【作者】 李国萍；

【导师】 何晓光；

【作者基本信息】 昆明医科大学 ， 头颈外科， 2012， 博士

【摘要】 鼻咽癌是我国发病率较高的十大恶性肿瘤之一,世界上有80%的鼻咽癌病例发生在中国,中国南方鼻咽癌的发病率明显高于欧洲。鼻咽癌发病与EB病毒(Epstein Barr Virus)感染、遗传因素、亚硝胺摄入及环境因素(饮用水中高含量的镉、镍)等因素密切相关。鼻咽癌早期即可出现颈部淋巴结转移。是否有颈淋巴结转移是影响鼻咽癌预后的重要指标之一。转移是鼻咽癌治疗失败的主要原因,尽管诊疗技术不断进步,但由于60-85%的患者确诊时已发生临床转移,因此鼻咽癌患者的5年生存率提升缓慢。探究鼻咽癌转移的分子机制,寻找新的治疗靶点,减少转移的发生,是我们面临的巨大挑战。利用细胞株来研究肿瘤细胞的生物学特性是现代医学研究的重要手段。1976年我国建成了第一个高分化鼻咽癌上皮样细胞株CNE-1,1980年我国建立了首个低分化鼻咽癌上皮细胞株CNE-2,2006年Qian通过有限稀释培养的方法从CNE-2中筛选到了29个亚克隆,并发现克隆18(S-18)与母代细胞及其它亚克隆相比具有较强的侵袭和迁移活性。由于S-18是CNE-2的亚克隆,除了侵袭迁移能力不同之外,它们具有类似的遗传学和病理学特性,从而在研究中可限制个体差异性、减小遗传差异性。因此,S-18的建立为更有针对性地研究不同侵袭迁移能力鼻咽癌细胞之间的内在差异提供了一个便利而可靠的平台。蛋白质组学是系统生物学的基础和组成部分之一,进入后基因组学时代以后,其地位尤为突出。双向电泳(two-dimensional gel electrophoresis,2-DE)是蛋白质组学研究的经典方法之一,是一种分析从细胞、组织或其他生物样本中提取的蛋白质混合物的有力手段,是目前唯一能将数千种蛋白质同时分离与展示的分离技术,其高分辨率、高重复性和兼具微量制备的性能是其它分离方法所无与伦比的,特别是对于表达蛋白质学的研究必不可少。双向电泳技术、计算机图像分析与大规模数据处理技术、质谱技术被称为蛋白质组研究的三大基本支撑技术。基于系统生物学、蛋白组学及相关技术的充分发展,我们希望通过这些新兴技术,从“发现的科学”的层面揭示S-18与其母代细胞之间的内在差别。在利用蛋白组学技术筛选到一系列差异表达蛋白之后,需要对这些蛋白进行深入的功能研究,才能充分揭示候选蛋白发挥生物活性的内在机制。基因过表达技术也就是通常所说的基因转染技术,是将外源基因导入靶细胞,使其表达相应蛋白质,从而实现对基因(或蛋白质)功能的研究或进行基因治疗的一种技术。以HIV-1为基础构建的慢病毒载体具有可感染静息状态细胞、可将目的基因整合至靶细胞基因组而长期表达、免疫反应小等优点,适于各种基因功能研究及体内基因治疗,是目前较为理想的基因转移载体。RNA干扰(RNA interference, RNAi)是与靶基因序列同源的双链RNA (double-stranded RNA, dsRNA)所诱导的一种特异性的转录后基因沉默现象(post-transcriptional gene silencing, PTGS)。自1998年Fire等发现RNA干扰(RNAi)现象以来,RNAi技术已被证实是一种特异、高效、经济的抑制基因表达手段,该技术与过表达技术互为佐证,能从另外一个角度研究基因及相应蛋白质的功能。本研究利用蛋白组学技术,筛查了低分化鼻咽癌细胞株CNE-2与其高转移亚克隆S-18之间的差异表达蛋白,共发现S-18细胞中有18个明显差异表达的蛋白(上调10个,下调8个)。通过生物信息学分析,确定了其中7个蛋白与肿瘤的转移有密切关系,并利用Western Blot对其中的4个蛋白(HSP27、Ezrirn、 Keratinl、VCP)在两种细胞中的表达水平进行了验证,证实了与CNE-2细胞相比,S-18细胞中HSP27和Ezrin呈明显高表达,而Keratinl8和VCP明显低表达。既往初步研究表明,HSP27和Ezrin可明显促进肿瘤细胞转移,Keratin18可稳定细胞骨架和细胞形态,因此,它们在S-18内异常表达,可能是影响S-18侵袭迁移能力的重要内在因素。HSP27是小HSP家族的主要成员之一,同时也是最明显而广泛诱导表达的分子伴侣之一,其对正常细胞具有保护作用,但在肿瘤细胞中通常是不良预后的标志。HSP27的致癌及促转移活性越来越受到研究者们的关注,通常认为其致癌性能可能与其抗凋亡性能相关,但对其发挥促转移活性的分子机制却知之甚少。为了进一步研究HSP27影响鼻咽癌细胞转移能力的分子机制,我们利用慢病毒载体过表达技术在鼻咽部上皮细胞NP-460(内源性HSP27水平较低)中过表达HSP27,利用RNA干扰技术沉默高转移鼻咽癌细胞S-18(内源性HSP27水平较高)中的HSP27,进一步利用Transwell实验检测了过表达或沉默相关基因后的细胞的侵袭迁移能力,并利用实时定量PCR技术检测了沉默HSP27后的S-18细胞内NF-к B、MMP2、MMP9、MMP11转录水平的变化。结果显示,过表达HSP27之后,鼻咽部上皮细胞NP-460的侵袭迁移能力明显增强；而沉默HSP27之后,S-18细胞的侵袭迁移能力明显减弱,并且细胞内的NF-к B、MMP9、MMP11转录水平明显下调,而MMP2的转录水平无明显变化。基于上述结果,我们确信HSP27在鼻咽癌细胞的侵袭迁移过程中发挥重要作用,并且推测HSP27促进鼻咽癌细胞侵袭迁移的机制存在以下三种可能：其一,HSP27可通过提高NF-к B的转录水平,并通过增加IKK的活性或直接促进I κ B的降解,从而强化NF-к B信号通路,引起MMP9、MMP11的活化和增强表达,从而增加鼻咽癌细胞的侵袭能力；其二,HSP27可在不影响MMP2的转录水平的情况下,直接增加鼻咽癌细胞内MMP2的活性,从而增加鼻咽癌细胞的侵袭能力；其三,HSP27可通过与F-acti、Keratin8、Keratin18等细胞骨架相关蛋白的相互作用,改变鼻咽癌细胞的变形和运动能力,从而在鼻咽癌细胞侵袭能力提升的基础上,进一步提升鼻咽癌细胞的迁移能力。综上所述,基于探究鼻咽癌细胞转移分子机制的目的,本研究以低分化鼻咽癌细胞株CNE-2及其高转移亚克隆S-18作为研究对象,利用蛋白组学技术比较了两种细胞的蛋白表达谱,共筛选到了18个明显差异表达蛋白,其中HSP27和Ezrin可明显促进肿瘤细胞转移,Keratin18可稳定细胞骨架和细胞形态。利用慢病毒载体过表达技术和RNA干扰技术,进一步证实了HSP27在鼻咽癌细胞转移的过程中发挥重要作用,而其促进鼻咽癌细胞转移的分子机制可能与NF-к B信号通路、基质金属蛋白酶家族(MMPs)及F-actin、Keratin8、Keratin18等细胞骨架相关蛋白密切相关。这些结果将为深入研究鼻咽癌转移的分子机制、寻找新的治疗靶点、减少转移的发生提供有益的线索。更多还原

【Abstract】 Nasopharyngeal Carcinoma (NPC) is one of the ten most common malignant tumors in China. Eighty percent of NPC cases in all over the world exist in China. The morbility of NPC in South China is almost100times than in Europe. The etiology of NPC involves infection of Epstein Barr Virus, heredity, ingestion of nitrosamine and environmental factors (such as high concentration of Cadmium or Niccolum in drinking water). Lymph nodes metastasis might occur even in the early stage of NPC. Whether accompanied with lymph nodes metastasis is one of the most important indicators determining the prognosis of NPC. Metastasis is the chief reason for failure in NPC treatment. Despite the diagnostic and therapeutic techniques is continuously developed,60-85%patients had suffered metastasis when they got the final diagnosis, so