本文選題：NDRG2　切入點(diǎn)：谷氨酰胺分解代謝　出處：《第四軍醫(yī)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：惡性腫瘤生長和增殖迅速,需要充足的營養(yǎng)物質(zhì)和能量供給。腫瘤細(xì)胞代謝出現(xiàn)明顯改變以適應(yīng)腫瘤的發(fā)生和發(fā)展,其主要表現(xiàn)為:葡萄糖有氧酵解代謝(glycolysis)增強(qiáng),谷氨酰胺分解代謝增強(qiáng),脂類和核苷酸生物合成增強(qiáng)等。谷氨酰胺分解代謝(glutaminolysis)是腫瘤細(xì)胞除Warburg效應(yīng)外另一重要的能量代謝方式。除了消耗葡萄糖供能外,腫瘤細(xì)胞還會通過谷氨酰胺分解代謝為其生長和增殖提供所需的能量和生物大分子原料,并且谷氨酰胺分解代謝還參與細(xì)胞內(nèi)氧化還原穩(wěn)態(tài)的維持和細(xì)胞內(nèi)信號通路的轉(zhuǎn)導(dǎo)。腫瘤細(xì)胞中原癌基因和抑癌基因的突變會導(dǎo)致谷氨酰胺代謝異常。抑癌基因NDRG2抑制結(jié)直腸腫瘤的惡性生長和增殖,但其在谷氨酰胺分解代謝中的作用尚不明確。人NDRG2(N-myc down-stream regulated gene 2,N-myc下游調(diào)節(jié)基因-2)基因?yàn)楸狙芯啃〗M首先發(fā)現(xiàn)并報道,研究發(fā)現(xiàn),該基因與細(xì)胞增殖和分化相關(guān),并且在多種腫瘤組織低表達(dá),發(fā)揮抑癌基因的作用。前期研究表明NDRG2能夠抑制腫瘤細(xì)胞糖酵解代謝產(chǎn)物乳酸的產(chǎn)生并降低葡萄糖的消耗,即NDRG2可以抑制腫瘤細(xì)胞糖酵解代謝。在此基礎(chǔ)上,本課題想要探討NDRG2對于腫瘤細(xì)胞谷氨酰胺分解代謝的調(diào)控作用與分子機(jī)制。目的:1、明確NDRG2是否在腫瘤谷氨酰胺分解代謝中發(fā)揮作用;2、研究NDRG2調(diào)控谷氨酰胺分解代謝途徑的靶點(diǎn);3、探討NDRG2調(diào)控谷氨酰胺分解代謝靶點(diǎn)的分子機(jī)制;;4、探討NDRG2參與調(diào)控腫瘤代謝重編程的臨床意義。方法:1、通過代謝組學(xué)的方法,分析NDRG2過表達(dá)細(xì)胞中代謝物種類及量的變化,明確NDRG2是否影響腫瘤細(xì)胞中谷氨酰胺和谷氨酸的含量。2、通過試劑盒檢測代謝物,分析NDRG2過表達(dá)與降低細(xì)胞系中谷氨酰胺和谷氨酸的變化,明確NDRG2對谷氨酰胺攝取及分解的調(diào)節(jié)作用。3、通過Real-time PCR、免疫印跡的方法,分析NDRG2過表達(dá)與降低細(xì)胞系中谷氨酰胺轉(zhuǎn)運(yùn)子(ASCT2)及谷氨酰胺酶1(GLS1)的表達(dá)水平;并進(jìn)一步,分析NDRG2與c-myc、β-catenin表達(dá)水平的相關(guān)性。4、通過細(xì)胞漿與細(xì)胞核的分離,免疫印跡檢測明確NDRG2與β-catenin亞細(xì)胞分布的關(guān)系。5、通過組織芯片免疫組織化學(xué)染色,分析NDRG2、c-myc在結(jié)直腸腫瘤的表達(dá)水平;通過新鮮、臨床結(jié)直腸腫瘤組織標(biāo)本免疫印跡檢測,分析NDRG2與c-myc、β-catenin、ASCT2、GLS1在臨床腫瘤組織中的表達(dá)水平及相關(guān)性。6、通過平板克隆形成實(shí)驗(yàn)、裸鼠成瘤實(shí)驗(yàn),分析NDRG2過表達(dá)細(xì)胞系的增殖能力,明確NDRG2對于細(xì)胞生長和增殖的影響。結(jié)果:1、代謝組學(xué)分析表明,與對照組相比,NDRG2過表達(dá)細(xì)胞系中谷氨酰胺及谷氨酸含量都出現(xiàn)顯著下降。2、代謝物檢測結(jié)果表明,與對照組相比,NDRG2過表達(dá)細(xì)胞系對谷氨酰胺的消耗降低、谷氨酸的產(chǎn)量也降低;NDRG2 RNA干涉細(xì)胞系對谷氨酰胺的消耗增加、谷氨酸的產(chǎn)量也增加。提示NDRG2通過抑制谷氨酰胺的攝取及分解,抑制谷氨酰胺分解代謝。3、轉(zhuǎn)錄和翻譯水平結(jié)果表明,與對照組相比,NDRG2過表達(dá)細(xì)胞系中ASCT2及GLS1的表達(dá)都出現(xiàn)顯著下調(diào),c-myc、β-catenin的水平也出現(xiàn)顯著下調(diào);NDRG2 RNA干涉細(xì)胞系中ASCT2及GLS1的表達(dá)都出現(xiàn)顯著上調(diào),c-myc及其上游調(diào)控元件β-catenin的水平也出現(xiàn)顯著上調(diào);降低c-Myc,將減弱NDRG2對谷氨酰胺分解代謝的抑制效應(yīng)。提示NDRG2抑制ASCT2及GLS1,并且NDRG2的對谷氨酰胺分解代謝的抑制作用是通過對c-myc及其上游分子β-catenin的抑制體現(xiàn)的。4、胞漿胞核分離檢測表明,與對照組相比,NDRG2過表達(dá)細(xì)胞系胞漿、胞核的β-catenin水平都出現(xiàn)了顯著下調(diào)。提示NDRG2通過抑制β-catenin的表達(dá)水平抑制c-myc,從而發(fā)揮對谷氨酰胺分解代謝的抑制作用。5、免疫組織化學(xué)結(jié)果表明,與瘤旁正常組織相比,結(jié)直腸腫瘤中NDRG2表達(dá)水平降低,c-myc表達(dá)水平升高,體現(xiàn)出負(fù)相關(guān)性。臨床結(jié)直腸腫瘤組織標(biāo)本免疫印跡檢測結(jié)果表明,與正常組織相比,結(jié)直腸腫瘤中NDRG2表達(dá)水平降低;c-myc、β-catenin、ASCT2、GLS1表達(dá)水平顯著升高,體現(xiàn)出顯著的負(fù)相關(guān)性。提示NDRG2對谷氨酰胺轉(zhuǎn)運(yùn)子ASCT2以及谷氨酰胺分解代謝的限速酶GLS1的調(diào)節(jié)與c-myc及其調(diào)控元件β-catenin密切相關(guān)。6、平板克隆形成實(shí)驗(yàn)、裸鼠成瘤實(shí)驗(yàn)結(jié)果表明,與對照組相比,NDRG2過表達(dá)細(xì)胞系克隆形成數(shù)減少;腫瘤體積及重量顯著降低。提示NDRG2通過對谷氨酰胺分解代謝的抑制作用顯著抑制細(xì)胞的增殖。結(jié)論:本研究揭示了NDRG2通過抑制癌基因c-myc從而發(fā)揮對結(jié)直腸腫瘤細(xì)胞谷氨酰胺分解代謝的抑制作用,并初步證實(shí)是這種抑制作用是通過抑制wnt通路的β-catenin發(fā)揮的。
