新抑癌基因SCNN1B通过降解GRP78对胃癌生长和转移的影响及机制研究

发布时间：2017-12-30 22:46

本文关键词：新抑癌基因SCNN1B通过降解GRP78对胃癌生长和转移的影响及机制研究　出处：《浙江大学》2017年博士论文　论文类型：学位论文

【摘要】：研究目的胃癌是目前世界上最常见的恶性肿瘤之一,2012年全球新增胃癌病例数95万,死亡病例达到72.3万例。其中在中国超过40%。由于胃癌不易早期发现,就诊时往往已经是中晚期,加之缺乏有效的治疗手段,胃癌仍然是严重影响人民生命健康的重要疾病。因此积极探索胃癌的发病机制,寻找能预测临床胃癌患者预后的分子标记物,将为胃癌的诊断和治疗提供重要思路和理论依据。癌基因的激活和抑癌基因的失活在肿瘤的发生发展过程中发挥重要作用。近年研究发现,抑癌基因表观遗传学的变化是导致肿瘤发生的重要机制,而DNA甲基化修饰是抑癌基因失活的主要原因之一,在胃癌发展的过程中起很重要的作用。基因启动子甲基化可以通过干扰转录起始和影响染色质结构从而减弱转录因子同该基因的结合能力并因此改变基因的表达,进而调节肿瘤细胞增殖、凋亡和转移等信号通路。识别受甲基化调控的抑癌基因有助于阐明胃癌发生发展规律和分子机制,为进一步的临床诊治研究提供理论基础。研究内容为了识别更多新的抑癌基因,我们使用InfiniumHumanMethylation450K甲基化芯片进行全基因组扫描:通过比对5株常见胃癌细胞AGS、HGC27、MGC803、MKN1、MKN45,永生化正常胃上皮细胞株GES1以及2例正常胃上皮组织标本,筛选出一组包括SCNN1B在内显著受甲基化调控的新型基因。甲基化芯片扫描结果提示,与正常胃上皮细胞和组织相比,SCNN1B基因启动子区CpG岛在人类胃癌中表现为高甲基化。因而推测其极有可能在胃癌的发生发展中发挥抑癌基因的功能。SCNN1B基因位于染色体16pl2.2-pl2.1,编码上皮钠通道(ENaC)的β亚单位,其由640个氨基酸组成,大小约为72659 Da,与αα及γ亚单位共同组成一复合体而行使功能。有越来越多的证据认为ENaC在癌症中异常表达。目前有关ENaC在癌症中作用的研究较多的集中在α亚单位(SCNN1A)上。比如在乳腺癌中观察到SCNN1A基因启动子区的高甲基化。在神经母细胞瘤中也发现了 SCNN1A基因启动子区的高甲基化现象,而且其甲基化程度与患者预后呈负相关。Dalgin等只发现了 SCNN1B在肾透明细胞癌中发生甲基化的现象,而Jacinto则发现对结肠癌细胞株HCT116行DNMT1和DNMT3B敲除后,SCNN1B甲基化程度明显降低。迄今为止,有关SCNN1B在肿瘤发生发展过程中的生物学功能及其机制研究尚未见报道。研究方法检测SCNN1B在临床胃癌组织与配对癌旁正常组织的表达及甲基化水平差异;免疫组化法检测胃癌组织芯片SCNN1B表达水平,评估SCNN1B的蛋白表达水平与胃癌患者临床特征及生存预后间的关系;基于外源性过表达和siRNA靶向干扰技术探讨SCNN1B对胃癌细胞增殖、存活、凋亡、细胞迁移和浸润过程的影响及其下游的通路调控机制;通过建立过表达SCNN1B胃癌动物模型实验来进一步验证SCNN1B对胃癌的抑制作用。应用免疫共沉淀联合液相色谱-串联质谱分析等现代分子技术筛选出SCNN1B的候选相互作用蛋白,并验证其相互关系及其在胃癌发生发展中的作用。研究结果研究结果发现,SCNN1B基因在81.3%的胃癌细胞株(13/16)和胃癌组织(P0.001)中表达沉默,而在人类正常胃组织中高表达。DNA启动子区高甲基化水平与SCNN1B的转录沉默相关。胃癌组织芯片检测结果显示SCNN1B高表达是预测胃癌患者更长疾病特异生存时间的预后因子(P=0.004)。在胃癌细胞中过表达SCNN1B能抑制细胞生长,诱导凋亡和G0/G1期细胞周期阻滞,抑制细胞迁移与侵袭,体内实验抑制裸鼠皮下移植瘤生长。蛋白SCNN1B与蛋白GRP78直接相互作用,促进泛素化途径介导的蛋白GRP78的降解。随后GRP78蛋白水平的下降引发折叠蛋白应答(unfolded protein response,UPR),顺序激活PERK、ATF4、CHOP和XBPls,导致caspase诱导的细胞凋亡以及对细胞迁移和侵袭的抑制。过表达SCNN1B使胃癌细胞对未折叠蛋白应答(UPR)诱导剂衣霉素(Tunicamy cin)细胞毒性的敏感性增强,说明SCNN1B能通过未折叠蛋白应答(UPR)介导肿瘤抑制。补救实验中过表达GRP78抵消SCNN1B对细胞生长和转移的抑制,而敲低GRP78则加强了SCNN1B对细胞生长的抑制。研究结论在胃癌中SCNN1B通过靶向GRP78蛋白、激活未折叠蛋白应答及其下游效应物,是一种新型肿瘤抑制剂。SCNN1B的表达状态可作为判断胃癌患者生存预后的独立生物学标志物。创新性成果我们首次确认SCNN1B能通过诱导细胞凋亡和细胞周期阻滞、抑制细胞迁移和侵袭以及促进细胞粘附,在胃癌行使抑癌基因的功能。我们还发现SCNN1B能通过与GRP78的直接相互作用、导致后者的降解及随后未折叠蛋白应答(UPR)的激活。从而在胃癌中发挥肿瘤抑制作用。SCNN1B的表达状态水平可以用来判断原发性胃癌的生存预后。因此我们还提出SCNN1B可作为胃癌患者判断生存预后的新型肿瘤标志物。理论与实际意义本课题的开展将不仅有助于进一步阐明胃癌发生发展的分子机制,还将有助于开发能预测胃癌患者预后的新型肿瘤标志物。总之,本课题是当前胃癌分子靶标研究领域创新性课题,其在胃癌发病机制和临床预后研究中具有重要的理论价值。
[Abstract]:Objective gastric cancer is one of the most common malignant tumor in the world, in 2012 the world's new number 950 thousand gastric cancer cases and death cases reaching 723 thousand. In China more than 40%. due to gastric cancer is difficult for early diagnosis and treatment is often already in advanced, and the lack of effective treatment, gastric cancer is still a serious important disease affecting people's life healthy. So exploring the pathogenesis of gastric cancer, to find molecular markers to predict the prognosis of patients with gastric cancer, will provide important theoretical basis and ideas for the diagnosis and treatment of gastric cancer. The activation and activity play an important role in the development and progression of tumor suppressor gene inactivation of oncogenes. Recent studies have found that tumor cancer gene changes of apparent genetics play an important role in the pathogenesis of cancer, while DNA