不同侵袭迁移能力鼻咽癌细胞蛋白组学及转移相关基因功能研究

发布时间：2018-06-25 09:59

本文选题：鼻咽癌 + 转移　；参考：《昆明医科大学》2012年博士论文

【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。
[Abstract]:Nasopharyngeal carcinoma is one of the ten major malignant tumors in China. There are 80% cases of nasopharyngeal carcinoma in China. The incidence of nasopharyngeal carcinoma in southern China is obviously higher than that in Europe. The incidence of nasopharyngeal carcinoma and EB virus (Epstein Barr Virus) infection, genetic factors, nitrosamine intake and environmental factors (high content of cadmium and nickel in drinking water) The metastasis of cervical lymph nodes is one of the most important factors affecting the prognosis of nasopharyngeal carcinoma. Metastasis is the main reason for the failure of nasopharyngeal carcinoma. Although the diagnosis and treatment technology is progressing, the 5 year of nasopharyngeal cancer patients have been diagnosed as the 5 year of nasopharyngeal carcinoma. It is a great challenge for us to explore the molecular mechanism of metastasis of nasopharyngeal carcinoma, search for new therapeutic targets and reduce the occurrence of metastasis.
The use of cell lines to study the biological characteristics of tumor cells is an important means of modern medical research. In.1976, China built the first highly differentiated nasopharyngeal carcinoma epithelioid cell line in CNE-11980, China established the first low differentiated nasopharyngeal carcinoma epithelial cell line CNE-22006 Qian through a limited dilute culture method from CNE-2 to 2 9 subclones, and found that clones 18 (S-18) have strong invasion and migration activity compared with parent and other subclones. Because S-18 is a subclone of CNE-2, they have similar genetic and pathological characteristics in addition to the invasion and migration ability, which can limit the individual differences and reduce genetic differences in the study. Therefore, the establishment of S-18 provides a convenient and reliable platform for more targeted study of the intrinsic differences between nasopharyngeal carcinoma cells with different invasiveness and migration ability.
Proteomics is one of the basic and component parts of system biology. After entering the post genomics era, its status is particularly prominent. Two-dimensional gel electrophoresis (2-DE) is one of the classical methods of proteomics research, and is an analysis of the protein mixture extracted from the cell, tissue, or other biological samples. A powerful means of material is the only separation technology that can separate and display thousands of proteins at the same time. Its high resolution, high repeatability and micro preparation are unparalleled in other separation methods, especially for the study of the expression of proteomics. Two dimensional electrophoresis, computer image analysis and big rules Model data processing technology, mass spectrometry has been known as the three basic support technology for proteome research. Based on the full development of system biology, proteomics and related technologies, we hope to reveal the internal differences between S-18 and its parent cells from the "Discovery Science" level through these new technologies.
After screening a series of differentially expressed proteins by using proteomics technology, we need to study these proteins thoroughly to reveal the inherent mechanism of the candidate proteins to play biological activity. Gene overexpression technology is the gene transfection technology commonly referred to as introducing foreign genes into target cells to express their corresponding expressions. A technique for the study of the function of gene (or protein) or gene therapy. The HIV-1 based lentivirus vector has the advantages of infecting the resting state cells, integrating the target genes into the target cell genome for long-term expression, and having small immune responses, and is suitable for various gene function studies and in vivo. Gene therapy is an ideal gene transfer carrier,.RNA interference (RNA interference, RNAi), a specific posttranscriptional gene silencing (post-transcriptional gene silencing, PTGS) induced by double-stranded RNA (dsRNA) homologous to the target gene sequence (post-transcriptional gene silencing, PTGS). Since the elephant, RNAi technology has been proved to be a specific, efficient and economical means of inhibiting gene expression. The technology and overexpression technology are supported by each other, and can study the functions of genes and corresponding proteins from another angle.
