过表达去泛素化酶USP44通过稳定促癌蛋白securin促进胶质瘤的恶性进展

发布时间：2018-03-14 11:34

本文选题：胶质母细胞瘤　切入点：泛素特异性蛋白酶44　出处：《第二军医大学》2017年博士论文　论文类型：学位论文

【摘要】：胶质母细胞瘤是恶性程度最高的中枢神经系统原发性肿瘤之一。由于肿瘤呈浸润性生长,与周围脑组织无明显分解且易于发生放化疗耐受,目前传统的治疗措施疗效均不甚理想。新兴的个体化基因治疗有望成为治愈胶质母细胞瘤的途径之一,但目前仍缺乏理想可靠的治疗靶标。因此寻找调控胶质母细胞瘤恶性生物学行为的分子靶标并明确其在GBM发病过程中的病理机制,已成为颅脑肿瘤研究领域的热点之一。细胞周期的进程中,姐妹染色单体准确适时地分离对细胞的遗传学稳定是至关重要的,纺锤体装配检查点(SAC)机制则是确保有丝分裂正常进行的重要监控机制,其异常是导致非整倍体细胞出现乃至肿瘤形成的重要原因。在之前的研究报道中我们发现纺锤体装配检查点机制中重要的调控蛋白——去泛素化酶USP44与肿瘤的发生密切相关。去泛素化酶USP44在正常的细胞周期进程中可以稳定保护素的蛋白水平直到全部动粒均与纺锤丝正确匹配,防止细胞出现不成熟的有丝分裂。抑制小鼠胚胎成纤维细胞中USP44的表达水品后能明显提高非整倍体细胞的比例及染色体的不稳定性,使之更易于发生恶性转化。USP44是肿瘤促进因子Ubc H10的拮抗蛋白,而在部分肿瘤细胞中USP44却表达上调,但因缺乏能有效识别内源性USP44的特异性抗体,表达上调的USP44是否参与维持肿瘤细胞的恶性增殖能力及其内在的调控机制目前尚不清楚。本实验从筛选能特异性识别内源性USP44的抗体入手,分析USP44在胶质瘤组织中的表达水平与肿瘤级别及预后的关系,USP44与GBM恶性生物学行为的关系以及USP44可能参与的部分信号通路。第一部分USP44特异性抗体的筛选与鉴定目的:利用分子生物学技术筛选并鉴定能有效识别内源性USP44的特异性抗体。方法:预选三种抗体:Abnova p Ab21808,Santa Cruz sc-337203,Origene TA801913。在U251MG、U87MG及A172等三种细胞系中利用免疫印迹技术检测抗体是否能特异性识别内源性USP44;在239T细胞中过表达Flag-USP44融合蛋白,分别检测USP44抗体与Flag抗体的荧光信号;在U251MG细胞中,利用免疫荧光技术分别检测USP44抗体与B23抗体的荧光信号;在U251MG细胞中利用免疫印迹及免疫荧光技术检测USP44敲减后抗体能否识别USP44的表达下调。结果:在免疫印迹实验中三种抗体均能特异性识别三组细胞中的内源性USP44并形成唯一印迹条带;抗体p Ab21808的荧光信号能与Flag的荧光信号完全重合;抗体p Ab21808与B23抗体的荧光信号在核仁内存在重合;三种抗体均能检测到USP44敲减后的表达下调,抗体在USP44敲减后的U251MG细胞中的信号明显减弱。结论:抗体p Ab21808识别内源性USP44的特异性最好,能准确的反映出USP44的亚细胞定位及si RNA的敲减效率,故我们采用抗体p Ab21808进行后续实验。第二部分USP44表达水平与胶质瘤病理级别及预后的关系目的:在蛋白和m RNA层面上,利用组织标本研究USP44表达水平与胶质瘤病理级别及预后的关系。方法:利用抗体p Ab21808进行免疫组化实验,分析含61例不同级别胶质瘤标本的组织芯片,检测USP44蛋白在组织标本中的表达情况;利用q RT-PCR技术分析40例不同级别胶质瘤冰冻组织中USP44m RNA的表达水平;结合患者随访资料及q RT-PCR数据进行生存分析。结果:USP44的蛋白表达水平与胶质瘤病理级别呈线性相关;在高级别胶质瘤组中USP44m RNA的表达水平明显高于低级别组;USP44m RNA高表达组患者的术后生存时间明显小于低表达组。结论:USP44的表达水平随胶质瘤病理级别的升高而升高,高表达的USP44提示患者预后较差。第三部分USP44对GBM细胞恶性生物学行为的影响目的:研究GBM细胞的增殖、侵袭、周期、凋亡等生物学行为在USP44敲减后的变化。方法:U251MG和A172细胞转染USP44-sh RNA慢病毒后进行WST-1实验和平板克隆形成实验,检测USP44表达下调对细胞增殖能力的影响;Transwell实验检测USP44敲减后U251MG和A172细胞迁移、侵袭的能力的变化;稳定转染的U251MG和A172细胞经碘化丙啶(PI)染色后进行流式细胞周期分析;流式细胞仪分析荧光染料PE/7-AAD双染的稳定转染细胞,检测USP44表达下调后GBM细胞凋亡比例的变化。结果:USP44表达下调能明显抑制U251MG和A172细胞的增殖和侵袭能力,同时显著提高G2/M期细胞及凋亡细胞的比例。结论:敲减USP44能明显抑制GBM细胞的恶性生物学行为,阻滞细胞周期于G2/M期并诱导细胞凋亡。第四部分USP44与促癌因子securin之间的相互作用目的:验证USP44与securin之间是否存在相互作用,USP44是否通过去泛素化作用稳定securin。方法:在U251细胞中利用免疫荧光技术检测USP44与securin的荧光信号在细胞内的定位关系;利用免疫共沉淀技术验证USP44能否与securin直接结合;USP44-sh RNA转染的U251MG细胞经不同浓度的蛋白酶体抑制剂MG132处理后,用western blot技术检测securin表达水平的变化。结果:USP44与securin的荧光信号在分裂相的细胞中明显重合;内源性USP44能与securin直接结合;MG132能逆转由敲减USP44引起的securin表达下调且不影响USP44本身的表达水平。结论:Securin是USP44的催化底物,USP44通过去泛素化作用稳定securin并抑制其被蛋白酶体降解。第五部分USP44表达下调对U87MG细胞体内成瘤能力的影响目的:验证USP44表达下调对GBM细胞体内成瘤能力的影响。方法:连续培养USP44-sh RNA稳定转染的U87MG细胞;选择5周龄雄性裸鼠5只,分别在裸鼠两侧后肢近端的内侧皮下注射上述细胞及正常U87MG细胞(107cells)。每2天用游标卡尺测量肿瘤的长短径一次。至肿瘤长径超过10mm时将裸鼠脱颈处死,取出肿瘤;用裸鼠肿瘤标本制作石蜡切片进行免疫组化实验,对细胞形态和Ki-67的表达进行检测。结果:USP44敲减组和对照组相比,肿瘤的终体积和生长速率均明显减小;免疫组化结果表明敲减组Ki-67的表达水平明显低于对照组。结论:USP44表达下调能明显抑制U87MG细胞的体内成瘤能力。
[Abstract]:Glioblastoma is one of the central nervous system of most malignant primary tumor. The tumor showed invasive growth, no significant decomposition and prone to chemotherapy tolerance and surrounding brain tissue, the curative effect of the traditional treatment measures are not ideal. Individual gene therapy is expected to become one of the new ways to cure glioma cells tumor, but there is still a lack of reliable ideal therapeutic target. So looking for molecular target for malignant biological behavior regulation of glioblastoma and clear in the pathogenesis of GBM pathogenesis, has become a hot topic in the research field of brain tumor. The process of cell cycle, sister chromatid separation timely accurate genetics the stable cell is crucial, spindle assembly checkpoint (SAC) is an important mechanism to ensure the normal