应用微阵列芯片研究蛋白质O-GlcNAc糖基化在肿瘤发生中的作用及其机制

Protein O-GlcNAc glycosylation is a new type of post-translational modification of proteins.It plays an essential role in various cellular processes including transcription, stress response, apoptosis, signal transduction, glucose sensing, and proteasomal degradation. It has been demonstrated that O-GlcNAc modification is closely related to some fatal diseases such as cancers. Since the lack of an efficient technology for O-GlcNAc analysis, the role of O-GlcNAc glycosylation in tumor progression is largely unknown till now. Based on the applicant's research experience in protein microarray chip, this project will first develop a powerful platform for a rapid, sensitive, specific, and high-throughput analysis of O-GlcNAc modification. Then,the array biochip-based tecnhonogy will be used to detect the level of O-GlcNAc modification in normal and prostate cancer cells, study the expression and O-GlcNAc glycosylation of tumor-related proteins, and investigate the effect of O-GlcNAc modification on expression and/or phosphorylation of key proteins such as FoxM1, AKT, PI3K, ERK, cyclin D1, Ki67, MMP-2, and MMP-9 in signaling pathways, which are related to cell viability, proliferation, metastasis,and apoptosis.Through above studies, we try to understand the role of O-GlcNAc modification in prostate cancer progression and its molecular mechanism.In a word, this project is not only very critical for understanding the fundamentals of protein O-GlcNAc glycosylation in tumor progression, but provides useful information for targeted tumor therapy and drug discovery.

蛋白O-GlcNAc糖基化修饰是新发现的一种重要的蛋白质翻译后修饰，在细胞生命活动中发挥极其重要的调控作用，近年来日益成为国内外研究的热点。尽管已有研究证明它与肿瘤密切相关，但是目前对O-GlcNAc修饰在肿瘤发生发展中的作用及调控机制知之甚少。本项目结合申请人在蛋白质微阵列芯片领域的研究基础，开发一种快速、灵敏、高特异性和高通量的O-GlcNAc糖基化芯片分析方法，并以不同类型的前列腺癌细胞系为模型，分别研究：O-GlcNAc修饰水平与前列腺癌发生和侵袭的关系；肿瘤相关蛋白的表达及O-GlcNAc糖基化；O-GlcNAc修饰对肿瘤细胞存活、增殖、迁移、凋亡相关信号通路上关键蛋白的表达和磷酸化的影响，以期掌握O-GlcNAc修饰在前列腺癌发生过程中的作用及调控机制。该项目的成功实施不仅可以丰富蛋白质糖基化和肿瘤生物学领域的基础理论，而且为肿瘤的靶向治疗和药物开发提供科学依据。

O-连接N-乙酰葡萄糖胺（O-linked-N-acetyglucosamine, O-GlcNAc）糖基化是一种重要的蛋白质翻译后修饰，它几乎参与了细胞的所有生物学功能。然而，受现有分析手段的制约，O-GlcNAc修饰相关领域的研究进展十分缓慢。本项目旨在开发灵敏、简单、高特异性的O-GlcNAc的检测方法，并进一步探讨O-GlcNAc糖基化修饰水平的改变与肿瘤细胞生物学效应的关系。在本项目的资助下，本课题组开发了包括微阵列生物芯片法、基于金纳米棒的表面等离子共振法、金纳米粒催化铜沉积介导的高灵敏比色法、基于微流控芯片的原位免疫荧光法等4种检测方法。应用上述方法对正常前列腺细胞和前列腺癌细胞内总O-GlcNAc糖基化修饰水平及O-连接N-乙酰葡萄糖胺转移酶（OGT）表达水平进行了分析评价，并进一步应用蛋白质微阵列生物芯片研究了特定肿瘤相关蛋白的O-GlcNAc修饰水平。另外，通过加入O-连接N-乙酰葡萄糖胺水解酶(OGA)抑制剂提高细胞内O-GlcNAc修饰水平研究了糖基化异常与细胞的生长、粘附和迁移的关系。结果显示：癌细胞内总O-GlcNAc糖基化水平和特定肿瘤相关蛋白的O-GlcNAc糖基化水平均显著高于正常细胞；同时癌细胞内OGT的表达水平也高于正常细胞；O-GlcNAc糖基化水平升高可抑制细胞增殖、粘附和迁移。本课题的实施建立了若干O-GlcNAc糖基化分析技术并探讨了其修饰水平与肿瘤细胞的生物学效应关系，对推动糖蛋白质组学的发展和掌握O-GlcNAc修饰在肿瘤发生发展过程中的作用具有重要意义。