神经导向蛋白Semaphorin 3E在胃癌发生发展中的作用和机制的研究

发布时间：2018-03-04 21:23

本文选题：Sema3E　切入点：胃癌　出处：《上海交通大学》2015年博士论文　论文类型：学位论文

【摘要】：目的:神经导向蛋白Semaphorin 3E(Sema3E)在多种肿瘤中表达上调并能够促进肿瘤细胞转移,抑制肿瘤细胞凋亡,但对其在胃癌发生过程中的研究尚属空白,且Sema3E在肿瘤中异常表达的机制目前还未被阐明。本研究主要探讨Sema3E在胃癌生长、转移和侵袭中的作用及其机制,并分析表观遗传学改变对Sema3E转录调控的影响。方法:1、采用聚合酶链式反应和免疫组织化学等技术方法分析胃癌组织中Sema3E mRNA和蛋白表达水平与正常组织相比的变化情况。2、使用DAC,TSA,MGCD0103,MC1568,C646等特异性化学抑制剂,过表达和基因敲减组蛋白乙酰化酶p300,荧光素酶报告系统等方法研究表观遗传学变化在SEMA3E转录调控中的作用机制。3、构建SEMA3E真核表达载体,研究过表达Sema3E对胃癌细胞增殖能力,克隆形成能力,裸鼠皮下成瘤能力,迁移能力以及体外重组基底膜侵袭能力的影响,并对其分子机制进行分析。结果:1、聚合酶链式反应和免疫组织化学等实验结果显示Sema3E的mRNA水平和蛋白水平在胃癌样本中表达明显下调。2、I类组蛋白去乙酰化酶(HDAC)和组蛋白乙酰化酶p300参与SEMA3E在胃癌细胞中的转录调控。3、过表达Sema3E能够有效抑制BGC-823、MGC-803、MKN-45等胃癌细胞株在体外的细胞增殖能力和克隆形成能力;抑制MGC-803体外迁移能力和重组基底膜侵袭能力;并在体内抑制裸鼠皮下移植瘤的生长。Sema3E通过细胞周期G0/G1期阻滞,促进肿瘤细胞凋亡和降低细胞信号通路中ERK、AKT等酶的磷酸化水平,发挥了对胃癌细胞增殖的抑制作用;通过抑制twist表达进而促进E-Cadherin表达上调,抑制ERK和AKT磷酸化进而抑制胃癌细胞的迁移和重组基底膜侵袭。结论:本研究表明,胃癌中Sema3E表达下调与表观遗传学调控的失衡密切相关,胃癌中组蛋白去乙酰化酶和组蛋白乙酰化酶表达异常导致Sema3E的表达下调;Sema3E对胃癌发生发展具有重要作用。综上所述,在胃癌中低表达的Sema3E可以作为诊断胃癌的候选标志物;同时,其抑癌基因的特点和功能,将为阐明表观遗传学异常与胃癌发生发展机制的关系奠定重要的理论基础,并为临床治疗提供新的思路。
[Abstract]:Objective: the expression of neurotropin Semaphorin _ 3E _ 2 Sema3E is up-regulated in many kinds of tumors and can promote the metastasis of tumor cells and inhibit the apoptosis of tumor cells, but the study on the expression of neurotropin Semaphorin _ (3E) Sema3E in gastric carcinogenesis is still blank. The mechanism of abnormal expression of Sema3E in gastric cancer has not been elucidated at present. This study mainly studies the role of Sema3E in the growth, metastasis and invasion of gastric cancer and its mechanism. The effects of epigenetic changes on Sema3E transcriptional regulation were analyzed. Methods the expression of Sema3E mRNA and protein in gastric cancer tissues was analyzed by polymerase chain reaction (PCR) and immunohistochemistry. Case .2Specific chemical inhibitors, such as Dacron TSAN MGCD0103, MC1568C646, were used. Overexpression and gene knockdown histone acetylase p300 and luciferase report system were used to study the mechanism of epigenetic changes in SEMA3E transcriptional regulation. The eukaryotic expression vector of SEMA3E was constructed and the ability of overexpression of Sema3E to gastric cancer cell proliferation was studied. The effects of clone forming ability, subcutaneous tumorigenesis ability, migration ability and invasion ability of recombinant basement membrane in nude mice in vitro. The molecular mechanism was analyzed. Results the mRNA and protein levels of Sema3E were significantly down-regulated in gastric cancer samples, including the expression of histone I deacetylase (HDAC) and histone type I deacetylase (HDAC-), and the results of immunohistochemistry and polymerase chain reaction showed that the expression of mRNA and protein in gastric cancer samples was significantly down-regulated. Histone acetylase p300 was involved in the transcriptional regulation of SEMA3E in gastric cancer cells. Overexpression of Sema3E could effectively inhibit the proliferation and clone formation of gastric cancer cell lines such as BGC-823, MGC-803 and MKN-45 in vitro. To inhibit the migration ability of MGC-803 in vitro and the invasion ability of recombinant basement membrane, and to inhibit the growth of subcutaneous xenograft tumor in nude mice in vivo. Sema3E could promote the apoptosis of tumor cells and decrease the phosphorylation level of ERKN AKT and other enzymes in cell signal pathway through G _ 0 / G _ 1 arrest of cell cycle. It can inhibit the proliferation of gastric cancer cells, promote the up-regulation of E-Cadherin expression by inhibiting the expression of twist, inhibit the phosphorylation of ERK and AKT, and thus inhibit the migration of gastric cancer cells and the invasion of recombinant basement membrane. The down-regulation of Sema3E expression in gastric cancer is closely related to the imbalance of epigenetic regulation. Abnormal expression of histone deacetylase and histone acetylase leads to down-regulation of Sema3E expression. The low expression of Sema3E in gastric cancer can be used as a candidate marker for the diagnosis of gastric cancer, and the characteristics and functions of its tumor suppressor gene will lay an important theoretical foundation for elucidating the relationship between epigenetic abnormalities and the mechanism of carcinogenesis and development of gastric cancer. And to provide new ideas for clinical treatment.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.2

【相似文献】