代谢应激条件下过表达NDRG2对肝癌细胞存活的抑制作用及其机制研究

发布时间：2018-04-01 20:22

  本文选题：NDRG2　切入点：脂肪酸氧化　出处：《第四军医大学》2017年博士论文

【摘要】：在肿瘤的形成和发展过程中,常常伴随着营养物质和氧气的供给不足[1]。特别是在实体瘤的迅速增长过程中,由于相对血供不足,常导致瘤体内的许多区域缺乏氧气和葡萄糖等营养物质[2]。在这种情况下,肿瘤细胞必须通过代谢模式的改变以适应恶劣的微环境。肿瘤细胞代谢重编程最为熟知的当属Warburg效应,其典型特征是主要通过增强产能效率低下的糖酵解途径来生产ATP并伴随大量乳酸的形成[3]。除此之外,肿瘤细胞也常常发生谷氨酰胺代谢和脂代谢的重编程[4],例如在缺乏葡萄糖等应激情况下,肿瘤细胞通过增强脂肪酸氧化分解来提供能量和营养物质,维持细胞存活[5,6]。由于这种对代谢应激的适应能力,在临床抗血管生成药物治疗后,复发的肿瘤细胞甚至表现出更强的增殖和转移能力[7]。因此,研究肿瘤代谢重编程的分子机制具有重要的理论意义,也能为肿瘤的分子靶向治疗提供新策略。NDRG2是我们课题组首先报道的抑癌基因,在人体多种肿瘤组织中低表达,过表达NDRG2后可以抑制多种肿瘤细胞系的增殖、迁移和侵袭[8]。另外,NDRG2还参与细胞的多种应激反应[9]。更为重要的是,越来越多的研究证实NDRG2与肿瘤细胞代谢有密切关系。我们前期的研究发现,NDRG2能够抑制乳腺癌细胞的葡萄糖摄取和结肠癌细胞的糖酵解和谷氨酰胺代谢[10,11]。然而NDRG2是否参与肿瘤细胞脂代谢还未见报道。基于此,本课题拟以过表达NDRG2的肝癌细胞为研究模型,探讨实体瘤因微环境异质性和基质脱落等代谢应激条件下NDRG2在肝癌细胞中的作用。目的:1.明确代谢应激时NDRG2对肝癌细胞存亡的影响2.研究缺糖应激时NDRG2对肝癌细胞能量及氧化还原状态的影响3.探究NDRG2在肝癌细胞缺糖应激中所起作用的分子机制方法:1.利用慢病毒感染构建过表达NDRG2的肝癌细胞系Huh7和Sk-hep-1及表达Cherry的对照细胞,q PCR和Western blot检测NDRG2的表达情况;CCK8实验检测和比较上述细胞在正常培养和代谢应激情况下的细胞活力;克隆形成实验检测正常培养和缺糖应激下的克隆形成能力;流式细胞术和TUNEL染色检测细胞凋亡。2.分别用ATP检测试剂盒、NADP/NADPH定量比色试剂盒和DCFH-DA探针检测NDRG2过表达和对照肝癌细胞在正常培养和缺糖应激情况下的细胞内ATP水平、NADPH含量和NADP+/NADPH比值以及细胞内ROS水平。3.q PCR检测过表达NDRG2和对照肝癌细胞在正常培养和缺糖应激下脂肪酸氧化(FAO)关键调控分子的表达情况。给予PPARα的激活剂BEZA(400μM)和CPT1A的抑制剂ETO(100μM)后,分别用ATP检测试剂盒、NADP/NADPH定量比色试剂盒和CCK-8检测上述细胞在低糖应激下的细胞内ATP水平、NADPH含量以及细胞存活情况。4.Western blot检测NDRG2过表达肝癌细胞在正常和缺糖情况下AMPK/ACC通路的活化情况(p-AMPKα,AMPKα,p-ACC和ACC);在NDRG2过表达细胞Huh7中强行表达组成性活化的AMPKα,Western blot比较AMPK信号通路的活化情况,流式细胞术比较细胞凋亡。结果:1.CCK-8实验、克隆形成实验和凋亡检测结果表明,缺糖应激条件下过表达NDRG2抑制肝癌细胞存活,降低肝癌细胞的克隆形成能力,促进缺糖应激中肝癌细胞的凋亡。2.细胞内ATP、NADPH和ROS检测结果表明,过表达NDRG2降低缺糖应激条件下肝癌细胞内ATP和NADPH含量,升高NADP+/NADPH比值和细胞内ROS水平,过表达NDRG2加剧了缺糖应激下肝癌细胞能量和氧化还原失衡。3.NDRG2过表达可抑制缺糖应激诱导的脂肪酸氧化相关分子PPARα、CPT1A和ACADM的表达上调及脂肪酸氧化的活化。PPARα的激动剂BEZA可增加缺糖应激时NDRG2过表达肝癌细胞内ATP和NADPH含量,从而削弱缺糖时NDRG2对细胞存活的抑制作用。反之,CPT1A的抑制剂ETO则显著降低细胞ATP和NADPH含量,抑制缺糖应激下的细胞存活,该作用在对照细胞中比NDRG2过表达细胞更明显。4.NDRG2过表达可抑制缺糖应激条件下肝癌细胞中AMPK信号通路的活化;组成性活化的AMPK可逆转NDRG2对AMPK信号通路的抑制作用并部分缓解缺糖应激诱导的肝癌细胞凋亡。结论:缺糖应激条件下过表达NDRG2通过抑制肝癌细胞AMPK信号通路的活化,干扰脂肪酸氧化的激活,加剧肝癌细胞的能量耗竭和氧化应激并导致细胞凋亡,最终降低缺糖应激时肝癌细胞的存活能力。
[Abstract]:In the process of formation and development of tumor, often accompanied by the insufficient supply of nutrients and oxygen to [1]. especially in the rapid growth of solid tumors in the process, due to the relatively insufficient blood supply, often leads to many areas of tumors lack of oxygen and glucose, nutrients such as [2]. in this case, tumor cells must through metabolic mode change in order to adapt to the harsh environment. The effect of Warburg micro is known as tumor cell metabolic reprogramming, its typical character is mainly by enhancing the capacity of low efficiency glycolysis to produce ATP and [3]. formed in addition with large amounts of lactic acid, tumor cells often occur and lipid metabolism of glutamine reprogramming [4], for example, in the absence of glucose stress tumor cells by enhanced fatty acid oxidation to provide energy and nutrients to maintain cell survival by [5,6]. in the The metabolic stress adaptation in clinical anti angiogenesis drugs after treatment, the recurrence of tumor cells showed even stronger proliferation and metastasis of [7]., therefore, has an important theoretical study on the molecular mechanism of tumor metabolic reprogramming, but also for tumor molecular targeted therapy provides a new strategy for.NDRG2 is an oncogene and our group first reported, low expression in a variety of human tumor tissues, overexpression of NDRG2 can inhibit tumor cell proliferation, migration and invasion of [8]. and NDRG2 were involved in various stress responses [9]. is even more important, more and more studies have confirmed that NDRG2 has a close relationship with the metabolism of tumor cells our previous study showed that NDRG2 can inhibit breast cancer cell glucose uptake in human colorectal cancer cells and glycolysis and glutamine metabolism in [10,11]. but NDRG2 is involved in tumor Cell lipid metabolism has not been reported. Based on this, this paper intends to over expression of NDRG2 in hepatocellular carcinoma cells as a model, to investigate the effect of solid tumors due to micro