本文關(guān)鍵詞： 胃癌 A1Up p53 細胞周期 磷酸化　出處：《北京協(xié)和醫(yī)學(xué)院》2016年博士論文　論文類型：學(xué)位論文

【摘要】：胃癌是常見的人消化道癌癥,發(fā)病率在全部人類癌癥中占據(jù)第四位,同時,胃癌的致死率達到70%,遠遠高于其他常見的人類癌癥。胃癌的高發(fā)病率與高致死率嚴重影響了人類健康和生活。近幾十年來,盡管新的診療技術(shù)不斷出現(xiàn),但由于胃癌早期診斷困難,且容易轉(zhuǎn)移,故其手術(shù)根治效果較差,術(shù)前術(shù)后放化療效果亦不理想。分子生物學(xué)研究的不斷深入,為胃癌的基因治療提供了廣闊前景,近些年來,人們越來越多地把目光投向癌癥的基因治療。因此,尋找可用于胃癌治療的靶基因?qū)ξ赴┑闹委熅哂兄匾獌r值。AlUp (ataxin-1 interacting ubiquitin-like protein)即人共濟失調(diào)蛋白-1泛素樣互作蛋白,屬于Ubiquilins家族的。Ubiquilins可以通過將蛋白連接到蛋白酶體,增強自噬介導(dǎo)的退化和參與內(nèi)質(zhì)網(wǎng)有關(guān)的蛋白降解。但A1Up在腫瘤與正常組織中的表達情況及在其腫瘤細胞中的功能相關(guān)研究還未見報道。在本研究中,我們對94對胃癌組織和癌旁組織進行免疫組化分析,結(jié)果發(fā)現(xiàn)A1Up在胃正常組織中表達高于胃癌組織(p0.01)。隨后通過體外實驗也進一步發(fā)現(xiàn)A1Up可抑制胃癌細胞增殖、克隆形成能力,誘導(dǎo)細胞G0/G1期阻滯、細胞衰老,以及抑制MKN45在裸鼠體內(nèi)的成瘤能力。我們對過表達及敲降A(chǔ)1Up的細胞進行基因表達譜分析,篩選到874個差異表達基因,其中上調(diào)基因450個,下調(diào)基因424個。為進一步驗證這些基因的功能,我們進行了GO分析和KEGG分析。結(jié)果顯示,A1Up很可能通過p53信號通路發(fā)揮生物學(xué)作用。Western blot實驗進一步驗證了A1Up可以調(diào)控p53、p21的表達,并且二者的表達量與A1Up的表達量呈正相關(guān)。用化療藥物Camptothecin誘導(dǎo)MKN45細胞中p53的表達,A1Up也呈表達增高趨勢,并且A1Up表達升高和下降的趨勢均早于p53、p21。而A1Up對p21的表達量的影響,除依賴p53的轉(zhuǎn)錄調(diào)控外,還有其它的調(diào)控方式。用蛋白合成抑制劑放線菌酮CHX處理細胞,與對照組相比,MKN45/pLVX-AlUp細胞中p21的降解速率明顯減慢。由此我們得出結(jié)論,A1Up可以抑制p21的泛素化降解,從而上調(diào)p21的表達水平。隨后我們對A1Up的相互作用蛋白進行了富集,通過質(zhì)譜分析尋找到相互作用蛋白——RNF114, Co-IP也進一步驗證了RNF114與A1Up有相互作用。在MKN45和BGC-823細胞中進行了A1Up的過表達和敲降,結(jié)果顯示RNF114與A1Up的表達量成反比。而RNF114作為p21的一個E3連接酶,能過RING結(jié)構(gòu)介導(dǎo)p21的降解,所以A1Up很有可能通過介導(dǎo)RNF114的泛素化降解來穩(wěn)定細胞中p21的表達。與胃癌細胞株MKN45、BGC823不同的是,A1Up可以誘導(dǎo)永生化胃粘膜細胞株GES-1發(fā)生凋亡。流式細胞術(shù)實驗結(jié)果顯示,感染空載病毒及A1Up病毒的兩組細胞早期凋亡比例分別為3.4±1.1%和13.1±1.8%,差異具有統(tǒng)計學(xué)意義(p0.01)。Western blot也檢測到了凋亡的執(zhí)行分子Caspase 3、PARP的活化。更有趣的是,我們分別將GES-1、MKN45、BGC823細胞同步化于G1/S、S、G2/M及M期,發(fā)現(xiàn)A1Up在M期發(fā)生了磷酸化修飾,并且該修飾依賴于激酶CDK1的活性。經(jīng)質(zhì)譜鑒定磷酸化位點,結(jié)果顯示A1Up在G1期的磷酸化位點有19個,在M期有32個,其中僅在M期發(fā)生磷酸化的位點有22個。綜上所述,我們對A1Up在胃癌中的表達情況進行了研究,首次發(fā)現(xiàn)了A1Up通過調(diào)控p53信號通路及其下游基因,在胃癌的發(fā)生發(fā)展中發(fā)揮著重要的抑癌作用。本文的研究結(jié)果具有創(chuàng)新意義,揭示了A1Up基因在胃癌細胞內(nèi)涉及的功能和的分子機制,不僅對今后理解并研究A1Up基因的功能和作用機制具有指導(dǎo)意義,還為胃癌的基因治療提供了新的靶點。
[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.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R735.2

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1 黃聲凱;AlUp對胃癌細胞增殖與細胞周期調(diào)控的影響及分子機制研究[D];北京協(xié)和醫(yī)學(xué)院;2016年

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本文編號：1540743