EMP1基因在头颈部鳞癌中的作用及机制研究

发布时间：2018-07-05 17:57

本文选题：EMP1 + 因在　；参考：《武汉大学》2016年博士论文

【摘要】：背景：头颈部恶性肿瘤在全球范围内都是一个威胁健康的重要问题,每年大约有60多万病例被诊断为头颈部恶性肿瘤,大约有35万人死于该疾病。近三十年以来,头颈部恶性肿瘤的发病率越来越高,已经成为了世界范围内的第五大肿瘤及肿瘤相关原因致死的第六大原因。其中头颈部鳞状细胞癌(HNSCC)在所有头颈部恶性肿瘤中所占比例高达85%,拥有多因素化和多相化的发病机制。探讨HNSCC的发病分子机制对充分认识多种致癌因素导致HNSCC发生的根本原因以及开发靶向治疗手段意义重大。EMP1基因与外周髓鞘蛋白-22 (PMP-22)同属一个跨膜家族,在细胞生长、增殖、分化及凋亡中起着重要作用,被认为与肿瘤发生发展具有相关性,但其在HNSCC中的具体作用机制尚缺乏全面深入的研究。近年来,高通量微阵列技术及测序技术快速发展,产生了大量的基因数据,生物信息数量迅速膨胀,为了适应这种高通量基因表达数据的不断增长和人们共享数据的需要,各种数据库应运而生,GEO数据库是NCBI创立的世界上最大的储存高通量分子丰度数据的公共数据库,涉及超过16亿个基因表达丰度数据,广泛覆盖各种生物学内容,且是完全免费共享,运用GEO数据库来分析特定基因表达及其相应的生物学功能已成为目前生物信息学的一个重要组成部分,本研究对GEO涉及HNSCC的数据集进行筛选分析,明确EMP1在HNSCC组织中的表达改变,并分析其与HNSCC患者TNM分期及肿瘤分级等的关系,进一步阐明EMP1在HNSCC发病过程中的作用及相关分子机制。研究方法：(1)我们先在GEO数据库中检索HNSCC相关数据集,并分类检索喉癌、舌癌、口腔癌及鼻咽癌等常见HNSCC相关数据集,仔细筛选相关数据集中的研究对象信息及平台信息,选取检测平台探针包含EMP1基因的数据集,同时数据集研究对象包括HNSCC及正常对照组织、HNSCC患者TNM分期及HNSCC患者肿瘤病理分级信息,将筛选得到的数据库中EMP1基因表达数据进行统计学分析,得出EMP1基因在HNSCC组织及正常对照组织中的表达差异、EMP1基因在不同TNM分期及肿瘤病理分级HNSCC组织中的表达差异； (2)我们选取包含多种头颈部常见部位恶性肿瘤及正常头颈部组织标本的HNSCC组织芯片,免疫组化法检测EMP1蛋白在HNSCC组织及正常对照组织中的表达差异、EMP1基因在不同TNM分期及肿瘤病理分级的HNSCC组织中的表达差异；(3)采用生物工程技术构建EMP1基因过表达载体,转染头颈部鳞癌细胞系,筛选阳性克隆,培养并鉴定其中EMP1基因表达是否明显增高,最终建立EMP1过表达的头颈部鳞状细胞癌细胞株； (4)检测第三部分建立的EMP1过表达细胞株与对照细胞在细胞增殖能力、体外迁移能力等细胞生物学行为的改变,之后,在CCLE数据库中分析并提取在1036肿瘤细胞中与EMP1基因co-expression的基因列表,并在TCGA HNSCC数据库中分析EMP1基因与co-expression基因mRNA水平的相关性,对EMP1 co-expression基因进行GO分析及KEGG pathway分析,最后采用免疫印迹检测EMP1 co-expression基因在EMP1过表达的HNSCC细胞株中的表达变化,明确过表达EMP1基因对头颈部鳞状细胞癌细胞增殖及迁移能力的影响,并初步探讨其中的分子机制。研究结果： (1)我们筛选出符合研究目的的数据集,分析EMP1在HNSCC组织及正常对照组织中的表达水平差异及在不同TNM分期、肿瘤分级的HNSCC组织中的表达差异,结果发现,在数据集GSE6631、GSE58911中, HNSCC组织和正常对照是配对的,EMP1在HNSCC组织中的表达水平显著低于正常对照,p值分别为：0.0001、0.0001,在数据集GSE33205、GSE59102、GSE51985及 GSE10774中,HNSCC组织及正常对照组织是来源于不同的患者,EMP1在HNSCC组织中的表达水平也是显著低于正常对照组织,p值分别为：0.0001、0.0001、0.0508及0.0114。在数据集GSE30788和GSE39366中分析EMP1在不同TNM分期及肿瘤分级的HNSCC组织中的表达水平差异,结果发现在不同T分期的HNSCC组织中EMP1的表达水平无统计学差异,不同N分期的HNSCC组织中EMP1的表达在两个数据集中结果不一致,在数据集GSE30788中,N+的HNSCC患者组织中EMP1表达水平高于在NO的HNSCC组织中的表达水平,而在数据集GSE39366中,NO与N+的HNSCC组织中EMP1表达水平无统计学差异,对不同肿瘤病理分级的HNSCC组织中EMP1的表达水平分析结果发现,随着分化程度增高,EMP1基因表达水平逐渐增高,差异具有统计学意义,p值分别为：0.0001和0.0064(2)免疫组化检测INSCC组织芯片中EMP1的表达水平,结果显示EMP1蛋白在正常组织中的表达水平显著高于在HNSCC组织中的表达水平,差异具有显著统计学差异,在不同T分期的(?) INSCC组织中的表达无明显统计学差异,在不同N分期的HNSCC组织中的表达也无明显统计学差异,在高分化HNSCC组织中的表达水平高于在中低分化的HNSCC组织中的表达水平,差异具有显著统计学差异； (3)通过基因工程技术成功构建EMP1过表达载体,转染HNSCC细胞系,筛选并鉴定EMP1高表达INSCC细胞系后,发现HNSCC细胞系的细胞增殖能力明显减弱,48h时吸光度值仅为对照组的50%,差异具有显著统计学意义,同时也发现过表达EMP1后,HNSCC细胞系的体外迁移能力显著减弱,Transwell法检测24h时穿过基底膜的细胞数目分别为：EMP1过表达组(34.8+10.6),空白对照组(83.5+12.1),阴性对照组(73.5+15.3),差异具有统计学意义, (4)在CCLE数据库中分析提取出与EMP1基因c o-expression的相关基因,并在T CGA HNSCC数据库中分析EMP1基因与co-expression基因的RNA表达水平的相关性,结果显示EPHA2、ITGA3及ANXA1 RNA表达水平与EMP1表达具有相关性,差异具有统计学意义,且在细胞实验中,过表达EMP1基因后,EPHA2、ITGA3及ANXA1蛋白表达水平也发生显著改变。研究结论-(1)EMP1基因在HNSCC中的表达水平明显低于在正常对照组织中的表达水平(2)EMP1基因在HNSCC组织中的表达水平随分化程度增高而增高；(3)过表达EMP1后,HNSCC细胞的增殖能力及迁移能力显著减弱,其可能的分子机制与EPHA2、ITGA3及ANXA1表达水平发生改变有关。
[Abstract]:Background: head and neck malignancies are an important global threat to health. About about 600000 cases are diagnosed as head and neck cancer each year. About 350 thousand people die of the disease. The incidence of head and neck malignancies has become more and more high and has become the fifth largest tumor in the world over the past thirty years. Sixth major causes of death of tumor related causes. The proportion of head and neck squamous cell carcinoma (HNSCC) in all head and neck malignant tumors is up to 85%, with the pathogenesis of multifactorization and multiphase. The molecular mechanism of the pathogenesis of HNSCC is discussed to fully understand the underlying causes of the occurrence of multiple carcinogens and the development of the targeting of HNSCC. Treatment means significant.EMP1 gene and peripheral myelin protein -22 (PMP-22) belong to a transmembrane family, which play an important role in cell growth, proliferation, differentiation