【作者】 李文娟；

【导师】 李小玲； 向波；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2013， 博士

【摘要】 本课题前期研究工作基础NOR1是本实验室自主克隆的鼻咽癌易感基因,在鼻咽癌组织中显著下调,但其下调机制和转录调控规律尚未阐明。NOR1蛋白定位于线粒体和胞浆中,与OSCP蛋白存在直接交互作用；NOR1表达抑制鼻咽癌细胞的生长与增殖,并促进缺氧引起的细胞凋亡。但该基因在肿瘤发生发展中的作用及机制研究的还未成熟。定位克隆NOR1基因的核心启动子采用预测软件PromoterScan、PromoterInspector和EMBOSS CpG Plot综合分析发现,NOR1基因启动子区可能位于转录起始位点(为+1)上游-517bp至下游+236bp以内。为了确定NOR1基因核心启动子序列,将NOR1基因的预测启动子调控区分为几段,构建出一系列NOR1基因不同调控区荧光素酶报告载体和绿色荧光蛋白报告载体。通过luciferase assay和荧光显微镜观察联合分析,发现NOR1核心启动子定位于NOR1基因5’端-258/+26区间内。NOR1基因启动子区存在一个典型的CpG岛,在鼻咽癌组织和细胞中因启动子高甲基化修饰而表达下调MatInspector软件分析发现NOR1基因启动子区序列无TATA盒和CAAT盒,GC含量高达58.8%,为一典型的CpG岛,提示NOR1启动子转录活性可能受到甲基化的影响。采用SssI甲基化酶体外甲基化修饰NOR1启动子报告载体,通过荧光素酶报告系统及绿色荧光蛋白报告系统检测发现,甲基化使NOR1启动子彻底丧失转录活性。进一步联合BSP和MSP两种经典甲基化检测方法分别对鼻咽癌组织、细胞及鼻咽正常上皮组织和细胞中NOR1的启动子甲基化状态进行检测,结果发现NOR1启动子在鼻咽癌组织、细胞系中呈不同程度的高甲基化状态,其中NOR1启动子在HSF1、NRF1结合部位含有CG位点,在鼻咽癌组织和细胞系中发生甲基化修饰,而在正常鼻咽组织或鼻咽上皮细胞系中呈完全非甲基化状态。免疫组化和RT-PCR检测发现,鼻咽癌组织和细胞系中NOR1mRNA和蛋白水平明显下调。采用甲基化酶抑制剂5-Aza-CdR处理NOR1启动子高甲基化细胞HL60和6-10B,RT-PCR及实时荧光定量PCR方法检测发现,5-Aza-CdR可以改变细胞中NOR1启动子的甲基化状态并恢复NOR1基因nRNA的表达。结果表明NOR1启动子的甲基化状态与其表达水平密切相关,启动子甲基化是使NOR1在鼻咽癌中表达下调的重要机制。应激相关转录因子HSF1和NRF1共同正向调控NOR1基因启动子活性采用转录因子预测软件MatInspector预测NOR1基因启动子区潜在的转录因子结合位点,结果显示在NOR1基因启动子区可能存在与应激密切相关的两个转录因子--热休克因子HSF1、核呼吸因子NRF1的结合位点。通过凝胶电泳迁移率实验(EMSA)和染色质免疫共沉淀技术(ChIP)证实HSF1和NRF1两个转录因子确实能与NOR1核心启动子序列特异性结合。进一步运用荧光素酶报告载体分析系统研究发现,转录因子HSF1蛋白能够增强细胞内NOR1启动子活性；Western blot检测发现瞬时转染HSF1和NRF1能够上调Hela细胞内NOR1蛋白表达水平；采用shRNA慢病毒感染293细胞,干扰HSF1或NRF1表达,通过PCR检测发现NOR1mRNA表达下调。以上结果均显示,转录因子HSF1、NRF1能够促进NOR1基因的转录。H202可以诱导鼻咽上皮细胞及恶性肿瘤细胞中NOR1基因的表达HSF1和NRF1能参与氧化应激调控,据此推测NOR1可能与氧化应激有关。采用H202处理鼻咽上皮细胞系NP69和乳腺癌细胞系MCF7,诱导氧化应激,通过实时荧光定量PCR、免疫荧光结合流式细胞分析证实,H202能够诱导鼻咽上皮细胞系NP69和乳腺癌细胞系MCF7中NOR1的表达,因此认为NOR1是一个氧化应激反应性蛋白。NOR1抑制肿瘤细胞自噬,影响能量代谢并抑制肿瘤细胞生长采用已建立的NOR1稳定表达HNE1细胞株,通过生长曲线实验发现,NOR1抑制了鼻咽癌细胞HNE1的生长和增殖,软琼脂集落形成实验发现,NORl明显抑制HNE1细胞的集落形成能力和增殖；而在NOR1表达相对较高的宫颈癌细胞HeLa中,干扰NOR1促进了HeLa的生长和增殖。电镜图片、Western blot等技术检测发现,过表达NOR1能够有效地抑制肿瘤细胞中基础水平的自噬行为,而在NOR1表达相对较高的HeLa细胞中使用RNAi干扰技术敲除NOR1的表达则能增强细胞内自噬水平。细胞自噬与肿瘤细胞能量代谢密切相关。乳酸生存率及葡糖糖消耗实验发现,NOR1基因能够抑制肿瘤细胞中活跃的糖酵解。氧耗量及DCFH-DA方法检测发现,NOR1表达可以降低鼻咽癌细胞线粒体呼吸水平及细胞内ROS的水平。NOR1增强了氧化应激状态下细胞毒性,降低了细胞活力；NOR1抑制细胞自噬水平,促进氧化应激状态下的细胞凋亡前期研究证实NOR1是一个氧化应激性蛋白,于是我们设想该基因在氧化应激状态下对肿瘤细胞是否产生影响。使用H202处理过表达NOR1的恶性鼻咽癌细胞NOR1/HNE1及对照细胞系Ctrl/HNE1,利用LDH技术及MTT方法检测发现,在肿瘤细胞中过表达NOR1会引起H2O2引起的细胞毒性的增加,以及细胞活力的减弱。于是进一步研究NOR1调节氧化应激引发的细胞毒性是否通过影响细胞在氧化应激状态的下的细胞自噬水平来实现。通过电子显微镜观察及Western blot检测发现,过表达NOR1显著地抑制了鼻咽癌细胞中的氧化应激状态下的白噬水平；而siRNA干扰NOR1后则增强了细胞内的自噬。通过TUNEL、Hoechst33258染色及电镜观察等方法检测发现,NORl促进了鼻咽癌细胞系HNE1中的细胞凋亡水平；相反,siRNA干扰NOR1后降低了对氧化应激诱导的凋亡敏感性。进一步分离细胞线粒体蛋白及胞浆蛋白,检测发现NOR1的表达增加了氧化应激状态下线粒体膜的通透性,促进了H202诱导的凋亡细胞中Smac/Diablo向胞浆中的释放,进而激活了caspase-9、caspase-3和PARP,激活了线粒体途径的细胞凋亡。NOR1显著增强肿瘤细胞的化疗敏感性研究发现,自噬的激活可能是一个抵抗化疗药物的保护机制,因此我们设想NOR1是否能增加肿瘤细胞对顺铂的化疗敏感性呢?于是使用经典鼻咽癌化疗药物顺铂处理过表达NOR1的鼻咽癌细胞NOR1/5-8F和对照组细胞Ctrl/5-8F, MTT实验发现NOR1显著增加了肿瘤细胞中顺铂的细胞毒性。Western blot及Hoechst33258染色检测发现NOR1也抑制了顺铂诱导的自噬,促进了顺铂诱导细胞凋亡,增强了肿瘤细胞的化疗敏感性。以上结果在HNE1细胞中也得到了验证。总之,抑癌基因的高甲基化异常修饰在鼻咽癌的发生发展中发挥了重要的作用,通过对鼻咽癌组织中NOR1甲基化状态的检测,明确了其在鼻咽癌中下调的分子机制,为鼻咽癌的早期诊断和预后分析提供了实验依据。同时,以上结果为抑癌基因NOR1在鼻咽癌中对细胞自噬和细胞凋亡的调节提供了新的实验依据,并提示NOR1可能是一个治疗肿瘤的新靶点。图53幅,表0个,参考文献125篇更多还原