methylation is one of the main reasons for the inactivation of tumor suppressor genes in gastric cancer development. Play a very important role in the process. The promoter methylation may interfere with transcription initiation and affect chromatin structure which weakens the binding ability with the transcription factor gene and thus alter gene expression and regulation of tumor cell proliferation, apoptosis and metastasis pathway. Tumor suppressor gene identification by methylation regulation of help to elucidate the molecular mechanism of regulation and the occurrence and development of gastric carcinoma, and provide a theoretical basis for clinical diagnosis and treatment of the further research. The research content in order to identify the new tumor suppressor gene, we use InfiniumHumanMethylation450K methylation chip for whole genome scan: by comparing 5 common strains of gastric cancer cells AGS, HGC27, MGC803, MKN1, MKN45, immortalized normal gastric epithelial cell line GES1 and 2 cases of normal gastric epithelial tissues were screened a group including SCNN1B gene methylation was significantly affected by the new regulation of methyl. Chip scanning results showed that compared with normal gastric epithelial cells and tissues, SCNN1B gene CpG island in the promoter region showed high methylation in human gastric cancer. So that it has great potential in the development of gastric cancer function of tumor suppressor gene.SCNN1B gene is located on chromosome 16pl2.2-pl2.1, encoding epithelial sodium channel (ENaC). The beta subunit, which is composed of 640 amino acids, the size is about 72659 Da, and alpha alpha and gamma subunit is composed of a complex function. There is increasing evidence that abnormal expression of ENaC in cancer. The present study more about role of ENaC in cancer on alpha subunit (SCNN1A) for example. In breast cancer was observed in the promoter region of SCNN1A gene hypermethylation. The hypermethylation of the promoter region of SCNN1A gene was also found in neuroblastoma, and the methylation level and prognosis of patients .Dalgin was negatively related to only found SCNN1B methylation in renal clear cell carcinoma phenomenon, while Jacinto is found on colon cancer cell line HCT116 DNMT1 and DNMT3B knockdown, SCNN1B methylation level decreased significantly. So far, the research of SCNN1B biological functions and mechanism in the process of tumor is still no report. The method of detection of SCNN1B in gastric cancer tissue and adjacent normal tissue expression and methylation level differences; immunohistochemical expression of SCNN1B in gastric cancer tissue chip assay, the expression of SCNN1B protein in relation to assessment level of patients with gastric cancer clinical characteristics and survival prognosis; exogenous overexpression and siRNA targeting jamming technique of SCNN1B on gastric cancer cell proliferation, survival, apoptosis based on the effect of cell migration and invasion process pathway and its downstream mechanism; by building up SCNN The experimental animal model of gastric cancer 1B to further confirm the inhibitory effect of SCNN1B on gastric cancer. Co immunoprecipitation combined with liquid chromatography tandem mass spectrometry analysis of modern molecular screening technology of