食管癌外泌体介导的恶性表型研究及肿瘤转移相关基因1（MTA1）相关外泌体的定量蛋白质组学分析

发布时间：2018-06-23 03:47

本文选题：MTA1 + 外泌体　；参考：《北京协和医学院》2017年硕士论文

【摘要】：目的探讨食管癌外泌体介导的肿瘤细胞之间的恶性表型传递以及MTA1对食管癌细胞外泌体特性及蛋白成分影响。方法采用差速超速离心法提取KYSE410细胞外泌体,通过透射电镜及免疫印迹法(Western blotting)观察所获得的外泌体的形态及其标志物;共聚焦显微镜观察KYSE410、KYSE510和YES2细胞摄取荧光染料标记的KYSE410外泌体;分别采用transwell实验、划痕愈合实验、细胞增殖实验、克隆形成实验检验KYSE410细胞外泌体对3种食管癌细胞迁移、侵袭、增殖和克隆形成的影响;免疫印迹法检测Wnt/β-catenin和PI3K/Akt信号通路相关蛋白的变化。分别对MTA1共表达基因和MTA1 Co-IP质谱相互作用蛋白作聚类分析;Nanosight检测不同MTA1表达水平的食管癌细胞分泌的外泌体粒径大小;Label-free质谱方法检测不同MTA1表达水平的食管癌细胞的外泌体蛋白并对差异蛋白作GO功能聚类分析;通过CCLE数据库和cBioPortal平台数据分析MTA1和目标差异蛋白在组织、细胞水平的相关性;分析目标差异蛋白与临床病理特征的相关性。结果透射电镜可以观察到具有膜结构的外泌体;免疫印迹方法能够检测到外泌体标志蛋白CD63和CD81;3种细胞均能够成功摄取荧光染料标记的KYSE410外泌体;3种食管癌细胞的迁移及侵袭能力从大到小依次为KYSE410、KYSE510、YES2,KYSE410外泌体能够促进3种食管癌细胞迁移、侵袭及克隆形成,但不影响3种食管癌细胞的增殖;外泌体处理能够增强3种食管癌细胞中β-catenin和p-Akt的表达。MTA1共表达基因聚类分析显示MTA1影响囊泡组织与生物合成;MTA1 Co-IP质谱相互作用蛋白聚类分析显示绝大多数MTA1相互作用蛋白在外泌体中有定位;不同MTA1表达水平的KYSE410细胞和KYSE450细胞分泌的外泌体的大小和数量没有显著差异;首次鉴定出25个新的外泌体蛋白成分;CytoScape软件分析两组外泌体蛋白连接度最高的节点均在UBC蛋白;对照/敲除MTA1的KYSE410细胞获得外泌体蛋白和对照/过表达KYSE450细胞获得的外泌体蛋白都主要定位在胞浆、细胞核、溶酶体、质膜等部位,主要的分子功能包括催化活性、分子伴侣活性、细胞骨架蛋白结合、GTPase活性等,主要影响的生物学进程包括蛋白代谢、能量通路、细胞生长和维持、免疫反应等;广泛参与整联蛋白家族细胞表面相互作用、β1整联蛋白细胞表面相互作用、蛋白多糖syndecan介导的信号事件等;对照/敲除MTA1的KYSE410细胞和对照/过表达KYSE450细胞的外泌体内MTA1调控的差异蛋白共同参与大分子代谢负调控、磷酸盐代谢、大分子分解代谢等生物学进程;MYH9、SLC7A5和CCT4均与MTA1在组织水平有中等强度相关性;MYH9、CCT4和食管癌病人T分期、临床分期有相关性;SLC7A5、CCT4高表达的食管癌病人总生存时间更短。结论高侵袭性食管癌细胞KYSE410来源的外泌体能够促进自身以及食管癌KYSE510、YES2细胞的迁移和侵袭、克隆形成,其可能是通过激活Wnt/β-catenin和PI3K/Akt信号通路发挥上述作用的。聚类分析结果表明MTA1可能参与外泌体调控;MTA1不影响食管癌细胞分泌外泌体的大小和总量;但MTA1影响食管癌细胞外泌体的蛋白质组成;关键的差异蛋白MYH9、SLC7A5、CCT4可能作为食管癌的预后评价指标。
[Abstract]:Objective to investigate the malignant phenotype transmission between tumor cells mediated by esophageal carcinoma and the effects of MTA1 on the extracellular secretory and protein components of esophageal cancer cells. Methods the extracellular secretory of KYSE410 cells was extracted by differential velocity centrifugation, and the morphology of the exosecrete obtained by transmission electron microscopy and immunoblotting (Western blotting) was observed and the morphology of the exosecrete was observed by transmission electron microscopy and immunoblotting (blotting). Its markers; confocal microscopy (confocal microscopy) to observe the KYSE410 Exocyst marked by KYSE410, KYSE510 and YES2 cells with fluorescent dye; Transwell experiment, scratch healing experiment, cell proliferation experiment, and clone formation test were used to test the effect of KYSE410 cell exocrine on the migration, invasion, proliferation and cloning of 3 kinds of esophageal cancer cells. The changes of Wnt/ beta -catenin and PI3K/Akt signal pathway related proteins were detected by trace method. The MTA1 coexpression gene and MTA1 Co-IP mass interaction protein were cluster analysis respectively; Nanosight was used to detect the outer secretory particle size of esophageal cancer cells with different MTA1 expression levels, and the Label-free spectrum method was used to detect the esophagus with different MTA1 expression levels. The exocrine protein of cancer cells was analyzed by GO function cluster analysis, and the correlation of MTA1 and target differential protein in tissue and cell level was analyzed by CCLE database and cBioPortal platform data, and the correlation between