LYRM2激活Wnt信号通路调控大肠发育和大肠癌起始的分子机制研究

It is believed that colorectal cancer stem cell (CCSC) are closely associated with the initiation, relapse and metastasis of colorectal cancer (CRC). Therefore, clarifying the molecular mechanisms of malignant phenotype of CCSC is the basis for improvement of diagnosis and treatment of CRC. Abnormal activation of Wnt signaling pathway is one of the most critical events in the development and progression of CRC. Our group firstly demonstrated that LYRM2 was upregulated in CCSC. Further experiment verified that LYRM2 could significantly activate Wnt signaling pathway, and promote the metastasis and tumorgenesis of CCSC. However, the molecular mechanisms of Wnt activation by LYRM2 and the role of LYRM2 in CRC development remain unclear. Based on previous study, this project aims to analyze the localization of LYRM2 using the whole-mount in situ hybridization technology in zebrafish, identify targeting Wnt molecules regulated by LYRM2 using co-immunoprecipitation and protein mass spectrometry assay, and to verify clinical significance of LYRM2 in the CRC initiation by use of GUT-GFP transgenic zebrafish model and LYRM2 knockdown zebrafish model. We aim to explore the possibility of LYRM2 and its regulatory molecules as therapeutic targets of CRC. This research will help to enrich knowledge of CCSC and has vital scientific significance and potential clinical value.

大肠癌的起始、转移及复发过程与大肠癌干细胞（CCSC）密切相关，阐明CCSC恶性表型的分子机制是提高大肠癌诊治水平的基础。Wnt信号通路的激活是肠道发育也是大肠癌发生发展过程中最关键的事件之一。我们前期研究首次发现LYRM2在CCSC中高表达，可显著激活Wnt信号通路，更证实LYRM2可促进大肠癌的转移、致瘤等干性能力。LYRM2激活Wnt的分子机制、肠道发育及大肠癌起始中的功能尚不明了。本课题在研究基础上，拟以斑马鱼整体原位杂交及共定位等技术进行LYRM2的定位分析；通过免疫共沉淀、蛋白质质谱等筛选鉴定LYRM2调控的Wnt相关分子；并以GUT-GFP转基因斑马鱼和Talen技术建立LYRM2敲除斑马鱼模型，从大肠癌起始的角度进一步阐明LYRM2的临床生物学意义，由此探讨LYRM2及其作用分子作为大肠癌治疗靶点的可能性，进一步丰富CCSC理论，具有重要科学意义和潜在临床应用价值。

大量研究表明肿瘤干细胞与肿瘤的发生、转移密切相关。先前大肠癌的研究中，课题组筛选出LYRM2为新的肿瘤干细胞相关分子，本项目在先前研究基础上，1、基于GEO上的芯片数据，分析了LYRM2在大肠癌、胃癌、肝癌、肺癌、乳腺癌中的表达情况，结果示其在肿瘤中呈现明显低表达；2、证实LYRM2能明显促进大肠癌的运动能力；3、甲基转移酶抑制剂处理大肠癌细胞系后real time PCR检测示LYRM2表达水平明显上升，提示其在肿瘤组织中的低表达与甲基化相关；4、通过Talen技术以及morpholino建立LYRM2敲除的斑马鱼模型，证实LYRM2并不改变肠道的早期胚胎发育阶段，并且整体原位杂交数据证实LYRM2在胚胎的早期发育阶段中高表达于多个组织、器官；5、免疫共沉淀技术-质谱分析LYRM2 的相互作用蛋白，从中筛选出Wnt信号通路相关分子；6、明确低密度脂蛋白胆固醇水平与大肠癌肝转移呈正相关，更进一步的体内外研究示胆固醇可促进大肠癌细胞的克隆球形成能力、运动迁移能力、干性相关基因的表达水平、致瘤能力；7、明确大肠癌组织中的LDLR水平明显升高，并且LDLR水平与大肠癌TNM分期中的N、M正相关。通过本研究可为大肠癌的诊断、治疗提供新标记物、靶点，具有较大的科学意义与临床转化应用价值。