[Abstract]:The growth and proliferation of malignant tumors rapidly, needed nutrients and energy supply. The metabolism of tumor cells showed obvious changes to adapt to the occurrence and development of tumor, its main performance is: aerobic glycolysis of glucose metabolism (glycolysis) enhanced glutamine enhanced catabolism, lipid and nucleotide biosynthesis enhancement of glutamine catabolism (glutaminolysis). In addition to the Warburg effect of tumor cells is another important energy metabolism. In addition to the consumption of glucose for energy, tumor cells but also through glutamine catabolism provides energy and biological macromolecules in the raw materials needed for its growth and proliferation, and glutamine metabolism is involved in cellular redox homeostasis and intracellular transduction signal the pathway of tumor cells. Mutations in oncogenes and tumor suppressor genes leads to glutamine metabolism. Tumor suppressor gene NDRG2 Malignant growth and proliferation of colorectal cancer, but the role of glutamine metabolism is not clear. NDRG2 (N-myc down-stream regulated gene 2 N-myc downstream regulated gene -2 gene) research for this research team first discovered and reported that the genes related to cell proliferation and differentiation, and in a variety of tumor tissue low expression, play the role of tumor suppressor genes. Previous studies showed that NDRG2 can inhibit tumor cell glycolysis metabolites of lactic acid production and decrease the consumption of glucose, NDRG2 can inhibit tumor cell glycolysis. On this basis, this paper wants to explore the regulatory effect and molecular mechanism of NDRG2 tumor cells for glutamine catabolism Objective: 1. To determine whether NDRG2 play a role in tumor glutamine metabolism decomposition; 2, study on the regulation of NDRG2 glutamine decomposition target metabolic pathways; 3, To investigate the molecular mechanism of the regulation of glutamine metabolic targets NDRG2 decomposition;; 4, to investigate the clinical significance of NDRG2 involved in the regulation of tumor metabolic reprogramming. Methods: 1, through the method of metabonomics analysis, overexpression of NDRG2 metabolite changes in the cell type and quantity, to determine whether NDRG2 affects.2 content of glutamine and glutamate in tumor cells the kit, through the analysis of metabolites, NDRG2 overexpression and decreased glutamine and glutamate in cell lines, clear NDRG2 on glutamine uptake and decomposition of the regulation of.3 by Real-time, PCR blotting method, analysis of NDRG2 expression and decreased glutamine transport in cell lines (ASCT2) and sub glutaminase 1 (GLS1) the expression level; and further, analysis of NDRG2 and c-myc, the level of correlation between the expression of.4 beta -catenin, by separating the cytoplasm and nucleus, Western blotting clear NDRG 2 and the subcellular distribution of beta -catenin.5, by immunohistochemical analysis, NDRG2, c-myc expression level in colorectal cancer; the clinical colorectal tumor tissue specimens of fresh, Western blotting analysis, NDRG2 and c-myc, beta -catenin, ASCT2 expression and correlation of.6 GLS1 in tumor tissues. By clone formation experiment, nude