新疆维、汉族胶质瘤临床特点及基因表达差异的研究

发布时间：2018-05-17 02:22

本文选题：胶质瘤 + 维吾尔族　；参考：《新疆医科大学》2014年博士论文

【摘要】：目的：总结新疆地区维、汉族胶质瘤患者的临床特点，分析相关临床因素的分布特点，结合随访资料，进行生存分析。通过对P53、MGMT、PTEN蛋白及Ki-67在胶质瘤中表达及其与胶质瘤临床特征的关系，探讨胶质瘤相关分子标记如P53、MGMT、PTEN、Ki-67蛋白在新疆维、汉族胶质瘤患者间的表达差异及其与胶质瘤发生、发展中的关系。首次进行新疆维、汉族患者胶质母细胞瘤的mRNA基因表达谱及全基因组甲基化表达谱差异检测，筛选并初步探讨差异表达的相关基因在新疆维、汉族患者胶质母细胞瘤的发病机制及预后转归中的意义。方法：1）收集新疆医科大学附属肿瘤医院及新疆医科大学第一附属医院神经外科自建院以来经治的成人脑胶质瘤患者共985例，其中维吾尔族、汉族胶质瘤患者共有843例，其他民族如哈萨克族、回族、蒙古族等共有142例。收集并整理842例胶质瘤患者的一般资料、临床表现、影像学特征、临床病理学特点等信息，初步比较维、汉族胶质瘤患者在发病和临床特点分布情况，进行统计学分析。选取2010年1月～2013年5月新疆医科大学附属肿瘤医院的111例胶质瘤患者进行回顾性电话随访及复诊随访，其中维吾尔族、汉族患者共有80例，其他民族共有31例。运用免疫组织化学SP法分别检测随访资料完整的80例胶质瘤组织的P53、MGMT、PTEN蛋白及Ki-67的表达情况；比较维、汉族胶质瘤中P53、MGMT、PTEN、Ki-67的表达差异以及P53、MGMT、PTEN、Ki-67蛋白表达与胶质瘤患者临床特征的关系；2）运用Illumina HT-12mRNA表达谱芯片检测6例胶质母细胞瘤（其中维、汉族各3例）组织中的基因表达情况，初步筛选维、汉族胶质母细胞瘤中存在的相同基因及差异表达基因。通过提取组织总RNA，反转录成cDNA，将标记的cDNA与基因表达谱芯片杂交，最后读取数据及获得图像，进行生物功能信息学分析。所有检测样品均通过6类内参质控检测；运用免疫组织化学SP法检测胶质母细胞瘤相关分子标记物，与筛选出来的表达谱差异表达基因进行综合分析和验证；3）运用Illumina Methylation450K全基因组甲基化芯片检测6例胶质母细胞瘤（其中维、汉族各3例）组织中的甲基化表达情况，初步筛选维、汉族胶质母细胞瘤中差异表达的甲基化位点及其对应的相关基因。通过提取组织DNA，经过亚硫酸盐处理，与甲基化芯片杂交，读取数据及获得图像，进行生物功能信息学分析。所有样品均通过11类内参质控检测；对维、汉族胶质母细胞瘤中筛选出来的差异甲基化位点对应的差异基因，与mRNA表达谱芯片筛选的差异基因的结果分别进行高甲基化且基因低表达及低甲基化且基因高表达的联合分析，筛选新疆维、汉族胶质母细胞瘤中可能存在的关键差异基因。结果：1）在所有入组842例胶质瘤病例中，男性481例，女性362例，年龄1～79岁，平均年龄40.14±18.03岁，其中维族342例，汉族501例。维、汉族胶质瘤患者在不同WHO组织学分级的分布差异有统计学意义（P＜0.05）。所选病例的组织学类型中，星形细胞瘤（含胶质母细胞瘤）的例数最多达554例（554/842，65.72%）；室管膜肿瘤共149例（149/842，17.67%）；少突胶质细胞肿瘤共50例（50/842，5.93%）；少突星形细胞肿瘤共12例（12/842，1.42%）；脉络丛肿瘤共7例（7/842，0.83％）；神经元混合性神经元-胶质瘤共11例（11/842，1.30%）；胚胎性肿瘤共54例（54/842，6.41％）；松果体肿瘤共6例（6/842，0.71%）。80例随访资料完整的胶质瘤病例中。胶质瘤患者中民族（P=0.000）、肿瘤病理分级（P=0.000）、是否行放、化疗（P=0.009）和肿瘤是否全切（P=0.003）这四项指标可能是影响患者预后的独立危险因素。P53、MGMT、PTEN蛋白和Ki-67的阳性率分别为66.25%（53/80）、65%（52/80）、45%（36/80）和47.5%（38/80）。维、汉族胶质瘤患者中MGMT、P53蛋白和Ki-67表达在不同WHO组织学分级组中的差异有统计学意义（P＜0.05），而在不同年龄、性别、民族及肿瘤部位、病灶大小、侵犯临近脑叶情况等各组中的差异均无统计学意义（P＞0.05）；PTEN蛋白表达在不同肿瘤部位及WHO组织学分级组中的差异有统计学差异（P＜0.05），而在不同年龄、性别、民族、病灶大小、是否侵犯临近脑叶情况等各组中的差异均无统计学意义（P＞0.05）。2）将芯片杂交扫描图片导入IlluminaGenomeStudio软件，得到校正后的数据，利用R语言lumi包进行标准化处理及差异基因筛选（P＜0.05）；利用Web Gestalt软件，对差异基因进行GO和KEGG分析（P＜0.05），维、汉族患者胶质母细胞瘤mRNA表达谱的比较分析发现，筛选出显著差异表达的基因共有1475个，其中表达上调的基因有669个（44.84％），表达下调的基因有807个（55.16％）（其中有1个基因（STRC）对应2个转录本，1个转录本表达上调1个转录本表达下调），差异基因主要参与了代谢过程、生物调节、对刺激反应、多细胞的有机过程等，其中包含小GTP酶调节信号通路、Ras信号通路、神经元反应蛋白调节、中枢神经系统髓鞘形成等多个功能节点；同时也参与了多条与肿瘤发生相关的信号通路，如：代谢通路、癌通路、MAPK信号通路、TGF-β信号通路、神经营养因子信号转导通路、mTOR信号通路等。3）将芯片杂交扫描图片导入Illumina GenomeStudio软件，得到校正后的数据，使用controls标准化；通过IlluminaCustom进行差异位点筛选，结果经过多重检验校正和筛选条件为，筛选条件为|DiffScore|≥13|DeltaBeta|≥0.2；利用WebGestalt软件，对差异甲基化位点对应的基因进行GO和KEGG的功能注释（P＜0.05），维、汉族患者胶质母细胞瘤全基因组甲基化表达谱的比较分析发现，筛选出显著差异表达的6406个差异位点所对应的1903个基因中，表达上调的位点有1953个（1953/6406，30.49％），表达下调的位点有4453个（4453/6406，69.51％），这些差异位点对应的相关基因主要参与了生物调节、代谢过程、多细胞的有机过程、对刺激反应、发育过程等，其中包含小GTP酶调节信号通路、Ras信号通路、神经元反应蛋白调节、中枢神经系统髓鞘形成等多个功能节点；同时也参与了多条与肿瘤发生相关的信号通路，如：神经分化、神经发育、MAPK信号通路、TGF-β信号通路、神经营养因子信号转导通路、mTOR信号通路等。4）通过对维、汉族胶质母细胞瘤mRNA基因表达谱及其全基因组甲基化状态的关联性分析，初步通过对差异表达基因及差异甲基化位点所对应的差异基因表达上调、下调关系进行比对发现，维、汉族胶质母细胞瘤中全基因组甲基化芯片筛选的高甲基化位点（表达上调）对应的基因中与表达谱筛选的差异基因中低表达（表达下调）的基因共同的有12个，分别是ADARB2，ATP11C，ATPGD1，C1orf59，GPR62，PIK3C2B，PIR，PLLP，SLC5A11，ZFYVE27，ZNF415，ZNF536；而维、汉族胶质母细胞瘤全基因组甲基化芯片筛选的低甲基化位点（表达下调）对应的基因中与表达谱筛选的差异基因中高表达（表达上调）的基因共同的有4个，分别是HSD17B10，MRPL3，ZBTB5，ZNF662。