and apoptosis, and are considered to be related to the development of tumor. However, the specific mechanism of its action in HNSCC is still lack of comprehensive and thorough research. In recent years, high throughput microarray technology has been used. The rapid development of technology and sequencing technology produces a large number of genetic data, and the number of biological information is expanding rapidly. In order to adapt to the growing number of high throughput gene expression data and the need for people to share data, various databases have come into being. The GEO database is the largest store of high flux molecular abundance data in the world created by NCBI. The common database, involving more than 1 billion 600 million gene expression abundance data, covers a wide range of biological contents and is completely free sharing. The use of GEO database to analyze specific gene expression and its corresponding biological functions has become an important component of bioinformatics. This study screened the data set for GEO involving HNSCC. Select and analyze the changes in the expression of EMP1 in the HNSCC tissue, and analyze the relationship between the TNM staging and the tumor classification of HNSCC patients, and further clarify the role and molecular mechanism of EMP1 in the pathogenesis of HNSCC. (1) we first retrieve the HNSCC related data set in the GEO database and classify the larynx, tongue, and oral cancer classification. HNSCC related data sets, such as nasopharyngeal carcinoma and other common data sets, are carefully screened for the information of research objects and platform information in the relevant data set, and the data set of the detection platform probes including the EMP1 gene is selected, and the data set includes the HNSCC and the normal control tissue, the TNM staging of the HNSCC patients and the pathological grading information of the HNSCC patients will be screened. The EMP1 gene expression data in the database were statistically analyzed, and the expression difference of EMP1 gene in HNSCC tissue and normal control tissue, the difference in the expression of EMP1 gene in different TNM stages and tumor pathological grading HNSCC tissues were found. (2) we selected a variety of head and neck malignant tumor and normal head and neck tissue specimens. HNSCC tissue chip was used to detect the difference in expression of EMP1 protein in HNSCC tissue and normal control tissue, and the difference in expression of EMP1 gene in HNSCC tissues of different TNM stages and tumor pathological grades. (3) bioengineering technique was used to construct EMP1 gene overexpressed body, transfection of head and neck squamous cell carcinoma cell lines, and screening positive clones. The expression of EMP1 gene was significantly increased and the expression of EMP1 overexpressed in the head and neck squamous cell carcinoma cell lines was established. (4) the changes in cell proliferation and in vitro migration ability of the third part of the EMP1 overexpressed cell lines and the control cells were detected and analyzed in the CCLE database. The gene list of the EMP1 gene co-expression in 1036 tumor cells was extracted and the correlation between the EMP1 gene and the mRNA level of the co-expression gene was analyzed in the TCGA HNSCC database. The GO analysis and KEGG pathway analysis of the EMP1 co-expression gene were carried out. The expression changes in the C cell line clearly express the effect of EMP1 gene on the proliferation and migration of squamous cell carcinoma cells in the head and neck, and discuss the molecular mechanism of it. (1) we screened the data set for the purpose of the study, and analyzed the difference in the expression level of EMP1 in the HNSCC tissue and the normal control tissue. The difference in the expression of HNSCC in the TNM