ACK1基因扩增调控胃癌发生和转移的机制及其与临床预后关系的研究

发布时间：2018-03-03 06:58

本文选题：ACK1　切入点：ECD　出处：《暨南大学》2016年博士论文　论文类型：学位论文

【摘要】：研究背景和意义:胃癌是目前全球高发的恶性肿瘤之一。手术切除以及放化疗是治疗胃癌患者的主要手段。胃癌早期发病症状隐匿,临床上一经确诊时,往往已经是晚期和发生了转移,从而导致胃癌患者的预后较差。靶向小分子/抗体特异抑制癌基因作为一种治疗策略受到广泛关注,此策略的关键在于找到胃癌发生和转移的靶点,以作为新的药物治疗靶标。前期研究发现ACK1基因在胃癌中可能存在扩增,提示ACK1与胃癌的发生发展相关。因此,本论文着重研究ACK1在胃癌中的临床意义以及分子机制。方法与结果:1.通过分析Oncomine数据库,发现ACK1基因的DNA拷贝数和m RNA水平在胃癌组织中显著上调。2.通过分析胃癌临床样品中ACK1蛋白的表达与预后的关系,发现ACK1蛋白在胃癌组织中上调且与胃癌患者预后差密切相关。3.通过使用Transwell小室、流式细胞仪等其它方法,我们检测了沉默ACK1和过表达ACK1后对胃癌细胞增殖、克隆形成、细胞周期、细胞凋亡、EMT以及细胞迁移侵袭能力的影响。过表达ACK1促进了胃癌细胞EMT和迁移侵袭。沉默ACK1抑制了胃癌细胞增殖、克隆形成、EMT和迁移侵袭。沉默ACK1诱导胃癌细胞发生G2/M阻滞和细胞凋亡。4.采用小鼠肿瘤异种移植裸鼠模型以及体内肺转移模型,证实稳定沉默ACK1抑制胃癌细胞的裸鼠成瘤能力和肺转移能力。5.通过采用Transwell、流式细胞仪、基因干扰等其它实验方法,证实ECD在ACK1促进胃癌细胞增殖、克隆形成、迁移侵袭以及EMT中发挥重要作用。6.采用双荧光报告基因分析、染色质免疫共沉淀技术、基因干扰技术以及其它方法,阐明了ACK1通过激活AKT-POU2F1-ECD通路促进胃癌发生和转移的分子机制。7.阐明了ACK1通过ECD依赖的方式促进p53蛋白泛素化降解。结论:1.ACK1基因扩增导致其蛋白在胃癌组织中上调,这一上调与胃癌临床预后差密切相关。2.证实了ACK1促进胃癌细胞增殖、克隆形成、EMT和侵袭转移的生物学功能。3.阐明了ACK1通过激活AKT-POU2F1-ECD通路,从而促进胃癌发生和转移。4.阐明了ACK1通过ECD依赖的方式促进p53蛋白的泛素化降解。
[Abstract]:Background and significance: gastric cancer is one of the most common malignant tumors in the world. Surgical resection and radiotherapy and chemotherapy are the main methods for the treatment of gastric cancer. The prognosis of patients with gastric cancer is often poor due to their late stage and metastasis. Targeted small molecule / antibody specific suppressor oncogenes have attracted wide attention as a therapeutic strategy. The key of this strategy is to find the target for carcinogenesis and metastasis of gastric cancer, which can be used as a new target for drug therapy. Previous studies have found that ACK1 gene may be amplified in gastric cancer, suggesting that ACK1 is related to the occurrence and development of gastric cancer. This paper focuses on the clinical significance and molecular mechanism of ACK1 in gastric cancer. Methods and results: 1. By analyzing the Oncomine database, It was found that the DNA copy number and m RNA level of ACK1 gene were significantly up-regulated in gastric cancer tissues. By analyzing the relationship between the expression of ACK1 protein and prognosis in clinical samples of gastric cancer, It was found that ACK1 protein was up-regulated in gastric cancer tissues and was closely related to the poor prognosis of gastric cancer patients. By using Transwell chamber, flow cytometry and other methods, we detected the proliferation and clone formation of gastric cancer cells after silencing ACK1 and over-expressing ACK1. The effects of cell cycle, apoptosis and the ability of cell migration and invasion. Overexpression of ACK1 promoted EMT and migration and invasion of gastric cancer cells. Silencing ACK1 inhibited the proliferation of gastric cancer cells. Silencing ACK1 induced G2 / M arrest and apoptosis of gastric cancer cells. 4. Xenotransplantation nude mice model and lung metastasis model in vivo were used. The ability of stable silencing ACK1 to inhibit tumorigenesis and lung metastasis of gastric cancer cells in nude mice was confirmed. 5. By using Transwell, flow cytometry, gene interference and other experimental methods, it was proved that ECD could promote the proliferation and clone formation of gastric cancer cells in ACK1. Migration, invasion and EMT play an important role. 6. Double fluorescence reporter gene analysis, chromatin immunoprecipitation, gene interference and other methods, The molecular mechanism of ACK1 promoting carcinogenesis and metastasis of gastric cancer by activating AKT-POU2F1-ECD pathway was elucidated. 7. It was clarified that ACK1 promoted the degradation of p53 protein ubiquitin by ECD dependent way. Conclusion: 1. The amplification of ACK1 gene leads to up-regulation of p53 protein in gastric cancer tissues. This upregulation is closely related to the poor clinical prognosis of gastric cancer .2.It is confirmed that ACK1 promotes the proliferation of gastric cancer cells, the biological function of clone formation and invasion and metastasis of gastric cancer cells. It is clear that ACK1 activates the AKT-POU2F1-ECD pathway. The results showed that ACK1 promoted the Ubiquitin degradation of p53 protein in ECD dependent manner.
【学位授予单位】：暨南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.2

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