结论：1）与新疆地区汉族胶质瘤患者相比，维族胶质瘤患者有发病年龄轻、男性比例高、低级别胶质瘤比例较高、星形细胞瘤比例高的趋势。胶质瘤患者临床特点中的民族、肿瘤病理分级、是否行放、化疗和肿瘤是否全切等指标可能是影响胶质瘤患者预后转归的因素。联合检测MGMT、P53、PTEN蛋白和Ki-67在胶质瘤中的表达可以作为判定胶质瘤生物学行为和预后的参考指标。2）新疆维、汉族患者胶质母细胞瘤mRNA表达谱芯片和全基因组甲基化芯片筛查出的众多相同基因及相同甲基化位点，也筛选出不同民族间的差异表达基因及差异甲基化位点。这些差异基因主要参与了代谢过程、生物调节、对刺激反应、多细胞的有机过程、发育过程等，其中包含小GTP酶调节信号通路、Ras信号通路、神经元反应蛋白调节、中枢神经系统髓鞘形成等多个功能节点；同时也参与了多条与肿瘤发生相关的信号通路，如：代谢通路、癌通路、神经分化、神经发育、MAPK信号通路、TGF-β信号通路、神经营养因子信号转导通路、mTOR信号通路等。3）mRNA表达谱及甲基化状态的关联性分析提示维、汉族胶质母细胞瘤全基因组甲基化芯片筛选的高甲基化位点（表达上调）对应的基因中与mRNA表达谱筛选的差异基因中低表达（表达下调）的基因共同的有12个，分别是ADARB2，ATP11C，ATPGD1，C1orf59，GPR62，PIK3C2B，PIR，PLLP，SLC5A11，ZFYVE27，ZNF415，ZNF536；而维、汉族胶质母细胞瘤全基因组甲基化芯片筛选的低甲基化位点（表达下调）对应的基因中与mRNA表达谱筛选的差异基因中高表达（表达上调）的基因共同的有4个，分别是HSD17B10，MRPL3，ZBTB5，ZNF662。对这些基因的进一步研究有助于在基因层面上揭示新疆地区维、汉族胶质瘤患者发病机制、预后转归等方面可能存在的差异，，也为将来的进一步在分子水平上对新疆胶质瘤患者的的研究和靶向治疗提供新的依据。
[Abstract]:Objective: To summarize the clinical characteristics of the patients with human glioma in Xinjiang, to analyze the distribution characteristics of the related clinical factors and to analyze the survival analysis with the follow-up data. The expression of P53, MGMT, PTEN protein and Ki-67 in glioma and the relationship between the glioma and the clinical characteristics of glioma are discussed, and the molecular markers of glioma, such as P53, MGMT, PTEN, Ki-67, are discussed. The expression of protein in Xinjiang Wei and Han glioma patients and their relationship with the development of glioma and glioma. The mRNA gene expression profiles and total genome methylation expression profiles of glioblastoma in Xinjiang and Han patients were detected for the first time, and the genes related to differential expression in Xinjiang dimension and Han patients were screened and preliminarily discussed. The significance of the pathogenesis and prognosis of the mass blastoma. Methods: 1) 985 cases of adult brain glioma were collected from the Affiliated Tumor Hospital of Xinjiang Medical University and the First Affiliated Hospital of Xinjiang Medical University since the establishment of the hospital. There were 843 cases of Uygur and Han Chinese gelatoma, and other ethnic groups such as Kazakh. There were 142 cases of ethnic, Hui, Mongolian, etc. collected and collate 842 cases of glioma patients general information, clinical manifestations, imaging features, clinicopathological features and other information, preliminary comparative dimension, Han glioma patients in the incidence and clinical characteristics of the distribution, statistical analysis, selected from January 2010 to May 2013 affiliated to the Xinjiang Medical University affiliated. 111 patients with glioma in the cancer hospital were followed up with retrospective telephone follow-up and follow-up visit. There were 80 cases in Uygur and Han people and 31 in other nationalities. The expression of P53, MGMT, PTEN protein and Ki-67 in 80 glioma tissues were detected by immunohistochemical SP method. The expression of P53, MGMT, PTEN, Ki-67 and the relationship between the expression of P53, MGMT, PTEN, Ki-67 protein and the clinical characteristics of the patients with glioma; 2) the detection of the gene expression in the tissues of 6 cases of glioblastoma (among