平分型N-糖链在乳腺癌细胞及临床样本中的分子调控及生物学功能研究

发布时间：2018-01-12 13:29

本文关键词：平分型N-糖链在乳腺癌细胞及临床样本中的分子调控及生物学功能研究　出处：《江南大学》2017年博士论文　论文类型：学位论文

【摘要】：糖基化是非常重要的蛋白质翻译后修饰之一,在多种生物过程中扮演重要角色,包括蛋白质的折叠、蛋白质的降解及细胞间的相互作用等。平分型N-乙酰葡萄糖胺(GlcNAc)结构是在β1,4-N-乙酰氨基葡萄糖转移酶Ⅲ(N-acetylglucosaminyltransferase Ⅲ,Gn T-Ⅲ or MAGT3)的催化下,将GlcNAc以β1,4-糖苷键连接到核心五糖中的甘露糖残基上形成的。蛋白质的平分型GlcNAc糖基化修饰对细胞内信号转导、细胞黏附、细胞迁移等生物过程都有重要作用。包括癌症在内的多种疾病中都存在异常的平分型GlcNAc糖基化。平分型GlcNAc结构及MGAT3与肿瘤的迁移及侵袭等过程密切相关。但在乳腺癌的发生发展过程中,平分型GlcNAc结构的变化及其生物学功能的全面性研究还鲜有报道,因此研究平分型GlcNAc结构与MGAT3有助于深入了解其在乳腺癌发生发展过程中的作用,为乳腺癌的生物标志物的研究及乳腺癌的治疗提供一定的理论基础。为此,本课题以乳腺癌为研究对象,全局性的分析平分型GlcNAc结构的含量及MGAT3的表达变化,并鉴定平分型GlcNAc糖基化修饰的糖蛋白质,研究平分型GlcNAc结构的生物学功能。主要研究结果如下:(1)EMT过程中糖组学分析。本实验首先建立了转化生长因子(TGFβ)诱导小鼠乳腺上皮(normal mouse mammary gland epithelial,NMu MG)细胞发生上皮间质转化(Epithelial-mesenchymal transition,EMT)的细胞模型,结合质谱、基因芯片及凝集素芯片等高通量技术,对EMT过程中异常糖基化及糖相关基因进行研究,并利用凝集素染色及荧光定量PCR等技术进行实验结果的验证。实验结果发现,在EMT过程中,高甘露糖型N-糖链结构含量升高;平分型GlcNAc结构含量降低;岩藻糖糖基化程度降低;N-糖相关基因表达发生显著变化,其中,糖相关基因ALG9与MGAT3表达下调,糖相关基因转录水平变化与N-糖链的变化相一致。(2)乳腺癌发生发展过程中平分型GlcNAc结构含量及MGAT3表达的异常变化。以人源、鼠源乳腺细胞株及人乳腺癌临床样本为实验材料,对乳腺癌中异常糖基化进行质谱检测,发现在乳腺癌细胞及乳腺癌临床样本中平分型GlcNAc结构含量显著降低;对含有30对乳腺癌组织的乳腺癌组织芯片进行PHA-E荧光染色,发现23对乳腺癌组织中平分型GlcNAc结构含量显著降低;利用ONCOMINE数据库、Western blot及免疫荧光染色等技术检测发现乳腺癌中MGAT3的转录水平及蛋白水平表达下调。利用在线数据库Meth HC分析发现MGAT3启动子区高甲基化,用去甲基化药物(地西他宾,decitabine)处理细胞可以大幅提高MGAT3的表达。利用在线绘制生存曲线的工具分析MGAT3对乳腺癌预后的影响,发现MGAT3高表达的乳腺癌患者无复发生存率较MGAT3低表达患者高。(3)利用PHA-E凝集素富集乳腺细胞系中平分型GlcNAc糖基化修饰的糖蛋白质,对蛋白质进行鉴定及功能解析。对人源及鼠源乳腺细胞系(人乳腺上皮细胞MCF10A、人乳腺癌细胞MCF7、SKBR3及MDA-MB-231;小鼠乳腺上皮细胞NMu MG、小鼠乳腺癌细胞4T1)中平分型GlcNAc糖基化修饰的靶蛋白进行鉴定及其进行生物信息学的分析,发现平分型GlcNAc糖基化修饰的靶蛋白有整合素(integrin)及表皮生长因子受体(EGFR)等,涉及到的信号通路有ERK、AKT等。(4)建立了一种凝集素辅助的N-糖链的分离、富集方法。利用凝集素将高峰度糖链从低峰度糖链中分离、富集出来,再对两个组分分别进行纯化、检测,以提高糖链检测的灵敏度及覆盖率。应用本方法对标准蛋白(鸡卵清白蛋白)及复杂生物样品(NMu MG细胞蛋白及人血清)中N-糖链进行分离、检测后发现此方法能够利用等量的蛋白质同时对低峰度糖链及高峰度糖链进行高灵敏度与高覆盖率的检测。
[Abstract]:Glycosylation is an important post-translational modification of proteins, play an important role in a variety of biological processes, including protein folding, protein degradation and cell interaction. N- bisecting GlcNAc transferase (GlcNAc) structure is in beta 1,4-N- acetylglucosamine (N-acetylglucosaminyltransferase III, Gn T- or MAGT3 III) under the catalysis of GlcNAc to beta 1,4- glycosidic bond to form mannose residues in the core of the five sugar. Bisecting GlcNAc protein glycosylation modification on intracellular signal transduction, cell adhesion, cell migration and other biological processes have an important role. Bisecting GlcNAc glycosylation there are a variety of unusual diseases including cancer. Closely related to the migration and invasion process of bisecting GlcNAc structure and MGAT3 with tumor. But in the development and progression of breast cancer, flat type Gl Study on the comprehensive changes in structure and biological function of cNAc has not been reported, so the study of bisecting GlcNAc structure and MGAT3 contribute to a deeper understanding of the process in its role in breast cancer development, providing a theoretical basis for the treatment of breast cancer and breast cancer biomarkers. Therefore, this topic with breast cancer as the research object, the expression change of MGAT3 content and analysis of split type GlcNAc structure of overall, sugar and protein identification bisecting GlcNAc glycosylation, biological function of bisecting GlcNAc structure. The main research results are as follows: (1) EMT in proteomics analysis. The first experiment the establishment of the