staging and tumor classification showed that in the data set GSE6631 and GSE58911, the HNSCC tissue was paired with the normal control, and the expression level of EMP1 in the HNSCC tissue was significantly lower than that of the normal control, and the p value was 0.0001,0.0001, respectively, in the data set GSE33205, GSE59102, GSE51985, and the tissues. The expression level of EMP1 in HNSCC tissues was also significantly lower than that of normal control tissue. The P values of 0.0001,0.0001,0.0508 and 0.0114. were respectively in the data sets GSE30788 and GSE39366 to analyze the difference in the expression level of EMP1 in HNSCC tissues of different TNM staging and tumor grading. There is no significant difference in the expression level of EMP1 in the HNSCC tissues of different T stages. The expression of EMP1 in the HNSCC tissues of different N staging is not consistent with the results of the two data sets. In the data set GSE30788, the EMP1 expression level of the HNSCC patients in N+ is higher than that in NO HNSCC tissue. There was no statistical difference in the expression level of EMP1 in tissue. The analysis of the expression level of EMP1 in HNSCC tissues of different tumor pathological grades showed that the expression level of EMP1 gene increased gradually with the increase of differentiation, and the P values were 0.0001 and 0.0064 (2) immunohistochemical detection of the expression of EMP1 in the INSCC tissue chip. The results showed that the expression level of EMP1 protein in normal tissues was significantly higher than that in HNSCC tissues, and there was significant difference in the difference. There was no significant difference in the expression of INSCC in the different T stages, and there was no significant difference in the expression of HNSCC in the different N stages, and in the highly differentiated HNSCC. The expression level in the tissue was higher than that in the medium and low differentiated HNSCC tissues, and there was significant difference in the difference. (3) the EMP1 overexpression vector was successfully constructed by gene engineering technology, and the transfection of HNSCC cell lines, and the screening and identification of the EMP1 high expression INSCC cell line, the cell proliferation ability of the present HNSCC cell line was significantly weakened, 48H At the same time, the absorbance was only 50% of the control group, and the difference was statistically significant. At the same time, after the expression of EMP1, the ability of the HNSCC cell line to migrate significantly decreased. The number of cells passing through the basement membrane in the Transwell assay was respectively: EMP1 overexpression group (34.8+10.6), blank control group (83.5+12.1), negative control group (73.5+15.3). The difference was statistically significant. (4) the related genes associated with the EMP1 gene C o-expression were analyzed and extracted in the CCLE database, and the correlation between the EMP1 gene and the RNA expression level of the co-expression gene was analyzed in the T CGA HNSCC database. The level of expression of EPHA2, ITGA3 and ANXA1 protein was also significantly changed in cell experiment. (1) the expression level of EMP1 gene in HNSCC was significantly lower than that in normal control tissue (2) the expression level of EMP1 gene in HNSCC tissues increased with the increase of differentiation. (1) the expression level of EMP1 gene in HNSCC was significantly lower than that in normal control tissue. 3) after over expression of EMP1, the proliferation and migration ability of HNSCC cells were significantly weakened, and the possible molecular mechanisms were related to the changes of EPHA2, ITGA3 and ANXA1 expression levels.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R739.91

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