本文選題：EMP1 + 因在　； 參考：《武漢大學(xué)》2016年博士論文

【摘要】：背景：頭頸部惡性腫瘤在全球范圍內(nèi)都是一個威脅健康的重要問題,每年大約有60多萬病例被診斷為頭頸部惡性腫瘤,大約有35萬人死于該疾病。近三十年以來,頭頸部惡性腫瘤的發(fā)病率越來越高,已經(jīng)成為了世界范圍內(nèi)的第五大腫瘤及腫瘤相關(guān)原因致死的第六大原因。其中頭頸部鱗狀細(xì)胞癌(HNSCC)在所有頭頸部惡性腫瘤中所占比例高達(dá)85%,擁有多因素化和多相化的發(fā)病機(jī)制。探討HNSCC的發(fā)病分子機(jī)制對充分認(rèn)識多種致癌因素導(dǎo)致HNSCC發(fā)生的根本原因以及開發(fā)靶向治療手段意義重大。EMP1基因與外周髓鞘蛋白-22 (PMP-22)同屬一個跨膜家族,在細(xì)胞生長、增殖、分化及凋亡中起著重要作用,被認(rèn)為與腫瘤發(fā)生發(fā)展具有相關(guān)性,但其在HNSCC中的具體作用機(jī)制尚缺乏全面深入的研究。近年來,高通量微陣列技術(shù)及測序技術(shù)快速發(fā)展,產(chǎn)生了大量的基因數(shù)據(jù),生物信息數(shù)量迅速膨脹,為了適應(yīng)這種高通量基因表達(dá)數(shù)據(jù)的不斷增長和人們共享數(shù)據(jù)的需要,各種數(shù)據(jù)庫應(yīng)運(yùn)而生,GEO數(shù)據(jù)庫是NCBI創(chuàng)立的世界上最大的儲存高通量分子豐度數(shù)據(jù)的公共數(shù)據(jù)庫,涉及超過16億個基因表達(dá)豐度數(shù)據(jù),廣泛覆蓋各種生物學(xué)內(nèi)容,且是完全免費(fèi)共享,運(yùn)用GEO數(shù)據(jù)庫來分析特定基因表達(dá)及其相應(yīng)的生物學(xué)功能已成為目前生物信息學(xué)的一個重要組成部分,本研究對GEO涉及HNSCC的數(shù)據(jù)集進(jìn)行篩選分析,明確EMP1在HNSCC組織中的表達(dá)改變,并分析其與HNSCC患者TNM分期及腫瘤分級等的關(guān)系,進(jìn)一步闡明EMP1在HNSCC發(fā)病過程中的作用及相關(guān)分子機(jī)制。研究方法：(1)我們先在GEO數(shù)據(jù)庫中檢索HNSCC相關(guān)數(shù)據(jù)集,并分類檢索喉癌、舌癌、口腔癌及鼻咽癌等常見HNSCC相關(guān)數(shù)據(jù)集,仔細(xì)篩選相關(guān)數(shù)據(jù)集中的研究對象信息及平臺信息,選取檢測平臺探針包含EMP1基因的數(shù)據(jù)集,同時數(shù)據(jù)集研究對象包括HNSCC及正常對照組織、HNSCC患者TNM分期及HNSCC患者腫瘤病理分級信息,將篩選得到的數(shù)據(jù)庫中EMP1基因表達(dá)數(shù)據(jù)進(jìn)行統(tǒng)計學(xué)分析,得出EMP1基因在HNSCC組織及正常對照組織中的表達(dá)差異、EMP1基因在不同TNM分期及腫瘤病理分級HNSCC組織中的表達(dá)差異； (2)我們選取包含多種頭頸部常見部位惡性腫瘤及正常頭頸部組織標(biāo)本的HNSCC組織芯片,免疫組化法檢測EMP1蛋白在HNSCC組織及正常對照組織中的表達(dá)差異、EMP1基因在不同TNM分期及腫瘤病理分級的HNSCC組織中的表達(dá)差異；(3)采用生物工程技術(shù)構(gòu)建EMP1基因過表達(dá)載體,轉(zhuǎn)染頭頸部鱗癌細(xì)胞系,篩選陽性克隆,培養(yǎng)并鑒定其中EMP1基因表達(dá)是否明顯增高,最終建立EMP1過表達(dá)的頭頸部鱗狀細(xì)胞癌細(xì)胞株； (4)檢測第三部分建立的EMP1過表達(dá)細(xì)胞株與對照細(xì)胞在細(xì)胞增殖能力、體外遷移能力等細(xì)胞生物學(xué)行為的改變,之后,在CCLE數(shù)據(jù)庫中分析并提取在1036腫瘤細(xì)胞中與EMP1基因co-expression的基因列表,并在TCGA HNSCC數(shù)據(jù)庫中分析EMP1基因與co-expression基因mRNA水平的相關(guān)性,對EMP1 co-expression基因進(jìn)行GO分析及KEGG pathway分析,最后采用免疫印跡檢測EMP1 co-expression基因在EMP1過表達(dá)的HNSCC細(xì)胞株中的表達(dá)變化,明確過表達(dá)EMP1基因?qū)︻^頸部鱗狀細(xì)胞癌細(xì)胞增殖及遷移能力的影響,并初步探討其中的分子機(jī)制。