environmental heterogeneity and matrix off metabolic stress under the condition of NDRG2 in hepatocellular carcinoma. Objective: 1. clear metabolic stress effects of NDRG2 on liver cancer cell survival of 2. glucose deprivation stress NDRG2 redox state effect on hepatoma cells and explore the 3. energy oxidation of NDRG2 in hepatoma cell glucosedeprivation plays stress molecular mechanism of the effect of 1. methods: using lentiviral infection constructed over expression of NDRG2 in hepatocellular carcinoma cell line Huh7 and Sk-hep-1 and Cherry expression in control cells, the expression of Q PCR and Western blot detection NDRG2; cell viability by CCK8 assay and compared the cells in normal culture and metabolic stress conditions; colony forming assay and cloning of cultured normal glucose deprivation stress formation Force; flow cytometry and TUNEL staining to detect the apoptosis of.2. cells were detected by ATP kit, NADP/NADPH quantitative colorimetric kit and DCFH-DA probe for detection of NDRG2 expression and control of hepatoma cells in normal culture and lack of sugar should be ATP level below passionate condition in cells, NADPH content and NADP +/NADPH ratio and intracellular ROS level.3.q PCR tested the expression of NDRG2 and control cells cultured under normal glucose deprivation stress and fatty acid oxidation (FAO) expression of key regulatory molecules. PPAR alpha activator BEZA (400 M) and CPT1A inhibitor ETO (100 M), were detected by ATP kit, NADP/NADPH quantitative colorimetric detection kit and CCK-8 cells in the low glucose stress under the intracellular ATP level, NADPH content and cell survival of.4.Western blot detection NDRG2 hepatocellular carcinoma cells in normal and low glucose conditions AMPK/ACC pathway activity Situation (p-AMPK alpha, alpha AMPK, p-ACC and ACC); in NDRG2 overexpressing Huh7 cells expressing a constitutively activated AMPK by activation of Western blot alpha, AMPK signaling pathway, flow cytometric analysis of cell apoptosis. Results: the 1.CCK-8 assay, clone formation assay and apoptosis detection results show that the lack of sugar stress overexpression of NDRG2 inhibits the survival of cancer cells, cancer cells reduced the colony forming ability, promote the lack of sugar stress in hepatocellular carcinoma cell apoptosis of.2. cells in ATP, NADPH and ROS results showed that overexpression of NDRG2 reduced glucose deprivation stress conditions in hepatocellular carcinoma cell line ATP and the content of NADPH, increase the level of ROS NADP+/NADPH ratio and in cells, overexpression of NDRG2 increased cell energy and oxidation of glucose deprivation stress reduction imbalance.3.NDRG2 overexpression inhibits glucose deprivation stress induced fatty acid oxidation related molecule PPAR alpha, CPT1A expression and ACADM. And fatty acid oxidation of activated.PPAR alpha agonist BEZA could increase glucose deprivation stress NDRG2 expression content of ATP and NADPH cells, thus weakening the lack of sugar when the inhibitory effect of NDRG2 on cell survival. On the contrary, the CPT1A inhibitor ETO significantly decreased the content of NADPH and ATP cells, inhibit glucose deprivation stress the role of cell survival in control cells than in NDRG2 overexpressing cells more obvious overexpression of.4.NDRG2 can inhibit glucose deprivation stress activation of AMPK signaling pathway in hepatocellular carcinoma cells; constitutively activated AMPK can reverse the NDRG2 of the AMPK signaling pathway inhibition and apoptosis of hepatocellular carcinoma cells with partial remission induced by glucose deprivation stress. Conclusion glucose deprivation stress conditions: overexpression of NDRG2 by inhibiting the activation of AMPK signaling in hepatocellular carcinoma cells, activate the interference of fatty acid oxidation, increased energy depletion and oxidative stress in liver cancer cells and induce cell apoptosis, The survival ability of hepatoma cells was reduced in the end.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.7
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本文编号：1697160