本文關鍵詞： UCHL1 NAIF1 胃癌 Akt通路 MAPK通路　出處：《北京協(xié)和醫(yī)學院》2015年博士論文　論文類型：學位論文

【摘要】：胃癌是常見的消化道腫瘤,嚴重威脅人類健康以及生活質量。根據(jù)2012年全球腫瘤流行病學統(tǒng)計結果,在全球范圍內,胃癌發(fā)病率在全部人類癌癥中排位第五,致死率占據(jù)第三位。由于早期胃癌癥狀隱匿,大部分患者在診斷胃癌時已經處于中晚期,伴有腫瘤遠端轉移,而轉移則是胃癌高致死率的主要因素。因此,尋找與胃癌發(fā)生、發(fā)展相關的基因,并對這些基因的功能加以研究,將有利于我們了解胃癌發(fā)生、侵襲轉移的分子機制,并從中鑒定新的診斷標志物與治療靶點,以提高胃癌患者生存率。UCHLl (ubiquitin C-terminal hydrolase-L1,泛素羧基端酯酶L1)是去泛素化酶家族成員之一,參與細胞內泛素依賴的蛋白水解過程,調控蛋白降解。UCHL1的去泛素化酶活性與第90位點半胱氨酸(C90)密切相關,當該位點被突變?yōu)榻z氨酸(S)時,UCHL1的去泛素化酶活性下降107倍。因此,UCHL1的突變體形式C90S幾乎不具備去泛素化酶活性。在本研究中,我們首先利用免疫組織化學技術,發(fā)現(xiàn)UCHL1在胃癌旁及胃癌遠端胃粘膜組織、胃癌原發(fā)灶、胃癌的肝轉移灶陽性表達率分別為56.5%,13%和70%。經Bonferroni校正x2檢驗,我們發(fā)現(xiàn)UCHL1分別在胃癌旁及胃癌遠端胃粘膜組織、胃癌原發(fā)灶之間(P=0.002),以及胃癌原發(fā)灶與胃癌的肝轉移灶之間(P=0.002)存在明顯的表達差異。隨后,我們選擇胃癌細胞系BGC823、MKN45,利用慢病毒系統(tǒng)建立6個穩(wěn)轉細胞系,分別為空載或者表達野生型UCHL1、UCHL1的突變體形式C90S蛋白。UCHL1、C90S均定位于細胞質。過表達UCHL1能夠促進胃癌細胞增殖、克隆形成、遷移和侵襲能力,并且上述功能均依賴于UCHL1的去泛素化酶活性。同時,過表達UCHL1促進Akt、Erkl/2的活化,該過程也依賴于去泛素化酶活性,并且通路的活化對于UCHL1介導的細胞遷移、侵襲功能的增強是必須的。依據(jù)UCHL1的前期研究與實驗結果,我們在文中解釋了其在胃相關組織的特殊表達模式,并認為UCHL1通過上述機制促進了胃癌的轉移過程,有希望成為針對于胃癌轉移的診斷標志物與治療靶點。NAIF1 (nuclear apoptosis-inducing factor 1,核凋亡誘導因子1)基因,也稱為C9orf90,能夠誘導細胞發(fā)生凋亡。前期的免疫組織化學研究顯示,在胃癌組織中, NAIF1的表達量顯著低于癌旁正常胃粘膜組織；并且,在分化良好的胃癌中,NAIF1的表達水平高于中等或低分化程度胃癌。該結果提示NAIF1在胃癌發(fā)生、發(fā)展過程中可能發(fā)揮抑制作用,但具體的分子機制有待于深入探討。我們的研究顯示,在胃癌細胞BGC823和MKN45中,NAIF1一方面通過下調Erk1/2的mRNA表達水平,下調Erk1/2的蛋白表達量,進而降低p-Erk1/2的表達量；另一方面,NAIF1通過泛素-蛋白酶體途徑加速JNK本底降解,從而下調p-JNK的表達量。通過對上述兩條MAPK通路的調控,NAIF1降低細胞的體外增殖、遷移、侵襲能力,以及MKN45在裸鼠體內的成瘤能力。由于NAIF1對胃癌細胞的惡性表型具有抑制作用,并且能夠負調控Erkl/2和JNK通路,因此,NAIF1具有潛在的臨床應用前景,有望成為胃癌診斷的分子標志物,并且可能作為針對于MAPK通路的治療靶點。本研究以我國常見的惡性腫瘤胃癌為研究對象,揭示了UCHL1、NAIF1在胃癌發(fā)生、發(fā)展中的作用和分子機制,為尋找胃癌診斷標志物、治療靶點提供新的線索。
[Abstract]:Gastric cancer is a common gastrointestinal tumor, a serious threat to human health and quality of life. According to the 2012 global cancer epidemiology statistics, in the global scope, the incidence of gastric cancer in all human cancer mortality rate ranked fifth, occupy third. Because of early gastric cancer symptoms hidden, most patients have been in advanced stage in the diagnosis of gastric cancer, accompanied by tumor distant metastasis, and gastric cancer metastasis is high death rate of main factor. Therefore, for the development and the development of gastric cancer related genes, and the gene function research, will help us to understand the molecular mechanism of gastric carcinogenesis, invasion and metastasis, and the identification of new diagnostic markers and therapeutic targets in order to improve the survival rate of patients with gastric cancer,.UCHLl (ubiquitin C-terminal hydrolase-L1, ubiquitin carboxyl terminal esterase L1) is one of the members of the family of enzymes to ubiquitination, and intracellular ubiquitin dependent parameters Protein hydrolysis process of lysine, regulation of protein degradation.UCHL1 deubiquitinase activity and ninetieth cysteine (C90) are closely related, when the site was mutated to serine (S), deubiquitinating enzyme activity decreased 107 times of UCHL1. Therefore, C90S mutant forms of UCHL1 almost does not have deubiquitinase activity in this study, we use immunohistochemical technique UCHL1 in gastric cancer and gastric cancer found distal gastric mucosa, primary foci of gastric