which 3 cases of Han nationality) was detected by Illumina HT-12mRNA expression chip, and the primary screening of the presence of the human glioblastoma in the Han nationality The same gene and differentially expressed genes were extracted by extracting the total tissue RNA and reverse transcriptional into cDNA. The labeled cDNA was hybridized with the gene expression chip. Finally, the data were read and the image was obtained, and the bioinformatics analysis was performed. All the samples were detected by 6 kinds of internal parameters, and the immunohistochemical SP method was used to detect the glioblastoma correlation. Molecular markers, integrated analysis and verification with the differentially expressed genes expressed by the screened expression profiles, and 3) detection of methylation in 6 cases of glioblastoma (including 3 of the Han people) by Illumina Methylation450K genome methylation chip, and preliminary screening of the differential expression in the human glioblastoma of the Han nationality. By extracting tissue DNA, using sulfite treatment, hybridization with methylation chips, reading data and obtaining images, bioinformatics analysis. All samples were detected by 11 types of internal parameters, and the differential methylation sites were screened in the Uygur and Han glioblastoma. The difference genes, respectively, were methylation, low gene expression, low methylation and high expression of genes, and the key differentially differentially expressed genes in Xinjiang and Han glioblastoma were screened by the results of differential genes screened by mRNA expression chip. Results: 1) in all 842 cases of glioma, 481 cases were male. There were 362 cases of female, aged 1~79 years, with an average age of 40.14 + 18.03 years old, including 342 cases of Uygur and 501 cases of Han nationality. There were significant differences in the distribution of WHO histology among the patients with glioma in the Han nationality (P < 0.05). The number of astrocytomas (including glioblastoma) was up to 554 cases (554/842,65.72%) in the histological type of the selected cases. 149 cases of ependyma tumor (149/842,17.67%); 50 cases of oligodendrocyte tumor (50/842,5.93%); 12 cases of oligodendroid astrocytoma (12/842,1.42%); 7 cases of choroid plexus tumor (7/842,0.83%); 11 cases of neuron mixed neuron glioma (11/ 842,1.30%); 54 cases of embryonal tumor (54/842,6.41%); pineal body, and pineal body. In a total of 6 cases (6/842,0.71%), a total of.80 cases were followed up with a complete follow-up of glioma cases. The four indexes of the group (P=0.000), tumor pathological grading (P=0.000), chemotherapy (P=0.009) and tumor total resection (P=0.003) may be the independent risk factors that affect the prognosis of the patients:.P53, MGMT, PTEN protein and Ki-67. The differences in MGMT, P53 and Ki-67 expressions in 66.25% (53/80), 65% (52/80), 45% (36/80) and 47.5% (38/80), and 47.5% (38/80). Human glioma were statistically significant (P < 0.05) in different WHO histology groups (P < 0.05). Study significance (P > 0.05); the difference of PTEN protein expression in different tumor sites and WHO histological grading groups was statistically