【作者】 李俊；

【导师】 崔红娟；

【作者基本信息】 西南大学 ， 细胞生物学， 2019， 硕士

【摘要】 按照世界卫生组织制定的肿瘤分级系统来看,胶质母细胞瘤(GBM)是恶性程度最高的一种脑胶质瘤,它的特点是高度扩散,能侵入周围正常组织形成血管从而发生转移。恶性胶质瘤年发病率约为1/20,000,每年新发病例超过14,000例,以老年人发病最为常见[1]。胶质母细胞瘤细胞的迁移侵袭能力很强,而且病灶位于大脑,所以患者预后较差,当前的IV级胶质瘤患者的平均生存期为13个月左右,临床治疗难度很大[2],所以寻找新的分子标记物对于胶质母细胞瘤的治疗来说是极其重要的。泛素特异性蛋白酶38(USP38)是泛素特异性蛋白酶USPs家族中的一员,它包含半胱氨酸内肽酶活性和硫醇依赖性泛素酰水解酶活性。此前有报道称,USP38与原发性乳腺癌的发生有关[3];此外,USP38还在慢性阻塞性肺疾病的肺组织中表达上调[4];最近又有报道,USP38是一种LSD1特异的去泛素化酶,通过与LSD1相互作用从而影响细胞生理功能[5]。虽然USP38在原发性乳腺癌、慢性阻塞性肺疾病等疾病中已有相关报道,但是USP38在胶质母细胞瘤等恶性实体瘤中却基本没有相关的报道。本文我们通过Western Blot及R2数据库分析发现等,USP38高表达,GBM病人的预后差,生存期短。同时我们检测了不同胶质母细胞瘤细胞系及星型胶质细胞系SVGP12中USP38的表达量,结果显示USP38在GBM细胞系中显著高表达。接下来我们对USP38进行了敲低和过表达,希望通过功能性实验方法来探究USP38的低表达以及高表达对胶质母细胞瘤细胞的增殖、迁移侵袭和体内外的成瘤能力等相关生理功能的影响,并且在可能的情况下进行更深入的机制探究,以便为临床应用提供更多可靠的理论依据。以下是我们的实验结果:1.在胶质母细胞瘤中USP38的表达与病人的预后密切相关我们通过R2等数据平台分析发现,USP38表达量与胶质母细胞瘤的分级成正相关。我们利用RT-PCR和Western blot实验检测到USP38在胶质母细胞瘤细胞系中普遍表达,而星型胶质细胞中却极少表达USP38,这提示我们USP38表达量与胶质母细胞瘤的恶性程度成正相关的。2.USP38促进胶质母细胞瘤的增殖及成瘤为了探究USP38在胶质母细胞瘤细胞增殖及成瘤中的作用,我们利用慢病毒介导的shRNA干涉技术对于USP38进行了敲低。Western Blot实验显示,我们成功敲低了USP38。细胞的形态学观察显示,USP38干涉组胶质母细胞瘤细胞系的生长显著地被抑制。接下来,我们利用BrdU掺入实验对于细胞DNA合成进行了检测,结果显示,USP38干涉组的DNA合成受到了显著的抑制。随后我们用流式细胞术和Western Blot实验分别对细胞周期及细胞周期相关蛋白进行了检测,结果发现,USP38干涉后,细胞周期阻滞在G0/G1期,与之相关的周期蛋白如CDK2,CDK4等都发生了显著的下调。然后我们利用软琼脂克隆实验、小鼠皮下成瘤实验和免疫组织化学(IHC)实验对胶质母细胞瘤细胞体内外成瘤能力进行了检测,结果发现,当我们敲低USP38后,胶质母细胞瘤细胞体内外成瘤能力都显著降低。虽然我们设计了两条干涉片段,但是由于shRNA易发生脱靶效应,所以我们对于敲低USP38的细胞系进行了全片段长度USP38的恢复,确保相关生理功能的变化是由敲低USP38引起,并且检测USP38恢复后细胞的相关生理功能是否得到了恢复。实验证实,在USP38得到恢复后,细胞的相关生理功能得到了恢复。以上实验说明,泛素特异性蛋白酶USP38能够促进胶质母细胞瘤细胞的增殖及成瘤。3.USP38促进胶质母细胞瘤的迁移侵袭肿瘤细胞极强的转移能力是其难以治愈的一个很重要的原因。为了检测USP38对胶质母细胞瘤细胞转移能力的影响,我们将敲低USP38以及敲低USP38后再恢复USP38的不同胶质母细胞瘤细胞系进行了迁移侵袭检测能力的实验。结果显示,在敲低USP38后,细胞的迁移及侵袭能力都受到了显著的抑制并且间充质标志物Snail,MMP2等蛋白的表达在干涉USP38后都发生明显的下调。同样的,当我们恢复USP38后,胶质母细胞瘤的迁移侵袭能力得到了恢复,并且Snail,MMP2等蛋白的表达量也得到了一定程度的恢复。以上实验说明,泛素特异性蛋白酶USP38能够促进胶质母细胞瘤细胞的迁移侵袭。4.USP38抑制胶质母细胞瘤细胞凋亡为了探究敲低USP38对胶质母细胞瘤细胞系细胞凋亡的影响,我们对干涉USP38后的LN-229及U-87 MG细胞系进行了流式细胞和Western Blot实验。实验结果显示,USP38被敲低后,抗凋亡家族中的重要抗凋亡因子Bcl2等蛋白的表达量明显下降,细胞发生了明显的凋亡;而线粒体凋亡途径相关蛋白C-caspase3、C-caspase9、C-PARP等都显著上调,说明细胞凋亡可能是通过线粒体凋亡途径进行。接下来,我们对USP38进行了全片段长度恢复,流式细胞术和Western Blot检测,实验结果显示,胶质母细胞瘤细胞系的细胞凋亡得到了一定程度的恢复,并且Bcl2蛋白的表达量发生了上调,线粒体凋亡途径相关蛋白C-caspase3、C-caspase9、C-PARP等都发生了下调。以上实验说明,泛素特异性蛋白酶USP38能够抑制胶质母细胞瘤细胞的凋亡发生。5.USP38调控原癌基因c-Myc在胶质母细胞瘤中的表达原癌基因c-Myc在胶质母细胞瘤被证实为原癌基因。在USP38干涉后,我们通过RT-PCR及Western Blot实验发现c-Myc的m RNA水平和蛋白水平均发生显著下调。我们查阅GeneCards时,其预测c-Myc可能与USP38的启动子区域相结合,而我们查阅有关USP38的文献时发现其功能发挥基本上都是在蛋白水平。所以在考虑敲低USP38后它是如何下调c-Myc的表达量的这一问题上,我们首先没有从转录水平去考虑这一问题,而是从转录后的水平来考虑的。接下来我们做了IP实验,检测USP38与c-Myc是否有会有相互作用。实验结果显示,USP38与c-Myc是有结合的和相互作用的。但是究竟USP38是如何通过调节泛素化与去泛素化的平衡来调节c-Myc的表达量还需要深入研究其机制来解释。综上所述,USP38可能通过影响c-Myc的泛素化,在转录后水平调控c-Myc的表达,从而影响胶质母细胞瘤的增殖及迁移侵袭能力。本研究为USP38作为胶质母细胞瘤标记物提供了重要的依据,也丰富了胶质母细胞瘤分子治疗的理论基础。更多还原