the5-year survival rate of NPC patients is hard to be enhanced. It is a huge challenge we have to face that to explore the molecular mechanism of NPC matastasis, to search new therapy target and to prevent NPC spread from situ.It is an important approach in the modern medical research to probe the biological property of tumor cells by using cell strain. The first high differentiated NPC epithelial cell strain, CNE-1, was established in1976, then the low differentiated one, CNE-2, was established in1980in China. In2006, Qian isolated29subclones from CNE-2by limited dilution methods. And it was found that clone18(S-18) possesses stronger capacity in invasion and migration comparing with the parental cell line and other subclones. Since S-18is derived from CNE-2, except the different metastastic ability, it has similar property in heredity, pathology and biology with CNE-2. The individual variant will be the minimum when investigation is performed between these two cell lines. So the isolation of S-18provides a convenient and reliable platform for exploring the underlying mechanism between NPC cells with different metastatic capacity.Proteomics is one of the basis and components of the systemic biology. Its role becomes more and more important with the coming of post genomic era. Two-dimensional gel electrophoresis (2-DE) is a classic method in proteomic researches, by which the protein mix detracted from cells, tissues or other bio-samples can be analyzed. So far,2-DE is the only technique which can isolate and indicate thousands of proteins from samples. It has higher resolution, better reproducibility and more feasible for micropreparation than other methods and is especially necessary for the expression proteomics.2-DE, computer based image analysis and large scale datamation are the supporting techniques in proteomics researches. By utilizing these advanced techniques in the system biology and proteomics, we hope to disclose the underlying differences between S-18cells and their parental cells on the level of "discovery science".After proteins significantly expressed are identified by proteomics screening, it is necessary to explore the functions of these proteins so that the underlying molecular mechanisms can be revealed. Gene-overexpression technique, also well known as gene-transfection technique, is a method that to study the functions of genes (proteins) or to perform gene therapy by transduction of exogenous genes into target cells so that the corresponding proteins are expressed. The lentivector based on HIV-1is an prospective gene-transfer vector for gene function study and gene therapy as it can transfect non-dividing cells, can stably and constantly express candidate proteins by integrating target gene into the host genome, and can avoid strong immune response. RNA interference (RNAi) is a phenomenon of post-transcriptional gene silencing (PTGS) which is induced by double-stranded RNA which is sequence-homologous with target gene. Since it was discovered by Fire in1998, RNAi has been proved to be a specific, efficient and economic approach to inhibit gene expression. Combined with gene-overexpression technique, RNAi can be used to further demonstrate functions of genes and corresponding proteins in an opposite direction.In the present study, proteomic technique was used to profile the differently expressed proteins between NPC cell line CNE-2and its high metastatic subclone S-18, and eighteen proteins were found (10proteins are up-regulated and8proteins are down-regulated in S-18cell line). Through bioinformatics analysis, seven of the eighteen proteins were believed concerning closely with cancer metastasis. And the expression levels of four proteins in the two cell lines