烟酰胺N甲基转移酶NNMT表观遗传调控促进胶质瘤增殖侵袭的作用机制研究

发布时间：2018-06-30 06:02

本文选题：NNMT + 胶质瘤　；参考：《第二军医大学》2016年博士论文

【摘要】：脑胶质瘤是中枢神经系统最常见的恶性肿瘤之一,约占全部脑肿瘤的45～55%。目前,脑胶质瘤的治疗手段主要包括手术治疗、放射治疗、化学治疗等,但由于肿瘤呈浸润性生长,与周围脑组织无明显分界且易于复发,上述传统治疗措施疗效均不甚理想。新的治疗手段如基因治疗等虽有望成为治愈胶质瘤的途径之一,但目前仍缺乏理想可靠的治疗靶标。因此寻找调控胶质瘤细胞恶性生物学行为的分子靶标,明确其在胶质瘤发病过程中的病理机理,已成为神经科学领域的研究热点之一。烟酰胺N-甲基转移酶NNMT是一种S-腺苷基-L-甲硫氨酸依赖性细胞质酶,催化烟酰胺及其它嘧啶的N-甲基单元,形成吡啶离子。NNMT在肝脏组织中有明显的表达,而在肾脏、肺脏、骨骼肌、胎盘、心脏等很少表达,在脑组织中低表达或不表达。目前研究发现,NNMT在多种肿瘤组织中异常表达,包括:胃腺癌、甲状腺乳头状癌、结肠癌、肝细胞癌、肺癌、胰腺癌等。在非肿瘤性病变中,NNMT被发现在慢性阻塞性肺疾病COPD、动脉粥样硬化、以及帕金森病中异常表达。NNMT参与肿瘤的代谢、甲基化、氧化应激反应,被认为在肿瘤的发生、发展中产生重要作用,但其具体机制尚不清楚。Kristian Ovaska等通过大数据框架整合技术Anduril,整合大规模分子及临床数据,分析筛选出一组与GBM增殖、生存以及多形性密切相关的基因改变,NNMT基因便是其中之一,初步发现在体外细胞中下调NNMT的表达可以显著抑制GBM的增殖,提示NNMT可能在胶质瘤的恶性生物学行为中扮演重要角色,但具体机制尚无研究。我们将从以下几个方面对NNMT在胶质瘤中的作用做系统的研究:制备了59例经手术切除的胶质瘤标本组织芯片,通过Western blot检测不同级别脑胶质瘤中NNMT的表达水平。制作生存曲线,观察NNMT表达水平与患者生存时间的关系。接着,进行体外细胞学实验,通过慢病毒敲减胶质瘤细胞系中NNMT的表达,检测其对胶质瘤细胞增殖、迁移、侵袭、凋亡等细胞恶性成为学行为的影响。通过成球实验,观察NNMT对胶质瘤干性的影响。随后,进行差异基因表达谱芯片检测,筛选NNMT基因下游靶标基因。由于NNMT是甲基化调控酶,我们还将进行甲基化检测,阐明NNMT甲基化调控靶标。最后,对NNMT调控下游靶标的方式做出初步研究,从而阐明NNMT促进胶质瘤增殖、侵袭的作用机制。本实验将分四个部分进行:1.NNMT在人脑胶质瘤中的表达及临床意义;2.NNMT对胶质瘤增殖、侵袭、凋亡等细胞功能及干性的影响;3.NNMT在胶质瘤中的下游靶标筛选及验证;4.NNMT是否通过表观遗传调控促进价胶质瘤增殖侵袭。第一部分NNMT在人脑胶质瘤中的表达及临床意义研究目的:探讨NNMT在胶质瘤中的表达情况及临床意义研究内容:对59例经手术切除的胶质瘤标本制作组织芯片,免疫组织化学检测NNMT在不同级别胶质瘤中的表达情况,同时对患者的随访资料进行分析,分析NNMT在不同级别胶质瘤中的表达情况与生存时间的关系,制作生存曲线。结果:NNMT在胶质瘤中过表达,其表对水平随着病理级别的增加而增加,与胶质瘤患者生存时间负相关,NNMT表达高的患者,生存时间缩短。结论:NNMT在人脑胶质瘤中过表达,表达水平与胶质瘤恶性程度正相关,与生存时间负相关,NNMT与胶质瘤的恶性行为相关。第二部分NNMT对胶质瘤增殖、侵袭、凋亡等细胞功能及干性的影响研究目的:体外细胞学实验研究NNMT对胶质瘤细胞系增殖、迁移、侵袭、凋亡等细胞功能及成球能力等干性的影响研究内容:构建经慢病毒转染SiRNA敲减NNMT的U87、U251细胞系,同时设立空白对照组,筛选稳转细胞株。Western blot验证慢病毒敲减效果,WST1法检测细胞增殖,划痕法检测细胞迁移,Transwell法检测细胞侵袭,流式细胞仪检测细胞凋亡,成球检测NNMT对干性的影响。结果:慢病毒转染SiRNA敲减NNMT后,U87、U251胶质瘤细胞的增殖、迁移、侵袭能力明显下降,成球能力下降,凋亡增加。结论:慢病毒转染SiRNA敲减NNMT在U87、U251胶质瘤细胞中的表达,能抑制胶质瘤细胞的增殖、迁移、凋亡等恶性表型,同时抑制成球能力(干性),并促进肿瘤细胞凋亡。第三部分NNMT在胶质瘤中的下游靶标筛选及验证研究目的:筛选NNMT促进胶质瘤细胞增殖、侵袭等恶性生物学行为的下游靶标基因。研究内容:构建慢病毒转染SiRNA敲减U251细胞系两组(U251-KD1,U251-KD2),同时设立空白对照组,筛选稳转细胞株。用基因表达谱芯片筛选下游靶标基因。对筛选出的基因进行作用分析,挑选感兴趣基因。PCR及Western Blot验证靶标基因。结果:U251-KD1和U251-KD2两个NNMT敲减细胞系和U251-NC对比,进行基因表达谱芯片分析,筛选出共同下调的基因共61个下调和32上调基因,进一步分析挑选出感兴趣的8个下调基因MMP2、MMP7、RSP03、SEMA3F、TNFRSF19、ANXA8、APOBEC3G、CXCR7,这8个基因与肿瘤的增殖、侵袭密切相关。同时挑选出5个上调基因PAK3、GAP43、ELAVL2、NEGR1、STAU2,这五个基因具有不同途径的抑癌作用。对筛选出的靶标基因进行PCR验证,PCR结果与基因表达谱芯片筛选出的结果一致。结论:NNMT在U251胶质瘤细胞中的下游上调靶标基因MMP2、MMP7、RSP03、SEMA3F、TNFRSF19、ANXA8、APOBEC3G、CXCR7等主要与增殖、侵袭、迁移相关,下游下调靶标主要与PAK3、GAP43、ELAVL2、NEGR1、STAU2抑癌相关。第四部分 NNMT是否通表观遗传调控促进胶质瘤增殖侵袭研究目的:初步探讨NNMT是否通过调控GAP-43促进胶质瘤的增殖、侵袭和凋亡。研究内容:构建慢病毒转染SiRNA敲减NNMT U251细胞系两组(U251-KD1,U251-KD2),同时设立空白对照组,筛选稳转细胞株。用甲基化芯片筛选下游作用靶标。对筛选出的基因进行文献学习,挑选感兴趣基因。结果:慢病毒转染SiRNA敲减NNMT U251细胞系两组(U251-KD1,U251-KD2),引起GAP-43的多个甲基化位点发生变化,GAP-43也是我们在基因表达谱芯片中筛选出的靶标基因。结论:NNMT促进胶质瘤增殖、侵袭的一个下游靶标是GAP-43,其作用可能是通过甲基化调控实现的。
[Abstract]:Glioma is one of the most common malignant tumors in the central nervous system, which accounts for about 45 to 55%. of all brain tumors. The treatment of glioma mainly includes surgical treatment, radiation therapy and chemical therapy. However, the tumor is infiltrative growth, and there is no clear demarcation between the surrounding brain tissue and easy to relapse. The above traditional therapeutic measures are effective. New therapies such as gene therapy, such as gene therapy, are expected to be one of the ways to cure glioma, but there is still a lack of ideal and reliable target for treatment. Therefore, it has become a research field in the field of neuroscience to find a molecular target to regulate the malignant biological behavior of glioma cells and to clarify its pathological mechanism in the pathogenesis of glioma. Nicotinamide N- methyltransferase NNMT is a S- adenosine based -L- methionine dependent cytoplasmic enzyme