PGRN促进宫颈癌细胞在血清饥饿压力下生存的抗氧化机制研究
发布时间：2018-10-14 18:24
【摘要】：宫颈癌是女性生殖系统最常见的恶性肿瘤之一。据统计数据显示,宫颈癌是女性第四常见的肿瘤,并已成为15-44岁女性中仅次于乳腺癌的第二杀手；2012年全球有527,624例新发宫颈癌,85%以上发生在发展中国家,死亡病例为265,653例,死亡率为50.3%。高危型人乳头瘤病毒(human papillomavirus, HPV)感染是宫颈癌的首要病因,但仅有少数HPV感染的女性发展为宫颈癌,提示其他因素也参与了宫颈癌的进展。组织侵袭与转移、生长信号自给、生长抑制信号耐受、逃脱程序性死亡、无限的复制潜能、持续的血管生成能力是癌细胞恶性进展的主要特征。癌细胞在机体内出现后,会遭受低氧、低糖、细胞与细胞之间或细胞与细胞外基质(extracellular matrix, ECM)之间的支持不足、营养和生长因子缺乏等微环境压力。只有成功适应这些压力,癌细胞才能在获得抗凋亡、促增殖活性的同时,维持血管生成、永生化、侵袭与转移等特性。氧化还原平衡是维持细胞正常功能、确保细胞生存的根本机制。代谢缺陷、低氧、内质网应激、癌基因均可引起活性氧(Reactive oxygen species, ROS)的产生。生长因子激活的PI3K/Akt途径具有刺激葡萄糖转运和糖酵解的功能,当生长因子缺乏时细胞PI3K/Akt信号途径活性降低,细胞葡萄糖摄入能力下降,间接引起葡萄糖代谢缺陷,引起ROS的产生。过多的ROS会引起蛋白质氧化、脂质过氧化、线粒体与DNA的损伤。红系衍生核因子相关因子2 (Nuclear factor (erythroid-derived 2)-related factor 2, Nrf2)是细胞抗氧化机制最重要的调控分子之一。在正常情况下Nrf2存在于胞浆中并与Keap1结合,经泛素化降解；在某些诱导因素作用下Nrf2与Keap1解离进入细胞核,结合多种抗氧化相关蛋白与Ⅱ相解毒酶基因调控区的抗氧化作用元件(antioxidant response element, ARE),驱动这些基因的表达,从而发挥其抗氧化作用。颗粒蛋白前体(progranulin, PGRN)是一种由71/2个富含半胱氨酸的GRN基序组成的生长因子,参与早期胚胎发育、骨发育、炎症、损伤修复、神经退行性病变、癌症等生理和疾病进程。本研究的前期工作证实PGRN在宫颈癌组织中高表达,并可通过PI3K/Akt与Erk信号途径促进宫颈癌细胞增殖与恶性转化。PGRN可以促进细胞增殖、生存与迁移等,是正常细胞与癌细胞公认的生存因子之一,能够促进细胞在化疗药物、低氧、酸性环境等压力下的存活,但PGRN在肿瘤微环境压力下癌细胞生存中的功能与调控机制尚不清楚。在明确PGRN在宫颈癌致癌机制中具有关键作用的基础七,本论文采用血清饥饿模拟肿瘤微环境压力,分析PGRN在宫颈癌细胞适应微环境压力中的功能,并探究PGRN促进微环境压力下宫颈癌细胞生存的抗氧化应激分子机制。本论文的主要发现包括：(1) PGRN为血清应答蛋白,血清饥饿导致宫颈癌细胞中PGRN蛋白的急剧下降；(2) PGRN能够抵御血清饥饿诱导的宫颈癌细胞死亡及凋亡信号,证明PGRN具有促进癌细胞在肿瘤微环境压力下生存的功能；(3) PGRN抑制血清饥饿处理的宫颈癌细胞内ROS水平,并减轻蛋白质氧化、脂质过氧化、DNA氧化损伤以及线粒体功能失调等,证明PGRN可介导抗氧化应激作用,促进癌细胞适应微环境压力；(4)PGRN可通过增强ROS清除剂系统多种抗氧化蛋白与Ⅱ相脱毒酶的表达与活性、提高细胞内关键还原性物质水平实现其抗氧化作用；(5)PGRN能够促进血清饥饿处理的宫颈癌细胞中重要的抗氧化相关转录因子Nrf2的表达,并促进Nrf2与Keap1的解离,进而转移入核,发挥其转录活性；(6)血清饥饿压力下PGRN的生存因子功能与抗氧化作用依赖于Nrf2/ARE信号途径。本论文首次证实PGRN在血清饥饿压力下胞内蛋白水平急剧下降的表达特征；首次发现PGRN通过增强细胞内ROS清除系统的活性,抵抗微环境压力诱导的氧化应激,促进癌细胞的生存。这些发现提示PGRN除通过促进细胞增殖与迁移外,还可介导癌细胞适应微环境压力驱动癌症的进展,进一步完善PGRN介导癌细胞恶性进展的机理,为以PGRN及其介导的Nrf2/ARE信号途径为靶点的包括宫颈癌的各类肿瘤的治疗提供新的治疗策略与研究证据；同时也为PGRN在炎症应答、神经退行性疾病及其他生理和病理进程中的功能提供抗氧化机制。
[Abstract]:Cervical cancer is one of the most common malignant tumors in female reproductive system. Cervical cancer is the fourth most common tumor in women and has become the second killer next to breast cancer among 15-44-year-olds, according to statistics. In 2012, 527,624 new cervical cancer, more than 85% occurred in developing countries, and death cases were 265, 653, with a mortality rate of 50.3%. High-risk human papillomavirus (HPV) infection is the primary cause of cervical cancer, but only a few HPV-infected women develop cervical cancer, suggesting that other factors are also involved in the progress of cervical cancer. Tissue invasion and metastasis, self-sufficiency of growth signals, tolerance of growth inhibition signals, escape of programmed death, unlimited replication potential, and sustained angiogenesis are the major features of malignant progression of cancer cells. In vivo, cancer cells suffer from hypoxia, low sugar, insufficient support between cells and cells or between cells and extracellular matrix (ECM), lack of nutritional and growth factors, and the like. Only by successfully adapting to these pressures, cancer cells can maintain the characteristics of angiogenesis, eternal life, invasion and metastasis while obtaining anti-apoptotic and pro-proliferative activity. The redox balance is the fundamental mechanism to maintain the normal function of cells and to ensure the survival of cells. Metabolic defects, hypoxia, ER stress and oncogene can cause reactive oxygen species (ROS) generation. The p38/ Akt pathway activated by the growth factor has the function of stimulating glucose transport and glycolysis, and when the growth factor