【Abstract】 Glioblastoma(GBM),the most malignant glioma,is characterized by a high degree of proliferation and invasion of surrounding normal tissue to form blood vessels.The annual incidence of glioblastoma is about 1/20,000,with more than 14,000 new cases annually,which is common in the olderly.Glioblastoma cells have strong migration and invasion ability,and the lesions are located in the brain,so the prognosis is poor.The current survival rate of grade IV gliomas is about 13 months,and the clinical treatment is very difficult.Therefore,further identification of useful biomarkers is critical for the treatment of GBM.USP38 is a member of the USPs family that includes cysteine-type endopeptidase activity and thiol-dependent ubiquitinyl hydrolase activity.Previously,USP38 has been reported to be associated with the occurrence of primary breast cancer;In addition,USP38 is upregulated in the lung tissue of copd.Recently,it has been reported that USP38 is a LSD1-specific deubiquitinase,which affects cell physiology by interacting with LSD1.Although USP38 has been reported in primary breast cancer,chronic obstructive pulmonary disease and other diseases,its role in the development of glioblastoma has hardly been reported.In this study,we analyzed R2 database and found that USP38 was highly expressed,and patients with GBM had poor prognosis and short survival.Meanwhile,we detected the expression levels of USP38 in different glioblastoma cell lines and astrocytes.Next,we knocked down and overexpressed USP38 to detect its influence on cell proliferation,apoptosis,migration and invasion,and tumorigenesis ability in vivo and in vitro,so as to provide new ideas for the treatment of glioblastoma.Our experimental results are as follows: 1.The expression of USP38 in glioblastoma is closely related to the prognosis of patientsAccording to the analysis of R2 data platform,the expression level of USP38 in glioblastoma with a higher degree of malignancy was higher.It was detected by qrt-pcr and Western blot that USP38 was generally expressed in glioblastoma,but not in normal glial cells,and the expression level was closely related to the degree of malignancy of the tumor.2.USP38 promotes the proliferation and tumorigenesis of glioblastomaTo explore the role of USP38 in the proliferation and tumorigenesis of glioblastoma cells,We knocked down USP38 using lentiviral-mediated shRNA interference.Western Blot showed that after we knocked down USP38 successfully,morphological observation of cells showed that the growth of glioblastoma cells was significantly inhibited.Then we used the BrdU incorporation assay to detect cellular DNA synthesis.The results showed that the DNA synthesis of the USP38 interference group was significantly inhibited.Next,we used flow cytometry and Western Blot to detect the cell cycle and related cyclins,respectively.It was found that after USP38 interference,cell cycle arrest was in G0/G1 phase,and related cyclins such as CDK2,CDK4,etc.have undergone significant downward adjustments.Then we used soft agar cloning experiments,mouse subcutaneous tumor formation experiments and immunohistochemistry(IHC)experiments to detect the tumorigenic ability of glioblastoma cells in vitro and in vivo,and found that when we knocked down USP38,the gelatinous The ability of tumor cells to form tumors in vitro and in vivo is reduced.Although we designed two interference fragments,since shRNA is prone to off-target effects,we performed a full-length USP38 recovery on cell lines that knock down USP38,ensuring that changes in physiological functions are caused by knocking down