that catalyzes the N- methyl unit of nicotinamide and other pyrimidine, forming a pyridine ion.NNMT in the liver tissue, while in the kidneys, lungs, skeletal muscles, placenta, heart and so on, low expression in the brain tissue or in the brain tissue is low. NNMT has been found to be abnormal in a variety of tumor tissues, including gastric adenocarcinoma, papillary thyroid carcinoma, colon cancer, hepatocellular carcinoma, lung cancer, pancreatic cancer, and so on. In non tumor lesions, NNMT is found in chronic obstructive pulmonary disease (COPD), atherosclerotic atherosclerosis, and the abnormal expression of.NNMT in the tumor in Parkinson's disease. Thanks, methylation, oxidative stress reactions are believed to play an important role in the development and development of tumor, but its specific mechanism is not clear about.Kristian Ovaska and other genetic changes that are closely related to GBM proliferation, survival and polymorphisms through the integration of large data frame integration technology Anduril, integration of large-scale molecular and clinical data. The NNMT gene is one of them. It is preliminarily found that down regulation of NNMT in vitro can significantly inhibit the proliferation of GBM, suggesting that NNMT may play an important role in the malignant biological behavior of glioma, but the specific mechanism is not yet studied. We will systematically study the role of NNMT in glioma from the following aspects: preparation The expression level of NNMT in different levels of glioma was detected by Western blot. The relationship between the expression of NNMT and the relationship between the expression of NNMT and the survival time of the patients was observed. Then, the cytological test was carried out in vitro, and the expression of NNMT in the lentivirus glioma cell line was detected. The effects of cell proliferation, migration, invasion, apoptosis and other cell malignancies on glioma cells were influenced by the study on the effects of NNMT on the dry character of glioma. Subsequently, differential gene expression spectrum chips were used to detect and screen the downstream target genes of the NNMT gene. As NNMT is the methylation regulator, we will also carry out methylation detection and clarify NNM T methylation targets the target. Finally, a preliminary study of the way NNMT regulates the downstream target is made to clarify the role of NNMT in promoting the proliferation and invasion of glioma. This experiment will be divided into four parts: the expression and clinical significance of 1.NNMT in human glioma; 2.NNMT on the proliferation, invasion, apoptosis and other cell functions of glioma. Sound; 3.NNMT's downstream target screening and validation in glioma; whether 4.NNMT can promote glioma proliferation and invasion through epigenetic regulation. Part 1 NNMT expression in human glioma and its clinical significance: To explore the expression and clinical meaning of NNMT in glioma: 59 cases of surgical resection of glioma The tumor specimens were made by tissue microarray, the expression of NNMT in different grade gliomas was detected by immunohistochemistry, and the follow-up data of the patients were analyzed. The relationship between the expression of NNMT in different grade gliomas and the survival time was analyzed, and the survival curve was made. Results: NNMT was overexpressed in glioma, and its table was associated with the disease. The increase in rational level was negatively related to the survival time of the patients with glioma, and the time of survival was shortened in patients with high NNMT expression. Conclusion: NNMT was overexpressed in human glioma, the level of expression was positively