is absent, the cell affinity/ Akt signal pathway activity is reduced, the cell glucose uptake capacity is reduced, the glucose metabolism defect is indirectly caused, and the generation of ROS is caused. Too much ROS can cause protein oxidation, lipid peroxidation, mitochondrial and DNA damage. Red-derived nuclear factor-related factor 2 (Nrf2) is one of the most important regulatory molecules in cell antioxidant mechanism. In normal cases, Nrf2 is present in cytoplasm and binds to Keap1, is degraded by ubiquitination, and Nrf2 dissociates into nucleus with Keap1 under the action of certain induction factors, and binds various anti-oxidation-related proteins and anti-oxidation active element (ARE) in the regulatory region of II-phase detoxication enzyme gene. The expression of these genes is driven so as to exert its anti-oxidation effect. Particle protein precursor (PGRN) is a growth factor composed of 71/ 2 cysteine-rich GRIN motifs, and is involved in the processes of early embryonic development, bone development, inflammation, injury repair, neurodegenerative diseases, and cancer. The preliminary work of this study confirms that PGRN is highly expressed in cervical cancer tissues, and can promote the proliferation and malignant transformation of cervical cancer cells by means of p38/ Akt and Erk signaling pathway. PGRN can promote cell proliferation, survival and migration and the like, is one of the well-known survival factors of normal cells and cancer cells, and can promote the survival of cells under pressure such as chemotherapy drugs, hypoxia, acidic environment and the like, However, the function and regulation mechanism of PGRN in cancer cell survival at micro-ambient pressure is unclear. In order to clarify the key role of PGRN in carcinogenesis of cervical cancer, this paper uses serum starvation to simulate the micro-environmental stress of tumor, and analyses the function of PGRN in cervical cancer cells to adapt to micro-environmental pressure. and explore the anti-oxidative stress molecular mechanism of PGRN for promoting the survival of cervical cancer cells under micro-ambient pressure. The main findings of this paper include: (1) PGRN is a serum-responsive protein, serum starvation leads to a sharp decrease in PGRN protein in cervical cancer cells; (2) PGRN is capable of resisting the death and apoptosis signals of cervical cancer cells induced by serum starvation, It is proved that PGRN has the function of promoting the survival of cancer cells in tumor microenvironment; (3) PGRN inhibits the level of ROS in cervical cancer cells treated by serum starvation, and reduces protein oxidation, lipid peroxidation, DNA oxidative damage and mitochondrial dysfunction, etc. It is proved that PGRN can mediate anti-oxidative stress and promote the adaptation of cancer cells to microambient pressure; (4) PGRN can increase the level of key reducing substances in cells by enhancing the expression and activity of various anti-oxidation proteins of ROS scavenger system and II-phase detoxification enzymes, and improve the level of key reducing substances in cells. (5) PGRN can promote the expression of important anti-oxidation-related transcription factor Nrf2 in cervical cancer cells treated by serum starvation, and promote the dissociation of Nrf2 and Keap1, and then transfer into the nucleus to exert its transcriptional activity; (6) The survival factor function and anti-oxidation effect of PGRN under serum starvation pressure depend on the Nrf2/ ARE signal pathway. For the first time, the expression of PGRN in the intracellular