本文關(guān)鍵詞：新抑癌基因SCNN1B通過降解GRP78對(duì)胃癌生長和轉(zhuǎn)移的影響及機(jī)制研究　出處：《浙江大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 胃癌 SCNN1B 腫瘤抑制劑 DNA甲基化 GRP78

【摘要】：研究目的胃癌是目前世界上最常見的惡性腫瘤之一,2012年全球新增胃癌病例數(shù)95萬,死亡病例達(dá)到72.3萬例。其中在中國超過40%。由于胃癌不易早期發(fā)現(xiàn),就診時(shí)往往已經(jīng)是中晚期,加之缺乏有效的治療手段,胃癌仍然是嚴(yán)重影響人民生命健康的重要疾病。因此積極探索胃癌的發(fā)病機(jī)制,尋找能預(yù)測臨床胃癌患者預(yù)后的分子標(biāo)記物,將為胃癌的診斷和治療提供重要思路和理論依據(jù)。癌基因的激活和抑癌基因的失活在腫瘤的發(fā)生發(fā)展過程中發(fā)揮重要作用。近年研究發(fā)現(xiàn),抑癌基因表觀遺傳學(xué)的變化是導(dǎo)致腫瘤發(fā)生的重要機(jī)制,而DNA甲基化修飾是抑癌基因失活的主要原因之一,在胃癌發(fā)展的過程中起很重要的作用�；騿�(dòng)子甲基化可以通過干擾轉(zhuǎn)錄起始和影響染色質(zhì)結(jié)構(gòu)從而減弱轉(zhuǎn)錄因子同該基因的結(jié)合能力并因此改變基因的表達(dá),進(jìn)而調(diào)節(jié)腫瘤細(xì)胞增殖、凋亡和轉(zhuǎn)移等信號(hào)通路。識(shí)別受甲基化調(diào)控的抑癌基因有助于闡明胃癌發(fā)生發(fā)展規(guī)律和分子機(jī)制,為進(jìn)一步的臨床診治研究提供理論基礎(chǔ)。研究內(nèi)容為了識(shí)別更多新的抑癌基因,我們使用InfiniumHumanMethylation450K甲基化芯片進(jìn)行全基因組掃描:通過比對(duì)5株常見胃癌細(xì)胞AGS、HGC27、MGC803、MKN1、MKN45,永生化正常胃上皮細(xì)胞株GES1以及2例正常胃上皮組織標(biāo)本,篩選出一組包括SCNN1B在內(nèi)顯著受甲基化調(diào)控的新型基因。甲基化芯片掃描結(jié)果提示,與正常胃上皮細(xì)胞和組織相比,SCNN1B基因啟動(dòng)子區(qū)CpG島在人類胃癌中表現(xiàn)為高甲基化。因而推測其極有可能在胃癌的發(fā)生發(fā)展中發(fā)揮抑癌基因的功能。SCNN1B基因位于染色體16pl2.2-pl2.1,編碼上皮鈉通道(ENaC)的β亞單位,其由640個(gè)氨基酸組成,大小約為72659 Da,與αα及γ亞單位共同組成一復(fù)合體而行使功能。有越來越多的證據(jù)認(rèn)為ENaC在癌癥中異常表達(dá)。目前有關(guān)ENaC在癌癥中作用的研究較多的集中在α亞單位(SCNN1A)上。比如在乳腺癌中觀察到SCNN1A基因啟動(dòng)子區(qū)的高甲基化。在神經(jīng)母細(xì)胞瘤中也發(fā)現(xiàn)了 SCNN1A基因啟動(dòng)子區(qū)的高甲基化現(xiàn)象,而且其甲基化程度與患者預(yù)后呈負(fù)相關(guān)。Dalgin等只發(fā)現(xiàn)了 SCNN1B在腎透明細(xì)胞癌中發(fā)生甲基化的現(xiàn)象,而Jacinto則發(fā)現(xiàn)對(duì)結(jié)腸癌細(xì)胞株HCT116行DNMT1和DNMT3B敲除后,SCNN1B甲基化程度明顯降低。迄今為止,有關(guān)SCNN1B在腫瘤發(fā)生發(fā)展過程中的生物學(xué)功能及其機(jī)制研究尚未見報(bào)道。研究方法檢測SCNN1B在臨床胃癌組織與配對(duì)癌旁正常組織的表達(dá)及甲基化水平差異;免疫組化法檢測胃癌組織芯片SCNN1B表達(dá)水平,評(píng)估SCNN1B的蛋白表達(dá)水平與胃癌患者臨床特征及生存預(yù)后間的關(guān)系;基于外源性過表達(dá)和siRNA靶向干擾技術(shù)探討SCNN1B對(duì)胃癌細(xì)胞增殖、存活、凋亡、細(xì)胞遷移和浸潤過程的影響及其下游的通路調(diào)控機(jī)制;通過建立過表達(dá)SCNN1B胃癌動(dòng)物模型實(shí)驗(yàn)來進(jìn)一步驗(yàn)證SCNN1B對(duì)胃癌的抑制作用。