related to the malignant degree of glioma, and it was negatively related to the survival time. NNMT was associated with the malignant behavior of glioma. Second part NNMT was the proliferation of glioma and invasion of glioma. Research objective: the effects of NNMT on proliferation, migration, invasion and apoptosis of glioma cell lines were studied in vitro: the construction of U87, U251 cell line by lentivirus transfection of SiRNA and U251 cell line, and a blank control group to screen the stability of the cell Cell strain.Western blot verified the effect of lentivirus knockout, cell proliferation was detected by WST1, cell migration was detected by scratch assay, cell invasion was detected by Transwell method, cell apoptosis was detected by flow cytometry, and the effect of NNMT on NNMT was detected. The results showed that the proliferation, migration, and invasion ability of U87 and U251 glioma cells were clear after the SiRNA transfected to NNMT. Conclusion: the expression of SiRNA knockout NNMT in U87, U251 glioma cells can inhibit the proliferation, migration, apoptosis and other malignant phenotype of glioma cells, and inhibit the ability to form the ball (dry), and promote the apoptosis of the tumor. The third part of NNMT is screened in the downstream target of glioma and Objective: to screen the downstream target genes of NNMT to promote glioma cell proliferation, invasion and other malignant biological behavior. Research contents: two groups of SiRNA knockout U251 cell lines (U251-KD1, U251-KD2) were constructed with lentivirus transfection, and a blank control group was set up to screen the stable cell lines. The selected genes were analyzed, the genes of interest gene.PCR and Western Blot were selected to verify the target genes. Results: U251-KD1 and U251-KD2 two NNMT knockout cell lines were compared with U251-NC, and the gene expression spectrum chip analysis was carried out to select 61 down regulated genes and 32 upper genes, and to further analyze 8 of the interest. Down regulated genes MMP2, MMP7, RSP03, SEMA3F, TNFRSF19, ANXA8, APOBEC3G, CXCR7, these 8 genes are closely related to the proliferation and invasion of the tumor. At the same time, 5 up-regulated genes, PAK3, GAP43, ELAVL2, NEGR1, have been selected, and these five genes have different ways of inhibiting cancer. The results were consistent. Conclusion: the downstream target genes of NNMT in U251 glioma cells are up regulation of target genes MMP2, MMP7, RSP03, SEMA3F, TNFRSF19, ANXA8, APOBEC3G, CXCR7, etc., which are mainly related to proliferation, invasion and migration. The downstream target is mainly related to PAK3, GAP43, metastasis and cancer suppression. The fourth part is whether epigenetic regulation is promoted. Study on the proliferation and invasion of glioma: whether NNMT can promote the proliferation, invasion and apoptosis of glioma by regulating GAP-43. Research contents: two groups (U251-KD1, U251-KD2) of SiRNA knockout NNMT U251 cell line were constructed by lentivirus transfection, and a blank control group was set up to screen the stable cell line. The downstream action target was screened by methylation chip. The selected genes were studied, and the genes of interest were selected. Results: the lentivirus transfected to SiRNA NNMT U251 cell line two (U251-KD1, U251-KD2), resulting in the change of multiple methylation sites in GAP-43, GAP-43 is the target gene we screened in the gene expression chip. Conclusion: NNMT promotes the proliferation and invasion of glioma. A downstream target is GAP-43, which may be regulated by methylation.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R739.41

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