protein level in serum starvation was confirmed; the first time we found that PGRN enhanced the activity of ROS scavenging system in the cell, resist oxidative stress induced by microenvironmental stress, and promote the survival of cancer cells. These findings suggest that, in addition to promoting cell proliferation and migration, PGRN can mediate cancer cell adaptation to micro-ambient pressure to drive cancer progression, and further improve the mechanism of PGRN-mediated malignant progression of cancer cells. To provide new therapeutic strategies and research evidence for the treatment of various tumors including cervical cancer with PGRN and its mediated Nrf2/ ARE signaling pathway; meanwhile, it also provides antioxidant mechanism for the function of PGRN in inflammatory response, neurodegenerative diseases and other physiological and pathological processes.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.33
[Abstract]:Cervical cancer is one of the most common malignant tumors in female reproductive system. Cervical cancer is the fourth most common tumor in women and has become the second killer next to breast cancer among 15-44-year-olds, according to statistics. In 2012, 527,624 new cervical cancer, more than 85% occurred in developing countries, and death cases were 265, 653, with a mortality rate of 50.3%. High-risk human papillomavirus (HPV) infection is the primary cause of cervical cancer, but only a few HPV-infected women develop cervical cancer, suggesting that other factors are also involved in the progress of cervical cancer. Tissue invasion and metastasis, self-sufficiency of growth signals, tolerance of growth inhibition signals, escape of programmed death, unlimited replication potential, and sustained angiogenesis are the major features of malignant progression of cancer cells. In vivo, cancer cells suffer from hypoxia, low sugar, insufficient support between cells and cells or between cells and extracellular matrix (ECM), lack of nutritional and growth factors, and the like. Only by successfully adapting to these pressures, cancer cells can maintain the characteristics of angiogenesis, eternal life, invasion and metastasis while obtaining anti-apoptotic and pro-proliferative activity. The redox balance is the fundamental mechanism to maintain the normal function of cells and to ensure the survival of cells. Metabolic defects, hypoxia, ER stress and oncogene can cause reactive oxygen species (ROS) generation. The p38/ Akt pathway activated by the growth factor has the function of stimulating glucose transport and glycolysis, and when the growth factor is absent, the cell affinity/ Akt signal pathway activity is reduced, the cell glucose uptake capacity is reduced, the glucose metabolism defect is indirectly caused, and the generation of ROS is caused. Too much ROS can cause protein oxidation, lipid peroxidation, mitochondrial and DNA damage. Red-derived nuclear factor-related factor 2 (Nrf2) is one of the most important regulatory molecules in cell antioxidant mechanism. In normal cases, Nrf2 is present in cytoplasm and binds to Keap1, is degraded by ubiquitination, and Nrf2 dissociates into nucleus with Keap1 under the action of certain induction factors, and binds various anti-oxidation-related proteins and anti-oxidation active element (ARE) in the regulatory region of II-phase detoxication enzyme gene. The expression of these genes is driven so as to exert its anti-oxidation effect. Particle protein precursor (PGRN) is a growth factor composed of 71/ 2 cysteine-rich GRIN motifs, and is involved in the processes