以去泛素化酶UCH37为靶点治疗脑胶质瘤恶性增殖的分子机理研究

发布时间：2018-05-01 06:03

本文选题：UCH37 + 胶质瘤　；参考：《第二军医大学》2017年硕士论文

【摘要】：研究背景及目的:脑胶质瘤是最常见的成人中枢神经系统原发性难治性恶性肿瘤,发病率约5—6/10万,是颅内肿瘤患者主要的死亡原因。随着人口老龄化脑胶质瘤发病率有进一步增加的趋势,给家庭、社会带来沉重负担,严重危害人类的健康。世界卫生组织(World Health Organization,WHO)将胶质瘤分为I-IV级,最高级别的胶质母细胞瘤(Glioblastoma,GBM)是恶性程度和发病率最高的胶质瘤,82%的恶性胶质瘤病理为胶质母细胞瘤。目前胶质瘤的标准治疗方案为手术切除联合术后辅助放化疗,标准化的诊疗使患者的总体生存时间(Overall survival,OS)在一定程度上得到延长。但因脑胶质瘤恶性增殖、高度复发、恶性侵袭的特点,替莫唑胺(Temozolomide,,TMZ)等化疗药耐药,化疗耐受性等因素GBM患者总体疗效没有突破性进展,几乎所有患者不可避免的复发,中位生存期仅12-15个月,5年生存率仅4.7%。改善胶质瘤患者的预后,开拓新的治疗思路和药物成为神经外科领域的难题和关注点。脑胶质瘤的发生、发展、侵袭、复发涉及很多基因和蛋白的改变。分子生物学、基因组学、蛋白组学的发展,为探索肿瘤恶性增殖的分子病理机制提供便利的工具。分子靶向治疗针对肿瘤发生的关键分子,促进凋亡,抑制肿瘤形成,成为目前肿瘤治疗研究的热点。新的胶质瘤病理分型将组织细胞形态和基因检测相结合,根据不同基因分型决定不同的靶向用药。单克隆抗体(贝伐单抗等)和小分子激酶受体抑制剂等是目前靶向药物的主体,其特异性强,毒副作用小,较传统细胞毒性化疗药物更有临床价值。在GBM发生发展中存在多基因的突变,靶向单独分子的疗效欠理想,长期使用易产生耐药性。所以阐明胶质瘤恶性增殖的分子机制,寻找调控胶质瘤发生发展的关键分子靶点非常重要。基因芯片、二代测序等技术和肿瘤基因数据库为研究肿瘤发生发展的分子机制提供有力的支持。泛素-蛋白酶体系统是真核生物大多数细胞内蛋白质降解的主要途径。去泛素化可作用于泛素化蛋白,解离出泛素分子,逆转泛素化过程,使底物不被蛋白酶体降解。去泛素化酶可对泛素前体进行修饰加工,对蛋白酶体复合物及细胞质内泛素链进行水解,从而使泛素分子在细胞内稳定存在和循环利用。去泛素化酶参与许多重要的生物过程,如细胞周期调控、DNA损伤修复、核染色质重组、调节细胞信号通路等。泛素化和去泛素化的相互作用参与调节癌基因、抑癌基因、致癌信号通路关键分子等蛋白的降解和稳定,调控肿瘤的发生、发展、迁移、侵袭、复发等过程。泛素羧基末端水解酶家族(Ubiquitin Carboxy Terminal Hydrolases,UCHs)是一种重要的去泛素化酶,包括UCH-L1、UCH-L3、UCH37、BRCA1相关蛋白1(BRCA1 Associated Protein 1,BAP1)。研究表明UCH37和卵巢癌、多发性骨髓瘤、肝癌等恶性肿瘤恶性增殖关系密切,促进肿瘤的复发并导致预后不佳,但是尚无报道关于UCH37和脑胶质瘤恶性增殖关系的实验研究。因此,本研究的目的是利用多样本信息验证UCH37在胶质瘤中的差异表达和对胶质瘤预后判断的价值,并利用体外实验探索UCH37在胶质瘤恶性增殖中的作用机制。本论文分为两部分:第一部分脑胶质瘤组织芯片中UCH37基因表达及与患者预后相关性验证收集来自上海长征医院经过病理证实的300例脑胶质瘤样本及16例正常脑组织对照样本,所有样本制备成高通量组织芯片。收集入组的患者的临床资料并对患者术后放化疗及生存时间进行随访,结合免疫组化分析UCH37表达对患者预后的影响。截止随访结束,脑胶质瘤患者总体生存期为31.96±26.82个月,无进展生存期为29.19±25.96个月;患者的存活率为39.1%,35.6%患者肿瘤未见影像学进展。免疫组化的结果显示UCH37在胶质瘤组织表达明显,对比正常脑组织在脑胶质瘤中表达显著增高。免疫组化UCH37在胶质瘤组织中表达评分1.905±1.935,在正常脑组织中评分为0.42±1.04,存在显著统计学意义(p0.0010)。通过Kaplan-Meier生存曲线研究UCH37表达和患者生存期发现,高、低表达UCH37的胶质瘤患者中位OS分别为20个月、40个月,中位无进展生存时间(Progression Free Survival,PFS)分别为19个月、30个月,高表达组的OS、PFS均明显较低表达组缩短(OS:p0.001;PFS:p0.001)。进一步在原发胶质母细胞瘤患者中研究发现UCH37显著高表达于原发GBM组织(p0.001),高表达UCH的原发GBM生存期较低表达的短(OS:10个月vs12个月,p0.001;PFS:11个月vs 8个月,p0.001)。Cox多因素风险回归发现UCH37是原发GBM患者OS(HR=2.050,95%CI 1.243-3.380,p=0.005)及PFS(HR=2.135,95%CI 1.291-3.531,p=0.003)预后判断的独立因子。接受化疗原发GBM患者中UCH37高表达组生存期较低表达组均缩短(OS:9个月vs15个月,p0.001;PFS:7个月vs11个月,p0.001),接受放疗的原发GBM患者中UCH37高表达组生存期较低表达缩短(OS:10个月vs15个月,p0.001;PFS:7个月vs12个月,p0.001)。结果提示UCH37表达和原发GBM患者对放化疗的敏感性具有相关性。第二部分:UCH37基因调控脑胶质瘤恶性增殖的机制研究为研究UCH37在胶质瘤增殖过程中发挥的细胞学功能,我们利用慢病毒转染的方法在脑胶质瘤细胞系U87中对UCH37进行干扰和过表达,并设置空载质粒作为对照。利用二代测序方法验证UCH37干扰和过表达的序列,通过定量聚合酶链式反应(Polymerase Chain Reaction,PCR)实验和Western Blot实验验证UCH37干扰和过表达后的m RNA和蛋白的表达效果,流式细胞仪检测细胞周期比例变化,通过CCK-8法绘制细胞增殖曲线,克隆形成率实验检测克隆能力。我们发现与空载质粒的对照组相比,UCH37过表达后胶质瘤细胞增殖、克隆能力显著提高;UCH37敲减后胶质瘤细胞增殖及克隆能力明显下降,且细胞周期被阻滞在S期。结论:综上所述,我们通过分析组织芯片结合临床组织样本筛选,验证基因UCH37表达于脑胶质瘤组织中并和患者临床预后具有相关性,可判断胶质瘤患者临床预后,并是原发GBM患者预后的独立预测因子并影响原发GBM患者对放化疗的敏感性。通过体外实验验证UCH37可促进胶质瘤细胞的恶性增殖。因此,UCH37可作为预测脑胶质瘤患者临床预后的分子靶点而进行深入的研究。
[Abstract]:Background and objective: glioma is the most common primary intractable malignant tumor in the adult central nervous system. The incidence of the disease is about 5 - 6/10 million. It is the main cause of death in the patients with intracranial tumors. With the increasing trend of the incidence of brain glioma in the population, it has brought a heavy burden to the family and society, which seriously endangers human beings. Health. The WHO (World Health Organization, WHO) divides the glioma into the I-IV grade, the highest grade of glioblastoma (Glioblastoma, GBM) is the highest malignancy and the highest incidence of glioma, and 82% of the malignant glioma is glioblastoma. The standard treatment for glioma is currently assisted by surgical resection combined with surgery. The overall survival time (Overall survival, OS) of patients was extended to a certain extent by radiotherapy and chemotherapy. However, the overall