BEX2敲除细胞系克隆成球能力，迁移侵袭能力和化疗耐药性均增强，BEX2过表达后干性特性减弱，同时，体内实验中发现BEX2敲除细胞系皮下成瘤率更高，并且我们在肠癌组织中验证发现BEX2低表达的患者无病生存时间较高表达组更短。进一步通过高通量测序对比BEX2敲除前后肠癌细胞系的转录水平基因表达情况，我们利用GO分析、KEGG信号通路分析发现Hedgehog信号通路关键基因显著激活。接着，我们利用Hedgehog通路的抑制剂GDC0449作用于BEX2敲除肠癌细胞系后发现，GDC0449抑制剂可以逆转BEX2低表达对肠癌细胞干性特征的影响。在前期基础中我们通过酵母双杂交技术筛选提示MCL1与BEX2基因存在相互作用，本研究中进一步在肠癌细胞系中通过免疫共沉淀和免疫荧光定位的方法证实BEX2与MCL1存在相互作用，因此，我们成功构建MCL1-Flag质粒，并在BEX2低表达细胞系上构建Mcl1过表达细胞系，并通过使用CHX和MG132，证实BEX2可促进泛素-蛋白酶体介导的MCL1发生降解，而BEX2低表达肠癌细胞系中敲除MCL1后，发现敲除BEX2的肠癌细胞系低表达MCL1后可抑制其干性特征的表达，以上结果证明BEX2通过MCL1调控Hedgehog通路维持大肠癌干细胞干性特征，为BEX2调控肠癌细胞的干性特征提供理论依据，提示BEX2和MCL1可作为肠癌耐药的潜在治疗靶点，为肠癌诊治提供一种新的治疗方案。