【Abstract】 The previous research of the projectOxidored-nitro domain-containing protein1(NOR1) is a candidate TSG that is downregulated in nasopharyngeal carcinoma (NPC). Despite these findings, little is known regarding mechanisms underlying downregulation of the NOR1gene in NPC cell lines and biopsies. NOR1is an interaction partner of the mitochondrial adenosine triphosphate synthase subunit OSCP/ATP5O protein;Ectopic expression of NOR1in NPC HNE1cells inhibited tumor cell colony formation and viability, and NOR1gene may be a critical tumor suppressor involved in the development of NPC. Despite these findings, little is known regarding the influence and mechanism of NOR1to tumor cell homeostasis and tumor progression.Identification of a NOR1promoterSeveral bioinformatics tools such as PromoterScan, Promoter Inspector and EMBOSS CpG Plot were used to identify and characterize the potential promoter region of the NOR1gene. A753bp region spanning positions-517to+236bp relative to the transcription start site was identified as the potential promoter region of the NOR1gene. To assess whether this sequence could function to drive transcription, different regions of the sequence were cloned and linked to a luciferase-or eGFP-based reporter construct. The results using luciferase assay and visible eGFP showed that NOR1promoter located in a region spanning positions-258to+26bp.The NOR1promoter is hypermethylated and NOR1expression is downregulated in NPC tissue samples A CpG island was detected using the EMBOSS CpGplot program, with a GC content of58.8%. We investigated the methylation status of the NOR1promoter in primary NPC biopsies and non-cancerous nasopharyngeal tissue samples, several NPC cell lines and normal nasopharyngeal epithelial cell line by MSP and BSP.The results showed that methylated NOR1promoter could be detected in NPC biopsies and cell lines. The data using immunohistochemical staining and RT-PCR indicate that there might be a negative correlation between NOR1methylation and gene expression levels. Semi-quantitative RT-PCR and real-time RT-PCR analysis showed that NOR1expression levels were restored by5-aza-dC treatment. Thus, these data strongly suggest that hypermethylation of the NOR1promoter might be responsible for transcription silencing of the NOR1gene in human malignancies.Transcription factors