significant (P < 0.05), and there was no statistical significance (P > 0.05).2) at different ages, sex, nationality, size of the focus, and the invasion of the adjacent lobes (P > 0.05).2). GenomeStudio software, obtained the corrected data, using the R language Lumi package for standardized processing and differential gene screening (P < 0.05). Using Web Gestalt software, GO and KEGG analysis (P < 0.05) of the differential genes (P < 0.05), and the comparison of mRNA expression profiles of the glioblastoma of the Han patients were compared and analyzed, and found that the significant differentially expressed genes were screened out. There are 1475 genes, of which 669 (44.84%) are up-regulated genes, and 807 (55.16%) down regulated genes (of which 1 genes (STRC) correspond to 2 transcripts, 1 transcripts are up regulated by 1 transcriptional downregulation), and the differential genes are mainly involved in metabolic processes, biological regulation, stimulus response, and multicellular organic processes. Several functional nodes, including small GTP enzyme regulation signaling pathway, Ras signaling pathway, neuron reactive protein regulation, central nervous system myelin formation, are also involved in a number of signaling pathways associated with tumor occurrence, such as metabolic pathways, cancer pathways, MAPK signaling pathways, TGF- beta signaling pathways, neurotrophic factor signal transduction pathways, mTOR .3 (signal path, etc.) introduced the chip hybrid scanning picture into Illumina GenomeStudio software, obtained the corrected data, used controls standardization, and screened the difference sites through IlluminaCustom, and the results were corrected by multiple tests and screening conditions, and the screening conditions were DiffScore| > 13|DeltaBeta| > 0.2; WebGestalt software was used. The functional annotation of GO and KEGG (P < 0.05) for the genes corresponding to the differential methylation sites, and the comparative analysis of the total genome methylation profiles of the glioblastoma in Han patients, found that of the 1903 genes corresponding to the 6406 differentially expressed differentially expressed heterotopic points, 1953 (1953/6406,30.49%) were expressed in the up regulated loci. There are 4453 down-regulated sites (4453/6406,69.51%). The related genes are involved in biological regulation, metabolic processes, multicellular organic processes, stimulation responses, development processes, including small GTP enzyme regulation signaling pathway, Ras signaling pathway, neuron reactive protein regulation, and central nervous system myelin shape There are multiple functional nodes, and they also participate in a number of signal pathways related to the occurrence of tumor, such as nerve differentiation, nerve development, MAPK signaling pathway, TGF- beta signaling pathway, neurotrophic factor signal transduction pathway, mTOR signaling pathway and other.4) through the mRNA gene expression profiles and full genome methylation of the Hans and grocin blastoma The correlation analysis of the state, the difference of the high methylation site (expression profile) selected by the whole genome methylation chip in the human