transforming growth factor (TGF) induced mouse mammary epithelial (normal mouse mammary gland epithelial, NMu MG) cells of epithelial mesenchymal transition (Epithelial-mesenchymal transition, EMT) cell model, Combined with mass spectrometry, gene chip and lectin microarray high-throughput technology, research on genes related to abnormal glucose and sugar based EMT process, and the experimental results were verified by lectin staining and fluorescence quantitative PCR technique. The experimental results showed that in the EMT process, high dew sugar N- sugar chain structure was increased; reduce the content structure of bisecting GlcNAc; fucose glycosylated sugar decreased; expression of N- related genes changed significantly, including sugar related gene ALG9 and MGAT3 expression, consistent with changes of sugar related gene transcription level and N- sugar chain. (2) the abnormal expression of GlcNAc and MGAT3 split type structure content development process the occurrence of breast cancer. The source of clinical samples of murine breast cell line and human breast cancer as the experimental materials for mass spectrometric detection of aberrant glycosylation in breast cancer found in breast cancer cells and breast cancer Pro The content of GlcNAc in the sample split type bed structure decreased significantly; PHA-E staining with 30 of breast cancer and breast cancer tissue microarray, found that 23 of the bisecting GlcNAc in breast cancer tissue structure was significantly lower; using ONCOMINE database, Western blot and immunofluorescence staining techniques detected the transcription and protein level of MGAT3 in breast cancer expression. Using the online database Meth HC analysis showed that MGAT3 promoter hypermethylation, with demethylation drugs (decitabine, decitabine) cells can significantly improve the expression of MGAT3. MGAT3 analysis tools to draw survival curves available online on the prognosis of breast cancer, found no recurrence the survival rate was lower than that of MGAT3 expression in patients with high expression of MGAT3 in patients with breast cancer. (3) by bisecting PHA-E lectin enriched breast cell lines GlcNAc glycosylated protein sugar Qualitative analysis, identification and function of protein. In human and mouse mammary cells (human breast epithelial cell MCF10A, human breast cancer cell line MCF7, SKBR3 and MDA-MB-231; mouse mammary epithelial cells NMu MG mouse breast cancer cell 4T1) target protein Zhongping type GlcNAc glycosylation of identification and analysis bioinformatics, found that the target protein bisecting GlcNAc glycosylation of integrin (integrin) and epidermal growth factor receptor (EGFR) signaling pathway, involving ERK, AKT et al. (4) a separation, a lectin assisted N- sugar chain by lectin concentration method. The peak of sugar chains separated from low kurtosis sugar chain is concentrated, then the two components were purified, detection, in order to improve the sensitivity of detection of sugar chain and coverage. The application of the method of standard protein (ovalbumin (NMu) and complex biological samples The N- sugar chain was separated from MG cell protein and human serum. After detection, it was found that this method could detect the high sensitivity and high coverage of the low kurtosis sugar chain and the peak sugar chain by using the same amount of protein.

【学位授予单位】：江南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.9

【参考文献】