HSF1and NRF1specifically bind to and activate the NOR1promoterSeveral bioinformatics tools MatInspector was used to characterize the potential promoter region of the NOR1gene. Within this promoter, one putative NRF1-binding site and ne putative HSF1-binding were identified at consecutive position. The results using EMAS and ChIP provide strong evidence demonstrating that transcription factors HSF1and NRF1specifically bind to and activate theNOR1promoter.Furthermore, cotransfection of the NOR1promoter constructs containing the HSF1-binding site with an HSF1-expressing vector showed that ecotopic expression of HSF1increased in the activity of the NOR1promoter. Western blot analyses showed that NOR1protein levels increased following exogenous expression of HSF1or NRF1. However, knockdown of endogenous HSF1or NRF1decreased NOR1mRNA levels. Taken together, our results provide strong evidence demonstrating that transcription factors HSF1and NRF1specifically bind to and activate the NOR1promoter.Hydrogen peroxide exposure upregulated NOR1expression in normal cells and cancer cells In previous study, NOR1gene promoter was showed to be regulated by HSF1and NRF1, both of which are critical mediators of oxidative stress response. Thus, in this study, we investigated expression of NOR1mRNA and protein in both cancer and non-cancerous cells after induced by hydrogen peroxide. Realtime PCR, FACS and immunofluorescence data showed that NOR1expression was induced by an acute oxidative stress in NP69normal human cells and MCF7tumor cells.NOR1suppress basal autophagy, mitochondrial oxidative phosphorylation and tumor cell viabilityIn this study, we investigated enforced expression of NOR1at physiologic levels in HNE1cells reduced HNE1tumor cell viability compared to vector-control cells. Overexpression of NOR1significantly suppressed an anchorage-independent growth of HNE1cells. In contrast, knockdown of NOR1expression using NOR1RNAi resulted in increase in cell viability.This study investigated effects of NOR1on autophagy under normal culture conditions. Western blot and transmission electron microscopy data showed overexpression of NOR1suppressed basal autophagy level in HNE1cells. In contrast, knockdown of NOR1expression using NOR1RNAi resulted in increase in autophagy level.Autophagy is required for tumor cell survival during starvation and tumorigenesis. We next investigated whether expression of NOR1reduced tumor cell glycolysis and mitochondrial oxidative phosphorylation. Our data showed that levels of lactate production and glucose consumption were decreased after NOR1expression in HNE1cells. Oxygen consumption was inhibited in NOR1expressing FNE1cells.Moreover, ROS generation