mice, analysis of NDRG2 overexpression cell line proliferation effect of clear NDRG2 for cell growth and proliferation. Results: 1, metabonomics analysis showed that compared with the control group, NDRG2 of glutamine and glutamate content expression cell lines were significantly decreased.2, metabolite test results show that, compared with the control group, NDRG2 over expression cell lines to reduce the consumption of glutamine, glutamate production also decreased; NDRG2 RNA interference cell line on glutamine consumption Increased glutamate production also increased. NDRG2 through inhibition of glutamine uptake and decomposition, inhibition of glutamine catabolism of.3, transcription and translation level. The results showed that compared with the control group, the expression of NDRG2 ASCT2 and GLS1 expression in cell lines were significantly reduced, c-myc, beta -catenin levels also significantly reduced; NDRG2 RNA interference expression of ASCT2 and GLS1 cells were significantly up-regulated, c-myc and its upstream regulatory element beta -catenin levels are significantly up-regulated; reduce c-Myc, will weaken the inhibitory effect of NDRG2 on glutamine catabolism. It suggests that the effect of NDRG2 ASCT2 and GLS1 on glutamine catabolism and the inhibitory effect of NDRG2 is reflected by inhibition the c-myc and its upstream molecular beta -catenin.4, showed that in both cytoplasm and nucleus were detected and compared with the control group, over expression of NDRG2 cell cytoplasm, the nucleus of the beta -caten The level of in had significantly decreased. NDRG2 inhibited c-myc expression by inhibiting beta -catenin, thereby inhibit.5 catabolism of glutamine, immunohistochemistry results showed that compared with the tumor adjacent normal tissues, colorectal cancer NDRG2 expression decreased, c-myc expression levels increased, reflecting the negative correlation between the clinical. Colorectal tumor tissues were detected by Western blot results showed that compared with normal tissue, reduce the level of NDRG2 expression in colorectal tumor; c-myc, beta -catenin, ASCT2, GLS1 expression level increased significantly, reflecting a significant negative correlation. It is suggested that the rate limiting enzyme of GLS1 catabolism of glutamine NDRG2 and glutamine transporter ASCT2 regulation is closely related to c-myc beta -catenin and its regulatory element.6, colony formation assay in nude mice, experimental results show that compared with the control group, over expression of NDRG2 cells Clone formation was decreased; the volume and weight of tumors were significantly decreased. NDRG2 significantly inhibited the cell through inhibition of catabolism of glutamine proliferation. Conclusion: This study reveals NDRG2 by inhibiting cancer gene c-myc to inhibit the catabolism of glutamine on colon cancer cells, and this inhibition is confirmed by inhibiting the Wnt pathway play a beta -catenin.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R735.34

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