In this study, proteomics was used to screen the differential expression proteins between low differentiated nasopharyngeal carcinoma cell line CNE-2 and its highly metastatic subcloned S-18. A total of 18 distinct differentially expressed proteins were found in S-18 cells (up 10, 8 down-regulation). Through bioinformatics analysis, 7 proteins were determined to be closely related to tumor metastasis. Western Blot was used to verify the expression level of 4 proteins (HSP27, Ezrirn, Keratinl, VCP) in two cells. It was proved that HSP27 and Ezrin were highly expressed in S-18 cells compared with CNE-2 cells, while Keratinl8 and VCP were obviously low expression. Migration, Keratin18 can stabilize cytoskeleton and cell morphology. Therefore, their abnormal expression in S-18 may be an important intrinsic factor affecting S-18 invasion and migration.
HSP27 is one of the main members of the small HSP family, and is also one of the most obvious and widely induced molecular chaperones. It has a protective effect on normal cells, but the carcinogenic and metastatic activity of.HSP27, a marker of bad prognosis in the tumor cells, is becoming more and more concerned by researchers. It is usually considered to be carcinogenic. In order to further study the molecular mechanism of HSP27 affecting the metastasis ability of nasopharyngeal carcinoma cells, we use the lentivirus vector overexpression technology to overexpress HSP27 in the nasopharyngeal epithelial cell NP-460 (the endogenous HSP27 level is low) and use RNA interference. The HSP27 of S-18 (high endogenous HSP27 level) in nasopharyngeal carcinoma cells was silenced by technique, and the invasion and migration ability of cells after overexpression or silencing related genes were further detected by Transwell test, and the changes of B, MMP2, MMP9, MMP11 transcriptional level were detected by real-time quantitative PCR technique. The results showed that after overexpression of HSP27, the invasion and migration of NP-460 in the nasopharyngeal epithelial cells increased obviously, and the invasion and migration ability of S-18 cells decreased obviously after HSP27 silencing, and the transcription level of NF- B, MMP9, MMP11 in the cells was obviously down, and the transcriptional water of MMP2 had no obvious change.
Based on the above results, we believe that HSP27 plays an important role in the invasion and migration of nasopharyngeal carcinoma cells, and we speculate that there are three possible mechanisms for HSP27 to promote the invasion and migration of nasopharyngeal carcinoma cells. One, HSP27 can increase the transcription level of NF- B and increase the activity of IKK or directly promote the degradation of I kappa B. The enhancement of NF- B signaling pathway induces the activation and enhanced expression of MMP9, MMP11, and increases the invasiveness of nasopharyngeal carcinoma cells; secondly, HSP27 can increase the activity of MMP2 in nasopharyngeal carcinoma cells without affecting the transcriptional level of MMP2, thus increasing the invasiveness of nasopharyngeal carcinoma cells; and third, HSP27 can be passed with F-acti, Keratin8. The interaction of Keratin18 and other cytoskeleton related proteins changes the deformation and movement ability of nasopharyngeal carcinoma cells, and further improves the migration ability of nasopharyngeal carcinoma cells on the basis of the enhancement of nasopharyngeal carcinoma cell invasiveness.
To sum up, based on the purpose of exploring the molecular mechanism of nasopharyngeal carcinoma cell metastasis, this study uses the low differentiated nasopharyngeal carcinoma cell line CNE-2 and its high metastatic subclone S-18 as the research object. The protein expression profiles of two cells are compared by proteomics technology, and 18 distinct differentially expressed proteins are screened, of which HSP27 and Ezrin can be significantly promoted. Keratin18 can stabilize the cytoskeleton and cell morphology of the tumor cells. Using the overexpression and RNA interference techniques of the lentivirus vector, it is further confirmed that HSP27 plays an important role in the metastasis of nasopharyngeal carcinoma cells, and the molecular mechanism for promoting the metastasis of nasopharyngeal carcinoma cells may be associated with the NF- signaling pathway, the matrix metalloproteinase. The family (MMPs) and F-actin, Keratin8, Keratin18 and other cytoskeleton related proteins are closely related. These results will provide useful clues for further study of the molecular mechanism of nasopharyngeal carcinoma metastasis, to find new therapeutic targets and to reduce the occurrence of metastasis.
【学位授予单位】：昆明医科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R739.63

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