monitoring mechanism of mitosis, the abnormality is caused by non An important reason for aneuploidy cells and tumor formation. Research reports before we found the important regulatory proteins of the spindle assembly checkpoint mechanism -- deubiquitinase USP44 closely associated with tumor. Deubiquitinase USP44 protein level could be stable until all kinetochores are ofosteoprotegerin and spindle right matching in the normal cell cycle progression, cells appeared to prevent premature mitosis. Inhibition of mouse embryonic expression of USP44 cells in the water can be significantly improved when the instability of non proportional and chromosome aneuploid cells, making it more prone to malignant transformation of.USP44 is a tumor promoting factor Ubc antagonist H10, and USP44 in tumor cells is up-regulated, but due to lack of specific antibodies can effectively identify endogenous USP44 expression, USP44 is involved in the maintenance of tumor Cell malignant proliferation and regulation mechanism is unclear. This experiment from screening antibody specific identification of endogenous USP44 with the analysis of the relationship between the level and the prognosis of USP44 expression in glioma tissues and tumor, part of the signal pathways of USP44 and GBM for the malignant biological behavior and the relationship between the USP44 may be involved in the the first part. Screening and identification of USP44 specific antibodies: using molecular biology techniques can effectively identify the screening and identification of endogenous USP44 specific antibody. Methods: three primary antibodies: Abnova P Ab21808, Santa Cruz sc-337203, Origene TA801913. in U251MG U87MG by Western blot and A172 three cell line detection whether the antibodies can specifically recognize endogenous USP44; overexpression of Flag-USP44 fusion protein in 239T cells, USP44 antibody and Flag antibody were detected by fluorescent signal No; in U251MG cells, were used to detect the fluorescence signal of USP44 antibody and B23 antibody by immunofluorescence technique; expression detection of USP44 antibody can recognize USP44 knockdown in U251MG cells by Western blot and immunofluorescence technique. Results: in immunoblotting experiments of three kinds of antibodies can specifically recognize endogenous USP44 three group of cells and the formation of the only blot bands; the fluorescence signal of Ab21808 and P antibody can completely overlapped fluorescence signal Flag; fluorescence signal Ab21808 and antibody P B23 antibodies in the presence of coincidence in the nucleoli of three; antibody was detected USP44 mRNA knockdown, antibody in USP44 knock signal after reduction the U251MG cells decreased significantly. Conclusion: P Ab21808 specific antibody recognition of endogenous USP44 best, can accurately reflect the subcellular localization of USP44 and Si RNA knockdown efficiency, so we adopt the anti P Ab2 1808 for subsequent experiments. The relation between the second part of the USP44 expression and the pathological grades of gliomas and prognosis in M protein and RNA level, the relationship between the expression level of USP44 of tissue samples with pathological grade and prognosis. Methods: immunohistochemical experiments using P antibody Ab21808 analysis of tissue microarray containing 61 patients with different grade gliomas. The expression of USP44 protein was detected in tissue samples; analysis of the expression level of 40 cases of different grade gliomas in frozen tissue using Q USP44m RNA RT-PCR; survival analysis combined with follow-up data and Q RT-PCR data. Results: the expression of USP44 was correlated with glioma