UCHL1、NAIF1基因在胃癌发生发展过程的功能研究

发布时间：2018-01-29 08:52

本文关键词： UCHL1 NAIF1 胃癌 Akt通路 MAPK通路　出处：《北京协和医学院》2015年博士论文　论文类型：学位论文

【摘要】：胃癌是常见的消化道肿瘤,严重威胁人类健康以及生活质量。根据2012年全球肿瘤流行病学统计结果,在全球范围内,胃癌发病率在全部人类癌症中排位第五,致死率占据第三位。由于早期胃癌症状隐匿,大部分患者在诊断胃癌时已经处于中晚期,伴有肿瘤远端转移,而转移则是胃癌高致死率的主要因素。因此,寻找与胃癌发生、发展相关的基因,并对这些基因的功能加以研究,将有利于我们了解胃癌发生、侵袭转移的分子机制,并从中鉴定新的诊断标志物与治疗靶点,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员之一,参与细胞内泛素依赖的蛋白水解过程,调控蛋白降解。UCHL1的去泛素化酶活性与第90位点半胱氨酸(C90)密切相关,当该位点被突变为丝氨酸(S)时,UCHL1的去泛素化酶活性下降107倍。因此,UCHL1的突变体形式C90S几乎不具备去泛素化酶活性。在本研究中,我们首先利用免疫组织化学技术,发现UCHL1在胃癌旁及胃癌远端胃粘膜组织、胃癌原发灶、胃癌的肝转移灶阳性表达率分别为56.5%,13%和70%。经Bonferroni校正x2检验,我们发现UCHL1分别在胃癌旁及胃癌远端胃粘膜组织、胃癌原发灶之间(P=0.002),以及胃癌原发灶与胃癌的肝转移灶之间(P=0.002)存在明显的表达差异。随后,我们选择胃癌细胞系BGC823、MKN45,利用慢病毒系统建立6个稳转细胞系,分别为空载或者表达野生型UCHL1、UCHL1的突变体形式C90S蛋白。UCHL1、C90S均定位于细胞质。过表达UCHL1能够促进胃癌细胞增殖、克隆形成、迁移和侵袭能力,并且上述功能均依赖于UCHL1的去泛素化酶活性。同时,过表达UCHL1促进Akt、Erkl/2的活化,该过程也依赖于去泛素化酶活性,并且通路的活化对于UCHL1介导的细胞迁移、侵袭功能的增强是必须的。依据UCHL1的前期研究与实验结果,我们在文中解释了其在胃相关组织的特殊表达模式,并认为UCHL1通过上述机制促进了胃癌的转移过程,有希望成为针对于胃癌转移的诊断标志物与治疗靶点。NAIF1 (nuclear apoptosis-inducing factor 1,核凋亡诱导因子1)基因,也称为C9orf90,能够诱导细胞发生凋亡。前期的免疫组织化学研究显示,在胃癌组织中, NAIF1的表达量显著低于癌旁正常胃粘膜组织；并且,在分化良好的胃癌中,NAIF1的表达水平高于中等或低分化程度胃癌。该结果提示NAIF1在胃癌发生、发展过程中可能发挥抑制作用,但具体的分子机制有待于深入探讨。我们的研究显示,在胃癌细胞BGC823和MKN45中,NAIF1一方面通过下调Erk1/2的mRNA表达水平,下调Erk1/2的蛋白表达量,进而降低p-Erk1/2的表达量；另一方面,NAIF1通过泛素-蛋白酶体途径加速JNK本底降解,从而下调p-JNK的表达量。通过对上述两条MAPK通路的调控,NAIF1降低细胞的体外增殖、迁移、侵袭能力,以及MKN45在裸鼠体内的成瘤能力。由于NAIF1对胃癌细胞的恶性表型具有抑制作用,并且能够负调控Erkl/2和JNK通路,因此,NAIF1具有潜在的临床应用前景,有望成为胃癌诊断的分子标志物,并且可能作为针对于MAPK通路的治疗靶点。本研究以我国常见的恶性肿瘤胃癌为研究对象,揭示了UCHL1、NAIF1在胃癌发生、发展中的作用和分子机制,为寻找胃癌诊断标志物、治疗靶点提供新的线索。
[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.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.2

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