were further verified by Western Blot. That is, compared with CNE-2, S-18has higher heat shock protein27(HSP27) and Ezrin level, and lower Keratin18and vasolin containing protein (VCP) level. Previous studies indicated that HSP27and Ezrin could significantly promote metastatic ability of cancer cells, and Keratinl8had the property to stabilize cell skeleton and cell morphology. The abnormal expression of these genes may be the important internal factors that result in the high metastatic potential of S-18.HSP27is the chief member of small HSP family, and is also the most obvious and extensive induced one of the chaperons. HSP27perform protective role in normal cells, however, it is usually a marker for bad prognostic in cancer patients. More and more researchers pay attention to the carcinogenetic and metastasis-enhancing potential of HSP27. It is commonly regarded that the carcinogenetic property of HSP27is associated with its anti-apoptosis capacity. But we know very little about the underlying mechanism of metastasis-enhancing potential of HSP27. In order to further the study about the molecular mechanism of HSP27in cancer metastasis, we utilized lentivector to over-expressed HSP27in NP-460, which is a normal nasopharyngeal epithelial cell line with low endogenous HSP27level; and used RNA interference technique to decrease the high HSP27level in S-18cell line. Then we used transwell test to determine the invasive and migratory ability overexpressed or silenced cells, and utilized real time PCR to detect the transcription levels of NF-κ B, MMP2, MMP9and MMP11in HSP27-silenced S-18cells. Data indicated that the invasive and migratory capacity of NP-460was significantly enhanced after HSP27was overexpressed, and the invasive and migratory capacity of S-18was significantly attenuated accompanying with the levels of NF-κ B, MMP9and MMP11were significantly downregulated after HSP27was inhibited. Interestingly, the transcriptional level of MMP2was not influenced with inhibition of HSP27.Based on the above data, we believe that HSP27plays a vital role in metastatic process of NPC cells, and we hypothesize it is via the following three probable mechanisms that HSP27promotes the metastatic ability of NPC cells. Firstly, HSP27can activate the NF-κ B signal pathway by enhancing the transcriptional level of NF-K B, by increasing the activity of IKK or by directly promoting the degradation of I κ B, then MMP9and MMP11will be upregulated and activated, so that the invasive capacity of NPC cells are enhanced. Secondly, HSP27can promote the activity of MMP2without influencing its transcriptional level. Thirdly, HSP27can enhance the deformability and motility of NPC cells by interacting with F-actin, Keratin8and Keratin18, which are cytoskeleton proteins.Altogether, for the goal to explore the molecular mechanisms of metastasis of NPC cells, NPC cell line CNE-2and its high metastatic subclone S-18were selected as the researching objects. Proteomics was utilized to profile the proteins differently expressed between the two cell lines and eighteen proteins were identified. Among these proteins, HSP27and Ezrin can promote metastasis of tumor cells, Keratin18has the property to stabilize cytoskeleton and cell shape. By using lentivectors and RNA interference technique, the metastasis-enhancing function of HSP27in NPC cells was verified. The molecular mechanisms of HSP27in promoting metastasis may be closely associated to NF-κ B signal pathway, activity of MMPs and its interaction with the cytoskeleton-related proteins such as F-actin, Keratin8and Keratin18. These findings will provide useful clues for further studying mechanisms of metastasis, finding new therapy targets and reducing metastasis of NPC.更多还原