USP38,and detection.Whether the relevant physiological functions of the cells were restored after USP38 was restored.Experiments confirmed that after USP38 was restored,the relevant physiological functions of the cells were restored.The above experiments indicate that the ubiquitin-specific protease USP38 can promote the proliferation and tumor formation of glioblastoma cells.3.USP38 promotes the migration and invasion of glioblastoma cellsThe extremely strong metastatic ability of tumor cells is a very important reason for its difficulty in curing.In order to examine the effect of USP38 on the metastatic ability of glioblastoma cells,we used different cell lines that knocked down USP38 and and then restore the USP38 for migration and invasion assays.The results showed that the migration and invasion ability of the cells were inhibited after interference with USP38.Moreover,the expression of mesenchymal markers Snail,Slug and other proteins were inhibited after interference with USP38.When USP38 was restored,the migration and invasion ability of glioblastoma was restored.4.USP38 inhibits apoptosis in glioblastoma cellsTo investigate the effect of knockdown of USP38 on apoptosis in glioblastoma cell lines,we performed flow cytometry and Western Blot experiments on LN-229 and U-87 MG cell lines that interfered with USP38.The results showed that after interfering with USP38,the expression of important anti-apoptotic factor Bcl2 in the anti-apoptotic family of cells decreased significantly,and the cells showed obvious apoptosis;while the mitochondrial apoptosis pathway-related protein C-caspase3,CCaspase9 and C-PARP were significantly up-regulated,indicating that apoptosis may be through the mitochondrial apoptotic pathway.Apoptosis of glioblastoma was restored when USP38 was restored.5.USP38 regulates the expression of the proto-oncogene c-Myc in glioblastomaThe proto-oncogene c-Myc was confirmed to be a proto-oncogene in glioblastoma.After USP38 interference,we found that m-RNA levels and protein levels of c-Myc were significantly down-regulated by qRT-PCR and Western Blot experiments.When we looked at GeneCards,we predicted that c-Myc might be combined with the promoter region of USP38,and we looked at the literature on USP38 and found that its function was essentially at the protein level.So on the question of how to down-regulate the expression of c-Myc after knocking down USP38,we first did not consider this issue from the transcription level,but considered it from the post-transcriptional level.Next we did an IP experiment to see if there is any interaction between USP38 and c-Myc.The experimental results show that USP38 and c-Myc are bound and interact.However,how USP38 regulates the balance between ubiquitination and deubiquitination of c-Myc requires further study of its mechanism to explain.In summary,USP38 may affect the expression of c-Myc at the post-transcriptional level by affecting the ubiquitination of c-Myc,thereby affecting the proliferation and migration of glioblastoma.This study provides an important basis for USP38 as a marker for glioblastoma and enriches the theoretical basis for molecular therapy of glioblastoma.更多还原