was also decreased in NOR1expressing HNE1cells.Effects of NOR1-inhibited hydrogen peroxide-induced autophagy on tumor cell cytotoxicity; NOR1induced HNE1cell apoptosis through inhibited autophagy Here we determined the effects of NOR1-inhibited hydrogen peroxide-induced autophagy on regulation of tumor cell cytotoxicity. LDH assay and MTT data showed that LDH release is increased and tumor cell toxicity was significantly induced in NOR1expressing HNE1cells with H2O2. Furthermore, we found that restoration of NOR1expression down-regulated autophagy after treating with H2O2assessed by using an autophagy marker LC3conversion and transmission electron microscopy. Western blot, TUNEL and Hoechst33258staining identified that induction of apoptosis in NOR1expressing HNE1cells treated with H2O2. In contract, Western blot showed that an induced autophagy evident and a decreased level of cleaved PARP in NOR1siRNA-transfected cells treated with H2O2. These results confirmed that autophagy inhibition by NOR1might confer sensitivity to oxidative stress-induced apoptosis in cancer cells.Western blot resulted in an increased Bax/Bcl-xL ratio in NOR1expressing cells treated with H2O2and then facilitated the release of Smac/Diablo from the mitochondria into the cytosol. Consequently, cleavaged caspase-9, caspase-3, and PARP were clearly increased in NOR1expressing HNE1cells after hydrogen peroxide treatment.These results suggested that NOR1mediated tumor cells apoptosis in oxidative stress was associated with upregulation of the pro-apoptotic pathway through increase in the ratio of mitochondrial Bax/Bcl-xL ratio and corresponding release of mitochondrial Smac/Diablo proteins.NOR1expression sensitized tumor cells to cisplatin-induced apoptosisSince autophagy may be activated as a protective mechanism against chemotherapy agents. We assess whether NOR1sensitizes tumor cells to cisplatin-induced apoptosis as a novel strategy in future cancer therapy. We treated NPC5-8F cells or HNE1cells with a series of concentrations of cisplatin. The data showed that expression of NOR1significantly inhibited the cisplatin-induced autophagy, resulting in increased cisplatin cytotoxicity and apoptosis. Thus, promoter hypermethylation has been recognized as a commonmechanism leading to inactivation of TSGs. These data revealed NOR1may be a critical TSG in NPC and represents a potential attractive target for epigenetic therapy of NPC and revealed novel aspects of the interplay between autophagy and apoptosis in nasopharyngeal carcinoma cells that underlie the tumor suppression function of NOR1, possibly providing a novel insight into development of a combinatorial therapy for nasopharyngeal carcinoma.更多还原