efficacy of GBM patients was almost no breakthrough due to the malignant proliferation, high recurrence, malignant invasion of glioma, the drug resistance of temozolomide (Temozolomide, TMZ) and other factors in chemical therapy. All patients have an inevitable recurrence. The median survival time is only 12-15 months. The 5 year survival rate is only 4.7%. to improve the prognosis of the glioma patients. The development of new treatment ideas and drugs has become a difficult problem and concern in the Department of neurosurgery. The occurrence, development, invasion, and recurrence of glioma involve many changes in genes and proteins. Molecular biology, genome The development of proteomics provides a convenient tool for the exploration of the molecular pathological mechanism of malignant tumor proliferation. Molecular targeting therapy is a hot spot in the research of tumor therapy, which is the key molecule of cancer, promoting apoptosis and inhibiting the formation of tumor. Different genotyping determines different targeting drugs. Monoclonal antibodies (bevacizumab) and small molecular kinase receptor inhibitors are the main body of target drugs at present. They are more specific and less toxic and side effects. They are more valuable than traditional cytotoxic chemotherapeutic drugs. There are multiple gene mutations in the development of GBM and the therapy target to individual molecules. Therefore, it is very important to elucidate the molecular mechanism of malignant proliferation of glioma and to find the key molecular targets to regulate the development of glioma. The gene chip, two generation sequencing and the tumor gene database provide strong support for the molecular mechanism of tumor development. The body system is the main pathway of protein degradation in most cells in eukaryotes. Deubiquitination can act on ubiquitin protein, dissociate ubiquitin molecules, reverse the ubiquitination process, and degrade the substrate by proteasome. Deubiquitination enzyme can modify the ubiquitin precursor and carry out the proteasome complex and the ubiquitin chain in the cytoplasm. Hydrolysis, thus making ubiquitin molecules stable and circulatory in cells. De ubiquitinase participates in many important biological processes, such as cell cycle regulation, DNA damage repair, nuclear chromatin recombination, regulation of cell signaling pathways, and so on. The interaction of ubiquitination and de generalization can regulate oncogene, tumor suppressor gene, and carcinogenic signal pathway. Ubiquitin Carboxy Terminal Hydrolases (UCHs) is an important deubiquitination enzyme, including UCH-L1, UCH-L3, UCH37, BRCA1 related protein 1 (BRCA1 Associated Protein 1,). H37 is closely related to malignant proliferation of malignant tumors such as ovarian cancer, multiple myeloma and liver cancer, which promotes the recurrence of tumor and leads to poor prognosis. However, there is no report about the relationship between UCH37 and glioma proliferation. Therefore, the purpose of this study is to verify the differential expression of UCH37 in glioma by using multi sample information. The prognostic value of glioma and the mechanism of UCH37 in the malignant proliferation of glioma were explored in vitro. This paper is divided into two parts: the first part is the expression of UCH37 gene in the tissue microarray of brain glioma and the correlation of the prognosis of the patients with the prognosis of the patients, which are collected from 300 cases of glioma confirmed by the Shanghai Changzheng Hospital and 1 6 normal brain tissue control samples, all samples were prepared into high throughput tissue chips. The clinical data of the patients in the group were collected and followed up for postoperative radiotherapy and chemotherapy and survival time. The effect of UCH37 expression