glioblastoma of the Han nationality by comparing the up regulation and down regulation of differentially expressed genes and differential methylation sites. ADARB2, ATP11C, ATPGD1, C1orf59, GPR62, PIK3C2B, PIR, PIK3C2B, PIR, PLLP, SLC5A11, ZFYVE27, ZNF415, ZNF536; and the differential genes of the low methylation site (down expression) of the whole genome of the Han glioblastoma selected by the whole genome of the human glioblastoma There are 4 common genes of high expression (up expression), which are HSD17B10, MRPL3, ZBTB5, ZNF662. conclusion: 1) compared with the patients of Xinjiang Han glioma, Uygur glioma patients have a high incidence of age, high male proportion, high ratio of low grade glioma, high proportion of astrocytoma, and the clinical characteristics of glioma patients. A combination of MGMT, P53, PTEN protein and Ki-67 in glioma can be used as a reference index to determine the biological behavior and prognosis of glioma,.2) in Xinjiang and Han patients, and the human glioblastoma in Han patients. Many of the same genes and methylation sites screened by mRNA and all genome methylation chips have also been screened for differentially expressed genes and differential methylation sites among different ethnic groups. These differentially expressed genes are mainly involved in metabolic processes, biological regulation, response to stimulation, multicellular organic processes, and development processes. A number of functional nodes including small GTP enzyme regulation signaling pathway, Ras signaling pathway, neuron reactive protein regulation, central nervous system myelin formation, and a number of signaling pathways associated with tumor occurrence, such as metabolic pathways, cancer pathways, neural differentiation, neurodevelopment, MAPK signaling pathways, TGF- beta signaling pathways, neurotrophic factors Subsignal transduction pathway, mTOR signaling pathway and other.3) mRNA expression profiles and methylation status correlation analysis suggested that the high methylation site (up expression) selected by the whole genome methylation chip of the Hans glioblastoma (up-regulated) corresponding genes with low expression of low expression (down expression) in the differential gene of mRNA expression profiles are 12 ADARB2, ATP11C, ATPGD1, ATPGD1, C1orf59, GPR62, PIK3C2B, PIR, PLLP, SLC5A11, ZFYVE27, ZNF415, ZNF536; and dimension, the low methylation site (down expression) of the whole genome methylation chip screened by the whole genome of the Han nationality glioblastoma (down regulation) of the corresponding genes with high expression (up-regulated expression) in the differential gene screened from the spectrum The further study of these genes, HSD17B10, MRPL3, ZBTB5, and ZNF662., will help to reveal the possible differences in the pathogenesis, prognosis and other aspects of the Xinjiang regional dimension, the prognosis and the prognosis of the Han glioma patients at the gene level, as well as to provide further research and targeted therapy for the patients of Xinjiang glioma at the molecular level. A new basis.
【学位授予单位】：新疆医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.41

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