AlUp对胃癌细胞增殖与细胞周期调控的影响及分子机制研究

发布时间：2018-02-27 02:27

  本文关键词： 胃癌 A1Up p53 细胞周期 磷酸化　出处：《北京协和医学院》2016年博士论文　论文类型：学位论文

【摘要】：胃癌是常见的人消化道癌症,发病率在全部人类癌症中占据第四位,同时,胃癌的致死率达到70%,远远高于其他常见的人类癌症。胃癌的高发病率与高致死率严重影响了人类健康和生活。近几十年来,尽管新的诊疗技术不断出现,但由于胃癌早期诊断困难,且容易转移,故其手术根治效果较差,术前术后放化疗效果亦不理想。分子生物学研究的不断深入,为胃癌的基因治疗提供了广阔前景,近些年来,人们越来越多地把目光投向癌症的基因治疗。因此,寻找可用于胃癌治疗的靶基因对胃癌的治疗具有重要价值。AlUp (ataxin-1 interacting ubiquitin-like protein)即人共济失调蛋白-1泛素样互作蛋白,属于Ubiquilins家族的。Ubiquilins可以通过将蛋白连接到蛋白酶体,增强自噬介导的退化和参与内质网有关的蛋白降解。但A1Up在肿瘤与正常组织中的表达情况及在其肿瘤细胞中的功能相关研究还未见报道。在本研究中,我们对94对胃癌组织和癌旁组织进行免疫组化分析,结果发现A1Up在胃正常组织中表达高于胃癌组织(p0.01)。随后通过体外实验也进一步发现A1Up可抑制胃癌细胞增殖、克隆形成能力,诱导细胞G0/G1期阻滞、细胞衰老,以及抑制MKN45在裸鼠体内的成瘤能力。我们对过表达及敲降A1Up的细胞进行基因表达谱分析,筛选到874个差异表达基因,其中上调基因450个,下调基因424个。为进一步验证这些基因的功能,我们进行了GO分析和KEGG分析。结果显示,A1Up很可能通过p53信号通路发挥生物学作用。Western blot实验进一步验证了A1Up可以调控p53、p21的表达,并且二者的表达量与A1Up的表达量呈正相关。用化疗药物Camptothecin诱导MKN45细胞中p53的表达,A1Up也呈表达增高趋势,并且A1Up表达升高和下降的趋势均早于p53、p21。而A1Up对p21的表达量的影响,除依赖p53的转录调控外,还有其它的调控方式。用蛋白合成抑制剂放线菌酮CHX处理细胞,与对照组相比,MKN45/pLVX-AlUp细胞中p21的降解速率明显减慢。由此我们得出结论,A1Up可以抑制p21的泛素化降解,从而上调p21的表达水平。随后我们对A1Up的相互作用蛋白进行了富集,通过质谱分析寻找到相互作用蛋白——RNF114, Co-IP也进一步验证了RNF114与A1Up有相互作用。在MKN45和BGC-823细胞中进行了A1Up的过表达和敲降,结果显示RNF114与A1Up的表达量成反比。而RNF114作为p21的一个E3连接酶,能过RING结构介导p21的降解,所以A1Up很有可能通过介导RNF114的泛素化降解来稳定细胞中p21的表达。与胃癌细胞株MKN45、BGC823不同的是,A1Up可以诱导永生化胃粘膜细胞株GES-1发生凋亡。流式细胞术实验结果显示,感染空载病毒及A1Up病毒的两组细胞早期凋亡比例分别为3.4±1.1%和13.1±1.8%,差异具有统计学意义(p0.01)。Western blot也检测到了凋亡的执行分子Caspase 3、PARP的活化。更有趣的是,我们分别将GES-1、MKN45、BGC823细胞同步化于G1/S、S、G2/M及M期,发现A1Up在M期发生了磷酸化修饰,并且该修饰依赖于激酶CDK1的活性。经质谱鉴定磷酸化位点,结果显示A1Up在G1期的磷酸化位点有19个,在M期有32个,其中仅在M期发生磷酸化的位点有22个。综上所述,我们对A1Up在胃癌中的表达情况进行了研究,首次发现了A1Up通过调控p53信号通路及其下游基因,在胃癌的发生发展中发挥着重要的抑癌作用。本文的研究结果具有创新意义,揭示了A1Up基因在胃癌细胞内涉及的功能和的分子机制,不仅对今后理解并研究A1Up基因的功能和作用机制具有指导意义,还为胃癌的基因治疗提供了新的靶点。
[Abstract]:Gastric cancer is a common gastrointestinal cancer incidence rate occupies the fourth position, in all human cancers and gastric cancer mortality rate reached 70%, far higher than other common human cancers. Gastric cancer high incidence and high death rate affects human health and life. In the past few decades, although the new treatment technology continue to appear, but because of difficulties in early diagnosis of gastric cancer, and easy to transfer, so the effect of surgical treatment is poor, preoperative and postoperative radiotherapy and chemotherapy effect is not ideal. Molecular biology further research for gene therapy of gastric cancer has provided broad prospects, in recent years, more and more people put their eyes on the gene therapy for cancer therefore, looking for the.AlUp is of great value in the treatment of gastric cancer gene therapeutic target for gastric cancer (ataxin-1 interacting ubiquitin-like protein) is human ataxia protein -1 ubiquitin like protein interaction, which belongs to The Ubiquilins family of.Ubiquilins protein can be connected to the proteasome, enhanced autophagy mediated degradation and protein degradation in the endoplasmic reticulum. But the expression of A1Up in tumor and normal tissues and its function in tumor cells has not been reported. In this study, we have 94 pairs of gastric cancer tissues and paracancerous tissues were examined by immunohistochemical analysis, the expression of A1Up in normal gastric tissues than gastric cancer (P0.01). Then through in vitro experiments further found that A1Up can inhibit the proliferation of gastric cancer cells, colony forming ability, inducing