pathological grade; in high grade glioma group in the expression level of USP44m RNA was significantly higher than low level group; the group of patients with high USP44m RNA expression after the survival time was less than the low expression group Conclusion: the expression level of USP44 increased with the pathological grade of glioma, the high expression of USP44 were associated with poor prognosis. The third part: the effect of USP44 on the malignant biological behavior of GBM cells Objective: To study the GBM cell proliferation, invasion, apoptosis in the cycle, the biological behavior changes after knockdown of USP44. Methods: U251MG USP44-sh RNA and A172 cells transfected with lentivirus after WST-1 assay and colony formation assay, detect the effect of USP44 knockdown on cell proliferation; Transwell USP44 and A172 U251MG assay on cell migration after reduction, change ability of invasion; stably transfected U251MG and A172 cells by propidium iodide (PI) staining after FCM analysis; analysis of transfected cells stable fluorescent dye PE/7-AAD double staining flow cytometry to detect the expression of USP44, change the ratio of GBM cell apoptosis after the cut. Results: the expression of USP44 Downregulation could significantly inhibit the proliferation and invasion of U251MG cells and A172 cells, and significantly improve the cell apoptosis and the ratio of G2/M phase cells. Conclusion: knockdown of USP44 can inhibit the malignant biological behavior of GBM cells, G2/M cell cycle arrest and apoptosis. The fourth part USP44 and promote the interaction between cancer factor securin the Objective: to verify the interaction between USP44 and securin, by USP44 to ubiquitination of stable securin. methods: located in U251 cells by fluorescence signal immunofluorescence detection of USP44 and securin in cells; verify whether USP44 directly combined with securin by CO immunoprecipitation; USP44-sh RNA transfection U251MG cells with proteasome inhibitor MG132 with different concentration, the expression of securin was detected by Western blot. Results: USP44 and securin Obviously the coincidence of the fluorescence signal in mitotic cells; endogenous USP44 can be directly combined with securin; MG132 can reverse caused by knockdown of USP44 securin expression without affecting the expression level of USP44 itself. Conclusion: Securin is catalyzed by USP44, USP44 by ubiquitination of stable securin and inhibited by the proteasome the fifth part of the USP44 expression. The degradation effect of down-regulation of tumorigenicity of U87MG cells Objective: to verify the effect of down-regulation of USP44 expression on the tumorigenicity of GBM cells in vivo. Methods: Cultured USP44-sh RNA transfected U87MG cells; 5 week old male nude mice 5, respectively, on both sides of the proximal hindlimb in nude mice subcutaneous inside the injection of cells and normal U87MG cells (107cells). Every 2 days measured with vernier caliper once. The length of tumor diameter to tumor size more than 10mm mice were sacrificed, remove the tumor; naked Paraffin sections of rat tumor specimens by immunohistochemical experiment, detect the expression on cell morphology and Ki-67. Results: USP44 knockdown group compared with the control group, the final volume and tumor growth rate were significantly decreased; immunohistochemistry showed that the expression level of knockdown of Ki-67 group was significantly lower than the control group. Conclusion: USP44 expression of U87MG cells was significantly inhibited in vivo tumorigenicity.

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

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