NDRG2在人结直肠癌谷氨酰胺分解代谢中的作用与分子机制研究

发布时间：2018-03-16 10:01

本文选题：NDRG2　切入点：谷氨酰胺分解代谢　出处：《第四军医大学》2015年硕士论文　论文类型：学位论文

【摘要】：恶性肿瘤生长和增殖迅速,需要充足的营养物质和能量供给。肿瘤细胞代谢出现明显改变以适应肿瘤的发生和发展,其主要表现为:葡萄糖有氧酵解代谢(glycolysis)增强,谷氨酰胺分解代谢增强,脂类和核苷酸生物合成增强等。谷氨酰胺分解代谢(glutaminolysis)是肿瘤细胞除Warburg效应外另一重要的能量代谢方式。除了消耗葡萄糖供能外,肿瘤细胞还会通过谷氨酰胺分解代谢为其生长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谢还参与细胞内氧化还原稳态的维持和细胞内信号通路的转导。肿瘤细胞中原癌基因和抑癌基因的突变会导致谷氨酰胺代谢异常。抑癌基因NDRG2抑制结直肠肿瘤的恶性生长和增殖,但其在谷氨酰胺分解代谢中的作用尚不明确。人NDRG2(N-myc down-stream regulated gene 2,N-myc下游调节基因-2)基因为本研究小组首先发现并报道,研究发现,该基因与细胞增殖和分化相关,并且在多种肿瘤组织低表达,发挥抑癌基因的作用。前期研究表明NDRG2能够抑制肿瘤细胞糖酵解代谢产物乳酸的产生并降低葡萄糖的消耗,即NDRG2可以抑制肿瘤细胞糖酵解代谢。在此基础上,本课题想要探讨NDRG2对于肿瘤细胞谷氨酰胺分解代谢的调控作用与分子机制。目的:1、明确NDRG2是否在肿瘤谷氨酰胺分解代谢中发挥作用;2、研究NDRG2调控谷氨酰胺分解代谢途径的靶点;3、探讨NDRG2调控谷氨酰胺分解代谢靶点的分子机制;;4、探讨NDRG2参与调控肿瘤代谢重编程的临床意义。方法:1、通过代谢组学的方法,分析NDRG2过表达细胞中代谢物种类及量的变化,明确NDRG2是否影响肿瘤细胞中谷氨酰胺和谷氨酸的含量。2、通过试剂盒检测代谢物,分析NDRG2过表达与降低细胞系中谷氨酰胺和谷氨酸的变化,明确NDRG2对谷氨酰胺摄取及分解的调节作用。3、通过Real-time PCR、免疫印迹的方法,分析NDRG2过表达与降低细胞系中谷氨酰胺转运子(ASCT2)及谷氨酰胺酶1(GLS1)的表达水平;并进一步,分析NDRG2与c-myc、β-catenin表达水平的相关性。4、通过细胞浆与细胞核的分离,免疫印迹检测明确NDRG2与β-catenin亚细胞分布的关系。5、通过组织芯片免疫组织化学染色,分析NDRG2、c-myc在结直肠肿瘤的表达水平;通过新鲜、临床结直肠肿瘤组织标本免疫印迹检测,分析NDRG2与c-myc、β-catenin、ASCT2、GLS1在临床肿瘤组织中的表达水平及相关性。6、通过平板克隆形成实验、裸鼠成瘤实验,分析NDRG2过表达细胞系的增殖能力,明确NDRG2对于细胞生长和增殖的影响。结果:1、代谢组学分析表明,与对照组相比,NDRG2过表达细胞系中谷氨酰胺及谷氨酸含量都出现显著下降。2、代谢物检测结果表明,与对照组相比,NDRG2过表达细胞系对谷氨酰胺的消耗降低、谷氨酸的产量也降低;NDRG2 RNA干涉细胞系对谷氨酰胺的消耗增加、谷氨酸的产量也增加。提示NDRG2通过抑制谷氨酰胺的摄取及分解,抑制谷氨酰胺分解代谢。3、转录和翻译水平结果表明,与对照组相比,NDRG2过表达细胞系中ASCT2及GLS1的表达都出现显著下调,c-myc、β-catenin的水平也出现显著下调;NDRG2 RNA干涉细胞系中ASCT2及GLS1的表达都出现显著上调,c-myc及其上游调控元件β-catenin的水平也出现显著上调;降低c-Myc,将减弱NDRG2对谷氨酰胺分解代谢的抑制效应。提示NDRG2抑制ASCT2及GLS1,并且NDRG2的对谷氨酰胺分解代谢的抑制作用是通过对c-myc及其上游分子β-catenin的抑制体现的。4、胞浆胞核分离检测表明,与对照组相比,NDRG2过表达细胞系胞浆、胞核的β-catenin水平都出现了显著下调。提示NDRG2通过抑制β-catenin的表达水平抑制c-myc,从而发挥对谷氨酰胺分解代谢的抑制作用。5、免疫组织化学结果表明,与瘤旁正常组织相比,结直肠肿瘤中NDRG2表达水平降低,c-myc表达水平升高,体现出负相关性。临床结直肠肿瘤组织标本免疫印迹检测结果表明,与正常组织相比,结直肠肿瘤中NDRG2表达水平降低;c-myc、β-catenin、ASCT2、GLS1表达水平显著升高,体现出显著的负相关性。提示NDRG2对谷氨酰胺转运子ASCT2以及谷氨酰胺分解代谢的限速酶GLS1的调节与c-myc及其调控元件β-catenin密切相关。6、平板克隆形成实验、裸鼠成瘤实验结果表明,与对照组相比,NDRG2过表达细胞系克隆形成数减少;肿瘤体积及重量显著降低。提示NDRG2通过对谷氨酰胺分解代谢的抑制作用显著抑制细胞的增殖。结论:本研究揭示了NDRG2通过抑制癌基因c-myc从而发挥对结直肠肿瘤细胞谷氨酰胺分解代谢的抑制作用,并初步证实是这种抑制作用是通过抑制wnt通路的β-catenin发挥的。
[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.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.34

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