cancer, the positive expression rate of 56.5% focal liver metastasis of gastric cancer, 13% and 70%. after Bonferroni correction x2 inspection, we found that UCHL1 in gastric cancer and gastric mucosa of distal gastric cancer and between the primary foci of gastric cancer (P=0.002), and the primary gastric cancer and gastric cancer liver metastases (P=0.002) between the obvious expression difference. Then, we select BGC823 gastric cancer cell line MKN45, established 6 using lentiviral system A stable cell lines were empty or expression of wild-type UCHL1, UCHL1 mutant forms of C90S protein.UCHL1 and C90S were localized in the cytoplasm. Overexpression of UCHL1 could promote the proliferation of gastric cancer cells, colony formation, migration and invasion, and these functions are dependent on the UCHL1 deubiquitinase activity. At the same time, over expression UCHL1 promotes Akt, Erkl/2 activation, the process also depends on the deubiquitinating enzyme activity, and pathway for cell migration mediated by UCHL1, enhance the invasion function is necessary. On the basis of previous research and experimental results of UCHL1, we explain the special expression patterns of the related organizations in the stomach in the. And that UCHL1 promotes the metastasis of gastric cancer through this mechanism, is expected to be for diagnosis in gastric cancer metastasis marker and therapeutic target of.NAIF1 (nuclear apoptosis-inducing factor 1, nuclear apoptosis induced by 1) gene, also known as C9orf90, can induce cell apoptosis. Immunohistochemical study study showed that in gastric cancer tissue. The expression of NAIF1 was significantly lower than that of adjacent normal gastric mucosa; and, in well differentiated gastric carcinoma, the expression level of NAIF1 was higher than that of medium or low differentiation of gastric carcinoma. The results NAIF1 in gastric cancer, may inhibit the process of development, but the exact molecular mechanism needs to be further discussed. Our research shows that in the BGC823 and MKN45 in gastric cancer cells, NAIF1 expression level by downregulating Erk1/2 mRNA, down-regulation of Erk1/2 protein, thereby reducing the expression of p-Erk1/2 is another; NAIF1, via the ubiquitin proteasome pathway JNK background accelerated degradation, thus reduced the expression of p-JNK. Through the regulation of the two MAPK pathway, NAIF1 decreased cell migration in vitro proliferation, The invasion ability of MKN45, and the tumorigenic ability in nude mice. The NAIF1 of gastric cancer cells can inhibit the malignant phenotype, and can negatively regulate Erkl/2 and JNK pathway, therefore, NAIF1 has a potential clinical application prospects, is expected to become a molecular marker for diagnosis of gastric cancer, and may serve as a therapeutic target for MAPK pathway in this paper, the most common malignant tumor of gastric cancer as the research object, revealed that UCHL1, NAIF1 in gastric cancer, the role and molecular mechanism of development, for the diagnosis of gastric cancer markers and therapeutic targets to provide new clues.

【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R735.2

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本文編號：1473032