β-1,4半乳糖基转移酶3表达升高引起的异常糖基化修饰对胃癌发展的作用机制研究

Abnormal glycosylation of proteins is associated with occurrence and development of tumors. Our previous study demonstrated that the expression of glycosyltransferase B4GALT3 and the glycopattern of LacNAc which was synthesized by this glycosyltransferase were up-regulated significantly in gastric cancer. Moreover, the glycopatterns of LacNAc on β1-integrin also showed increased in tumor progression. However, the mechanism of transcription regulation and function of aberrant glycosylation for the progression of gastric cancer is remains unknown yet. This project intends to unravel the mechanism of abnormal glycosylation caused by up-regulated of B4GALT3 for the progression of gastric cancer with the following research: ChIP and luciferase assay will be used to investigate the regulatory mechanism of B4GALT3 in gastric cancer. To understand the function of the aberrant glycosylation of β1-integrin in tumor development, Lectin blotting and MS will be used to character the altered of glycan on β1-integrin. The influence of the alteration on interaction between cell-ECM, formation of integrin dimer as well as the activation of β1-integrin and integrin signal pathway will be investigated. In addition, the B4GALT3 will be knocked down or over-expressed in gastric cell lines, the level of protein glycosylation and signal pathways associated with metastasis will be investigated, and EMT(Epithelial-mesenchymal transition), VM(vasculogenic mimicry), cytoskeletal rearrangement, anoikis and the abilities of adhesion, infection and migration will be evaluated. Furtherly, the gastric cancer mouse model will be constructed to investigate the role of abnormal glycosylation in development of gastric cancer in vivo. This will provide us a new insight into function of the abnormal glycosylation in development of gastric cancer.

蛋白质的异常糖基化修饰对肿瘤的发生发展具有重要作用，我们前期研究证实β-1, 4半乳糖糖基转移酶3（B4GALT3）及其合成的N-乙酰乳糖胺在胃癌组织和细胞中表达均显著升高，并与中晚期胃癌及淋巴结转移相关，但其相关的调控机制及功能尚不清楚。本课题拟在此基础上，深入研究B4GALT3表达升高引起的异常糖基化修饰的转录调控机制及功能。通过筛选B4GALT3基因表达调控相关的转录因子及信号通路，发掘异常糖基化修饰产生的转录调控机制；构建细胞及动物模型，分析B4GALT3介导β1-整合素上糖基化修饰改变对细胞连接，整合素异二聚体形成以及整合素及其相关信号通路激活的影响；结合糖组学、细胞生物学以及分子遗传技术评估异常糖基化修饰在胃癌发生进程中的作用。本研究将阐明胃癌发展过程中B4GALT3表达升高引起的异常糖基化产生的分子机制及对胃癌发生发展的作用，为胃癌的治疗和抗肿瘤药物的研发提供新思路和新靶点。

胃癌是全球最常见的五种恶性肿瘤之一，严重威胁着人类的生命安全。蛋白质的异常糖基化修饰对肿瘤的发生发展具有重要作用，我们前期的研究证实β-1, 4半乳糖糖基转移酶3（B4GALT3）及其合成的N-乙酰乳糖胺（LacNAc）在胃癌组织和细胞中表达均显著升高，并与中晚期胃癌及淋巴结转移相关，但其相关的调控机制及功能尚不清楚。本项目通过建立B4GALT3基因沉默/过表达稳转细胞，确定B4GALT3表达影响胃癌细胞中LacNAc的合成。虽然B4GALT3表达对胃癌细胞的增殖能力无明显影响，但是与胃癌细胞的迁移及侵袭能力显著相关。进一步的分析发现，B4GALT3催化整合素β1的LacNAc糖基化修饰进而促进细胞的迁移和侵袭能力并增强细胞克隆形成能力。我们的研究提示，整合素β1的LacNAc糖基化修饰不仅能够用于肿瘤细胞转移潜能的评估，而且为胃癌治疗药物及单克隆抗体研发提供有效的靶点。对B4GALT3的转录调控研究发现，miR-27a-3p通过结合B4GALT3的3’UTR调节B4GALT3表达，进而影响胃癌细胞的迁移及侵袭能力。在整个工作进展期间，根据项目课题组发展情况以及国内外相关的研究进展，我们适当调整了研究策略，在发挥优势技术的基础上进一步建立了全新的肿瘤相关糖鞘脂糖链的分析方法，与传统的质谱技术相比，该方法能够简便快捷的分析糖鞘脂糖链类型。与此同时，我们以唾液为研究对象，筛选及鉴定乳腺癌，食管鳞细胞癌及肺癌患者唾液异常糖蛋白糖链，评估唾液异常糖蛋白糖链在肿瘤辅助诊断中的应用价值。我们的研究显示，唾液糖蛋白糖链在肿瘤的早期诊断中具有巨大的应用潜力，并且该方法以唾液为检测材料，具有取材方便，并且对患者无侵入性损伤等优势。在过去四年间本项目取得了一系列重要成果，相关的研究工作分别发表在《Nature protocol》等杂志上，目前共计发表SCI论文11篇，申请发明专利2项，培养4名博士研究生及2名硕士研究生，总体完成了预期设定的目标。