應(yīng)用免疫共沉淀聯(lián)合液相色譜-串聯(lián)質(zhì)譜分析等現(xiàn)代分子技術(shù)篩選出SCNN1B的候選相互作用蛋白,并驗(yàn)證其相互關(guān)系及其在胃癌發(fā)生發(fā)展中的作用。研究結(jié)果研究結(jié)果發(fā)現(xiàn),SCNN1B基因在81.3%的胃癌細(xì)胞株(13/16)和胃癌組織(P0.001)中表達(dá)沉默,而在人類正常胃組織中高表達(dá)。DNA啟動(dòng)子區(qū)高甲基化水平與SCNN1B的轉(zhuǎn)錄沉默相關(guān)。胃癌組織芯片檢測結(jié)果顯示SCNN1B高表達(dá)是預(yù)測胃癌患者更長疾病特異生存時(shí)間的預(yù)后因子(P=0.004)。在胃癌細(xì)胞中過表達(dá)SCNN1B能抑制細(xì)胞生長,誘導(dǎo)凋亡和G0/G1期細(xì)胞周期阻滯,抑制細(xì)胞遷移與侵襲,體內(nèi)實(shí)驗(yàn)抑制裸鼠皮下移植瘤生長。蛋白SCNN1B與蛋白GRP78直接相互作用,促進(jìn)泛素化途徑介導(dǎo)的蛋白GRP78的降解。隨后GRP78蛋白水平的下降引發(fā)折疊蛋白應(yīng)答(unfolded protein response,UPR),順序激活PERK、ATF4、CHOP和XBPls,導(dǎo)致caspase誘導(dǎo)的細(xì)胞凋亡以及對(duì)細(xì)胞遷移和侵襲的抑制。過表達(dá)SCNN1B使胃癌細(xì)胞對(duì)未折疊蛋白應(yīng)答(UPR)誘導(dǎo)劑衣霉素(Tunicamy cin)細(xì)胞毒性的敏感性增強(qiáng),說明SCNN1B能通過未折疊蛋白應(yīng)答(UPR)介導(dǎo)腫瘤抑制。補(bǔ)救實(shí)驗(yàn)中過表達(dá)GRP78抵消SCNN1B對(duì)細(xì)胞生長和轉(zhuǎn)移的抑制,而敲低GRP78則加強(qiáng)了SCNN1B對(duì)細(xì)胞生長的抑制。研究結(jié)論在胃癌中SCNN1B通過靶向GRP78蛋白、激活未折疊蛋白應(yīng)答及其下游效應(yīng)物,是一種新型腫瘤抑制劑。SCNN1B的表達(dá)狀態(tài)可作為判斷胃癌患者生存預(yù)后的獨(dú)立生物學(xué)標(biāo)志物。創(chuàng)新性成果我們首次確認(rèn)SCNN1B能通過誘導(dǎo)細(xì)胞凋亡和細(xì)胞周期阻滯、抑制細(xì)胞遷移和侵襲以及促進(jìn)細(xì)胞粘附,在胃癌行使抑癌基因的功能。我們還發(fā)現(xiàn)SCNN1B能通過與GRP78的直接相互作用、導(dǎo)致后者的降解及隨后未折疊蛋白應(yīng)答(UPR)的激活。從而在胃癌中發(fā)揮腫瘤抑制作用。SCNN1B的表達(dá)狀態(tài)水平可以用來判斷原發(fā)性胃癌的生存預(yù)后。因此我們還提出SCNN1B可作為胃癌患者判斷生存預(yù)后的新型腫瘤標(biāo)志物。理論與實(shí)際意義本課題的開展將不僅有助于進(jìn)一步闡明胃癌發(fā)生發(fā)展的分子機(jī)制,還將有助于開發(fā)能預(yù)測胃癌患者預(yù)后的新型腫瘤標(biāo)志物�？傊�,本課題是當(dāng)前胃癌分子靶標(biāo)研究領(lǐng)域創(chuàng)新性課題,其在胃癌發(fā)病機(jī)制和臨床預(yù)后研究中具有重要的理論價(jià)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.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.2

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