SCNN1B candidate interacting proteins, and to verify the relationship and its role in the occurrence and development of gastric cancer. Results the results of the study showed that the SCNN1B gene in gastric cancer cell line 81.3% (13/16) and gastric cancer (P0.001) expression in silence, and in normal human gastric tissues with high expression of transcriptional silencing of.DNA hypermethylation of the promoter and SCNN1B sub area. The detection results showed that the expression of SCNN1B in gastric cancer tissue microarray is a prognostic factor to predict the survival time of gastric cancer patients with longer disease specific (P=0.004). Overexpression of SCNN1B can inhibit cell growth in gastric cancer cells, apoptosis and cell cycle arrest in G0/G1 phase, inhibit cell migration and invasion in vivo, inhibition experiment The growth of tumor transplanted subcutaneously in nude mice. Preparation of protein SCNN1B and protein GRP78 direct interaction, promote the degradation of ubiquitin mediated protein mediated pathway. GRP78 then decreased GRP78 protein levels induced unfolded protein response (unfolded protein, response, UPR), ATF4, CHOP activate PERK, and XBPls, leading to caspase induced apoptosis and inhibition the migration and invasion of cells. The overexpression of SCNN1B in gastric cancer cells of the unfolded protein response (UPR) induced by tunicamycin (Tunicamy CIN) enhanced the cytotoxic sensitivity, SCNN1B can through the unfolded protein response (UPR) mediated by tumor suppressor GRP78. SCNN1B offset the inhibition of cell growth and metastasis over expression recovery experiments, while knockdown of GRP78 was enhanced the inhibition of SCNN1B on cell growth. The conclusion of the study in SCNN1B in gastric cancer by targeting GRP78 protein activates the unfolded protein response and its downstream effectors, Is the expression of a new tumor inhibitor.SCNN1B could be used as an independent prognostic judgment of gastric cancer patients with biological markers. Innovative results we confirmed for the first time SCNN1B can induce cell apoptosis and cell cycle arrest, inhibit cell migration and invasion and promote cell adhesion in gastric cancer, to exercise the function of tumor suppressor gene. We also found that SCNN1B can through direct interaction with GRP78, which led to the degradation and then the unfolded protein response (UPR) activation. Thus the expression level of play state of tumor inhibitory effect of.SCNN1B can be used to judge the prognosis of primary gastric cancer in gastric cancer. So we also proposed SCNN1B can be used as a new tumor survival prognosis in patients with gastric cancer judgment sign the theoretical and practical significance. The development of this project will not only help to elucidate the molecular mechanism of the occurrence and development of gastric cancer, there will be It helps to develop new tumor markers that can predict the prognosis of patients with gastric cancer. In short, this topic is an innovative topic in the field of molecular targeted research of gastric cancer. It has important theoretical value in the research of gastric cancer pathogenesis and clinical prognosis.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.2

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