研究結(jié)果： (1)我們篩選出符合研究目的的數(shù)據(jù)集,分析EMP1在HNSCC組織及正常對照組織中的表達(dá)水平差異及在不同TNM分期、腫瘤分級的HNSCC組織中的表達(dá)差異,結(jié)果發(fā)現(xiàn),在數(shù)據(jù)集GSE6631、GSE58911中, HNSCC組織和正常對照是配對的,EMP1在HNSCC組織中的表達(dá)水平顯著低于正常對照,p值分別為：0.0001、0.0001,在數(shù)據(jù)集GSE33205、GSE59102、GSE51985及 GSE10774中,HNSCC組織及正常對照組織是來源于不同的患者,EMP1在HNSCC組織中的表達(dá)水平也是顯著低于正常對照組織,p值分別為：0.0001、0.0001、0.0508及0.0114。在數(shù)據(jù)集GSE30788和GSE39366中分析EMP1在不同TNM分期及腫瘤分級的HNSCC組織中的表達(dá)水平差異,結(jié)果發(fā)現(xiàn)在不同T分期的HNSCC組織中EMP1的表達(dá)水平無統(tǒng)計學(xué)差異,不同N分期的HNSCC組織中EMP1的表達(dá)在兩個數(shù)據(jù)集中結(jié)果不一致,在數(shù)據(jù)集GSE30788中,N+的HNSCC患者組織中EMP1表達(dá)水平高于在NO的HNSCC組織中的表達(dá)水平,而在數(shù)據(jù)集GSE39366中,NO與N+的HNSCC組織中EMP1表達(dá)水平無統(tǒng)計學(xué)差異,對不同腫瘤病理分級的HNSCC組織中EMP1的表達(dá)水平分析結(jié)果發(fā)現(xiàn),隨著分化程度增高,EMP1基因表達(dá)水平逐漸增高,差異具有統(tǒng)計學(xué)意義,p值分別為：0.0001和0.0064(2)免疫組化檢測INSCC組織芯片中EMP1的表達(dá)水平,結(jié)果顯示EMP1蛋白在正常組織中的表達(dá)水平顯著高于在HNSCC組織中的表達(dá)水平,差異具有顯著統(tǒng)計學(xué)差異,在不同T分期的(?) INSCC組織中的表達(dá)無明顯統(tǒng)計學(xué)差異,在不同N分期的HNSCC組織中的表達(dá)也無明顯統(tǒng)計學(xué)差異,在高分化HNSCC組織中的表達(dá)水平高于在中低分化的HNSCC組織中的表達(dá)水平,差異具有顯著統(tǒng)計學(xué)差異； (3)通過基因工程技術(shù)成功構(gòu)建EMP1過表達(dá)載體,轉(zhuǎn)染HNSCC細(xì)胞系,篩選并鑒定EMP1高表達(dá)INSCC細(xì)胞系后,發(fā)現(xiàn)HNSCC細(xì)胞系的細(xì)胞增殖能力明顯減弱,48h時吸光度值僅為對照組的50%,差異具有顯著統(tǒng)計學(xué)意義,同時也發(fā)現(xiàn)過表達(dá)EMP1后,HNSCC細(xì)胞系的體外遷移能力顯著減弱,Transwell法檢測24h時穿過基底膜的細(xì)胞數(shù)目分別為：EMP1過表達(dá)組(34.8+10.6),空白對照組(83.5+12.1),陰性對照組(73.5+15.3),差異具有統(tǒng)計學(xué)意義, (4)在CCLE數(shù)據(jù)庫中分析提取出與EMP1基因c o-expression的相關(guān)基因,并在T CGA HNSCC數(shù)據(jù)庫中分析EMP1基因與co-expression基因的RNA表達(dá)水平的相關(guān)性,結(jié)果顯示EPHA2、ITGA3及ANXA1 RNA表達(dá)水平與EMP1表達(dá)具有相關(guān)性,差異具有統(tǒng)計學(xué)意義,且在細(xì)胞實(shí)驗(yàn)中,過表達(dá)EMP1基因后,EPHA2、ITGA3及ANXA1蛋白表達(dá)水平也發(fā)生顯著改變。研究結(jié)論-(1)EMP1基因在HNSCC中的表達(dá)水平明顯低于在正常對照組織中的表達(dá)水平(2)EMP1基因在HNSCC組織中的表達(dá)水平隨分化程度增高而增高；(3)過表達(dá)EMP1后,HNSCC細(xì)胞的增殖能力及遷移能力顯著減弱,其可能的分子機(jī)制與EPHA2、ITGA3及ANXA1表達(dá)水平發(fā)生改變有關(guān)。
[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.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R739.91

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