of early embryonic development, bone development, inflammation, injury repair, neurodegenerative diseases, and cancer. The preliminary work of this study confirms that PGRN is highly expressed in cervical cancer tissues, and can promote the proliferation and malignant transformation of cervical cancer cells by means of p38/ Akt and Erk signaling pathway. PGRN can promote cell proliferation, survival and migration and the like, is one of the well-known survival factors of normal cells and cancer cells, and can promote the survival of cells under pressure such as chemotherapy drugs, hypoxia, acidic environment and the like, However, the function and regulation mechanism of PGRN in cancer cell survival at micro-ambient pressure is unclear. In order to clarify the key role of PGRN in carcinogenesis of cervical cancer, this paper uses serum starvation to simulate the micro-environmental stress of tumor, and analyses the function of PGRN in cervical cancer cells to adapt to micro-environmental pressure. and explore the anti-oxidative stress molecular mechanism of PGRN for promoting the survival of cervical cancer cells under micro-ambient pressure. The main findings of this paper include: (1) PGRN is a serum-responsive protein, serum starvation leads to a sharp decrease in PGRN protein in cervical cancer cells; (2) PGRN is capable of resisting the death and apoptosis signals of cervical cancer cells induced by serum starvation, It is proved that PGRN has the function of promoting the survival of cancer cells in tumor microenvironment; (3) PGRN inhibits the level of ROS in cervical cancer cells treated by serum starvation, and reduces protein oxidation, lipid peroxidation, DNA oxidative damage and mitochondrial dysfunction, etc. It is proved that PGRN can mediate anti-oxidative stress and promote the adaptation of cancer cells to microambient pressure; (4) PGRN can increase the level of key reducing substances in cells by enhancing the expression and activity of various anti-oxidation proteins of ROS scavenger system and II-phase detoxification enzymes, and improve the level of key reducing substances in cells. (5) PGRN can promote the expression of important anti-oxidation-related transcription factor Nrf2 in cervical cancer cells treated by serum starvation, and promote the dissociation of Nrf2 and Keap1, and then transfer into the nucleus to exert its transcriptional activity; (6) The survival factor function and anti-oxidation effect of PGRN under serum starvation pressure depend on the Nrf2/ ARE signal pathway. For the first time, the expression of PGRN in the intracellular protein level in serum starvation was confirmed; the first time we found that PGRN enhanced the activity of ROS scavenging system in the cell, resist oxidative stress induced by microenvironmental stress, and promote the survival of cancer cells. These findings suggest that, in addition to promoting cell proliferation and migration, PGRN can mediate cancer cell adaptation to micro-ambient pressure to drive cancer progression, and further improve the mechanism of PGRN-mediated malignant progression of cancer cells. To provide new therapeutic strategies and research evidence for the treatment of various tumors including cervical cancer with PGRN and its mediated Nrf2/ ARE signaling pathway; meanwhile, it also provides antioxidant mechanism for the function of PGRN in inflammatory response, neurodegenerative diseases and other physiological and pathological processes.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.33
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