on the prognosis of the patients was analyzed with immunohistochemical analysis. The total survival period of the patients with glioma was 31.96 + 26.82 months by the end of the follow-up. The survival rate of progression free was 29.19 + 25.96 months, the survival rate of the patients was 39.1%, and there was no imaging progress in the 35.6% patients. The results of immunohistochemistry showed that the expression of UCH37 in glioma tissues was obvious, and the expression of normal brain tissue in glioma was significantly higher than that of normal brain tissue. The expression of immuno group UCH37 in glioma tissues was 1.905 + 1.935. The score of the normal brain tissue was 0.42 + 1.04, and there was a significant statistical significance (p0.0010). Through the Kaplan-Meier survival curve, the UCH37 expression and the patient's survival time were found. The middle OS of the patients with glioma with high and low expression of UCH37 was 20 months, 40 months, and the median progression free survival time (Progression Free Survival, PFS) was 19 months, 30 months, respectively. The OS and PFS in the high expression group were significantly shorter in the lower expression group (OS:p0.001; PFS:p0.001). Further studies in the patients with primary glioblastoma showed that UCH37 was highly expressed in the primary GBM tissue (p0.001), and the primary GBM survival period of the high expression UCH was shorter (OS:10 month vs12 month, p0.001; 8 months of UCH months) Factor risk regression found that UCH37 was an independent factor in predicting the prognosis of OS (HR=2.050,95%CI 1.243-3.380, p=0.005) and PFS (HR=2.135,95%CI 1.291-3.531, p=0.003) in the primary GBM patients. The low expression of UCH37 high expression group was shortened in the high expression group of GBM patients with radiotherapy (OS:10 months vs15 months, p0.001; PFS:7 month vs12 months, p0.001). The results suggest that the expression of UCH37 expression and the primary GBM patients are related to the sensitivity of radiotherapy and chemotherapy. The second part: the mechanism of UCH37 gene regulating the malignant proliferation of glioma is to study UCH37 In the proliferation process of glioma, we used the method of lentivirus transfection to interfere and overexpress UCH37 in the glioma cell line U87, and set the empty plasmid as the control. The two generation sequencing method was used to verify the UCH37 interference and overexpressed sequence, and the quantitative polymerase chain reaction (Polymerase Chain) was used. Reaction, PCR) experiment and Western Blot test verified the expression of M RNA and protein after UCH37 interference and overexpression. Flow cytometry was used to detect cell cycle ratio. Cell proliferation curve was plotted by CCK-8 method and clone formation rate test was used to detect cloning ability. The proliferation and cloning ability of the tumor cells increased significantly, and the proliferation and clone ability of glioma cells decreased significantly after UCH37 knockout, and the cell cycle was blocked in the S phase. Conclusion: in summary, we screened the tissue chip and clinical tissue samples to verify that the gene UCH37 was expressed in the brain glioma tissue and had a phase with the patient's clinical prognosis. The clinical prognosis of the patients with glioma can be judged and the independent predictor of the prognosis of the primary GBM patients and the sensitivity of the primary GBM patients to radiotherapy and chemotherapy. It is proved that UCH37 can promote the malignant proliferation of glioma cells through in vitro experiments. Therefore, UCH37 can be used as a molecular target to predict the clinical prognosis of glioma patients. Research.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.41

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