G0/G1 cell cycle arrest, cell senescence, and inhibit MKN45 tumorigenic ability in nude mice. We on the overexpression and knockdown of A1Up cells by gene expression profiling, screened 874 differentially expressed genes, including 450 up-regulated genes and 424 down regulated genes in order to further verify this. Some genes, we conducted analysis of GO and KEGG. The results showed that A1Up probably through p53 signaling pathway play a biological role of.Western blot experiment verified that A1Up can regulate the expression of p21, p53, A1Up expression and expression of Cheng Zhengxiang and the two. Induced expression of MKN45 cells with p53 the chemotherapy drug Camptothecin, A1Up expression was also increased, and the expression of A1Up increased and decreased earlier than p53, p21. and A1Up on expression of p21 the influence of transcriptional regulation of p53 dependent, in addition, there are other methods of regulation. With the protein synthesis inhibitor cycloheximide treated CHX cells compared with the control. The degradation rate of MKN45/pLVX-AlUp cells in group p21 decreased significantly. Thus we conclude that ubiquitination of A1Up can inhibit p21, and thus increase the expression level of p21. Then we A1Up the interaction of eggs White was enriched by mass spectrometry analysis to find the proteins interacting with RNF114, Co-IP also further confirmed that RNF114 can interact with A1Up. In MKN45 and BGC-823 cells for overexpression and knockdown of A1Up, the results showed that the expression level of RNF114 and A1Up is inversely proportional to the RNF114 as an E3 ligase p21 that can degrade the RING structure mediated by p21, the expression of ubiquitin degradation so A1Up is likely mediated by RNF114 to stabilize the p21 in cells. MKN45 and gastric cancer cell lines, BGC823 is different, A1Up can induce immortalized gastric mucosa apoptosis in GES-1 cells. Flow cytometry results showed that the early apoptosis rate of A1Up virus infection and virus load of cells in two groups were 3.4 + 1.1% and 13.1 + 1.8%, the difference was statistically significant (P0.01).Western blot also detected the apoptosis of Caspase 3 molecules, the activation of PARP. Interestingly, we were GES-1, MKN45, BGC823 cell synchronization in G1/S, S, G2/M and M, found that the phosphorylation of A1Up in M phase, and the modification depends on the kinase activity of CDK1. After identification of sites of phosphorylation by mass spectrometry, the results showed that A1Up in G1 phosphorylation sites 19, in the period of M 32, which only occurred in the M phase of the phosphorylation sites were 22. In summary, we investigated the expression of A1Up in gastric carcinoma, A1Up was first identified by p53 signal pathway and its downstream genes in the development of gastric cancer plays a tumor suppressor role important. The result of this paper has innovation significance, reveals a molecular mechanism involving A1Up gene in gastric cancer cells and the function, not only to the future understanding and instructive function and mechanism of A1Up gene for gene therapy of gastric cancer, also provide a new target Point.

【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】：2016
【分类号】：R735.2

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1 黄声凯;AlUp对胃癌细胞增殖与细胞周期调控的影响及分子机制研究[D];北京协和医学院;2016年

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