target differential protein and clinicopathological features was analyzed. The immunoblotting method can detect the exosecreting protein CD63 and CD81, and 3 kinds of cells can successfully absorb the KYSE410 exote marked by fluorescent dyes; the migration and invasion ability of the 3 esophageal cancer cells from large to small are KYSE410, KYSE510, YES2, and the KYSE410 exote can promote the migration, invasion and cloning of 3 kinds of esophageal cancer cells. The proliferation of 3 esophageal cancer cells was not affected; exosecretory treatment enhanced the expression of.MTA1 co expression genes in 3 esophageal cancer cells and.MTA1 co expression gene cluster analysis showed that MTA1 affects vesicle tissue and biosynthesis; MTA1 Co-IP mass spectrometry interaction protein cluster analysis showed that most of MTA1 interacting proteins were determined in exosecrete. There was no significant difference in the size and quantity of exocrine secreted by KYSE410 and KYSE450 cells at different levels of MTA1 expression; 25 new exocrine proteins were identified for the first time; CytoScape software analyzed the nodes with the highest degree of exocytosis of the two groups in UBC protein; and the exocrine protein was obtained for KYSE410 cells that were illuminated / knocked out of MTA1. The exosecreting proteins obtained from the controlled / overexpressed KYSE450 cells are mainly located in the cytoplasm, nucleus, lysosome, plasma membrane and other parts. The main molecular functions include catalytic activity, molecular chaperone activity, cytoskeleton binding, and GTPase activity. The main effects of biological processes include protein metabolism, energy pathway, cell growth and dimension. Holding, immune responses, and so on; widely involved in the cell surface interaction of the integrin family, the surface interaction of the beta 1 integrin cells, the signaling events mediated by the proteoglycan syndecan, and the negative modulation of the MTA1 KYSE410 cells and the control / overexpressed KYSE450 cells in the MTA1 regulation of the exocrine KYSE450 cells. Biological processes such as control, phosphate metabolism, macromolecular catabolism and other biological processes; MYH9, SLC7A5 and CCT4 have moderate correlation with MTA1 at the tissue level; MYH9, CCT4 and esophageal cancer patients have a correlation of T staging and clinical staging; SLC7A5 and CCT4 high expression of esophageal cancer have shorter total survival time. Conclusion high invasive esophageal cancer cells are of KYSE410 origin. Exosecrete can promote the migration and invasion of KYSE510 and YES2 cells in the carcinoma of the esophagus and the formation of the clone formation, which may be played by activating the Wnt/ beta -catenin and PI3K/Akt signaling pathway. Cluster analysis results show that MTA1 may be involved in exocrine regulation; MTA1 does not affect the size and total amount of exocrine secreted by cancer cells; but MTA 1 the protein composition of exocrine cells in esophageal cancer may be affected. The key differentially expressed proteins MYH9, SLC7A5 and CCT4 may be used as prognostic indicators for esophageal cancer.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.1

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