【作者】 周勇；

【导师】 周东辉；

【作者基本信息】 浙江大学 ， 肿瘤学， 2019， 硕士

【摘要】 背景胃癌是导致人类死亡最主要的恶性肿瘤之一,据统计2018年全世界胃癌病人的发病率和死亡率均位列所有恶性肿瘤前五位。在我国,胃癌患者确诊时往往已经是中晚期,现有的治疗手段效果有限,因此亟需探索新的发病机制和寻找早期诊断标志物及治疗靶点。近年来由于质谱技术的出现及快速发展,蛋白质组学已大规模地在肿瘤研究中应用,为探索肿瘤的发病机制及寻找早期诊断标志物和治疗靶点提供了新的有力手段。本研究通过质谱技术,采取无标定量蛋白组学的方法来探索胃癌新的发病机制及潜在生物标志物。方法本课题通过收集胃癌患者的14例胃癌组织及6例胃正常粘膜上皮组织,利用质谱技术和无标记定量蛋白组学方法获得两者间的差异表达蛋白谱,并对这些蛋白进行生物信息学功能注释及相关分析。之后对候选的生物标志物进行蛋白印迹和免疫组织化学染色等多层次技术手段加以验证其具体表达水平。利用小干扰RNA、CCK8试验、流式细胞术与蛋白印迹等技术在体外细胞水平上探讨候选的生物标志物对胃癌细胞系增殖及凋亡等恶性生物学行为的影响及其相关机制。结果通过以上一系列研究,我们获得以下结果:1.通过质谱技术和无标记定量蛋白质组学方法分析胃癌组织标本,鉴定到多达3400种蛋白质,其中294种蛋白为差异表达蛋白;2.对差异表达蛋白进行生物信息学分析发现这部分蛋白中核酸结合蛋白富集显著,与RNA合成、剪切及代谢等过程密切相关;3.利用蛋白印迹及免疫组织化学染色技术证实所候选的蛋白MAGOH及MAGOHB在胃癌组织及多种胃癌细胞系中高表达,而在胃正常粘膜上皮组织及胃正常细胞系中低表达;4.在胃癌细胞系中利用小干扰RNA同时敲低这两种蛋白后发现,胃癌细胞的增殖及克隆形成受到极大抑制,凋亡比例显著增加,并且ERK1/2信号通路上的相关蛋白表达明显减少。结论通过胃癌-胃正常粘膜上皮组织的无标记定量蛋白组学研究发现MAGOH及MAGOHB蛋白在胃癌组织中高表达,这两种蛋白协同参与调节胃癌细胞的增殖及凋亡,有望成为胃癌临床诊断及靶向治疗的新型分子生物标志物。更多还原

【Abstract】 BackgroundGastric cancer is one of the most malignant tumors,and its morbidity and mortality rank among the top five in the world.In China,because the patient diagnosed as gastric cancer are often in the advanced stage,and the effect of existing treatments is limited,it is urgent to explore its underlying mechnisms and find new biomarkers and possible therapeutic targets.In recent years,due to the emergence and rapid development of mass spectrometry,proteomics has been widely applied in cancer research,providing a new powerful means for exploring the pathogenesis of cancer and finding early diagnostic biomarkers and therapeutic targets.In this study,we used mass spectrometry and label-free quantitative proteomics to explore the new mechnism and potential biomarkers for the diagnosis and therapy of gastric cancer.MethodsIn this study,14 gastric cancer tissues and 6 normal gastric tissues were collected and analysized by label-free quantitative proteomics.The bio-informatics function annotations were performed on these differentially expressed proteins.Subsequently,candidate biomarkers were verified by western blot and immunehistochemistry.Finally,using small interfering RNA,CCK8 assay,flow cytometry and Western blot to evaluate the role of candidate biomarkers in cell proliferation and apoptosis.ResultsThrough a series of experiments,we obtained the following results.First,we analyzed up to 3400 proteins and 294 differentially expressed proteins,and bio-informatics analysis revealed that the enrichment of nucleic acid-binding proteins was particularly prominent,which were also related to the process of RNA synthesis,splicing and metabolism.Then,we used Western blot and immunohistochemistry to verify that the expressions of MAGOH and MAGOHB were higher in gastric cancer tissues than normal gastric tissues.Finally,after down-regulating MAGOH and MAGOHB together in the gastric cancer cell,the cell proliferation was greatly inhibited,the rate of cell apoptosis was significantly increased,and the related proteins on the ERK1/2 signaling pathway were significantly decreased.ConclusionWe found that MAGOH and MAGOHB proteins were highly expressed in gastric cancer tissues by lable-free quantitative proteomics.Both two proteins cooperate in regulating the proliferation and apoptosis of gastric cancer cells,and are expected to become new biomarkers for clinical diagnosis and therapeutic of gastric cancer.更多还原