【作者】 江涛；

【导师】 万经海；

【作者基本信息】 安徽医科大学 ， 外科学（神经外科）， 2016， 博士

【摘要】 第一部分:ARPP-19在神经胶质瘤中的表达和意义目的神经胶质瘤是人类中枢神经系统最常见的原发肿瘤,其中高级别胶质瘤恶性程度高,预后很差,5年生存率不到10%。至今为止,神经胶质瘤的发生、发展和生长的具体分子机制尚不清楚。ARPP-19是c AMP调节的磷蛋白19(c AMP-regulated phosphoprotein 19)的简称。它是alpha-endosulfine(ENSA阿尔法-内磺肽)家族体系中重要的成员之一,是所有哺乳动物脑部分布比较广泛的一个蛋白,作为蛋白激酶A的底物发挥生理作用。ARPP-19在中枢神经系统中发挥着重要信号调节的作用。研究表明,ARPP-19的异常表达会导致中枢神经系统疾病的发生,如导致神经系统肿瘤的产生、诱发阿尔兹海默症。近些年,人们逐步认识ARPP-19的生理功能,发现它对多种恶性肿瘤的发生发展具有促进作用。尽管最近的研究证明了ARPP-19在细胞有丝分裂以及肿瘤发生发展中发挥着一定作用,但是有关ARPP-19具体到在人神经胶质瘤中的作用及其异常表达的临床意义至今尚无报道。特异性周期蛋白D1(cyclin D1)可以促进细胞增殖,如过度表达则会导致细胞增殖失控。c-myc基因定位与8q24,是myc基因家族一员。c-myc与正调节的生长因子协同促进细胞增殖,而与具有负调控的生长抑制基因结合后可加速细胞凋亡。cyclin D1和c-myc均与细胞生长周期相关,故我们选择两者以了解ARPP-19如何调节cyclin D1和c-myc基因的表达,来推测ARPP-19在肿瘤细胞增殖、转移过程中的作用。为此,本研究旨在明确ARPP-19在人神经胶质瘤中的表达及其临床意义;为了探索ARPP-19在神经胶质瘤中发挥作用的机制。方法选择人的神经胶质瘤细胞系A172用来做体外细胞实验研究。将合成ARPP-19的si RNA转染A172人神经胶质瘤细胞系。通过实时定量RT-PCR定量检测转染ARPP-19的si RNA以后的ARPP-19 m RNA水平的的表达;通过细胞软琼脂克隆形成实验检测转染ARPP-19 si RNA的A172细胞的增殖、非锚定依赖生长和肿瘤细胞的恶性行为,通过Transwell小室和细胞划痕创伤愈合实验检测转染了ARPP-19的si RNA的A172细胞迁移及侵袭能力,应用Western Blot实验检测转染了ARPP-19的si RNA的A172细胞和对照组NC的神经胶质瘤细胞中Cyclin D1和c-myc蛋白表达的变化,同时选择β-ACTIN作为内部参照基因作蛋白上样量平衡。利用免疫组织化学技术检测安医大二附院2009-2015年间81例神经胶质瘤石蜡标本的ARPP-19蛋白的表达水平,通过门诊或电话随访患者并收集无病生存和总生存的相关数据,使用皮尔森卡方检验和卡普兰-迈耶曲线(Kaplan-Meier curves)分析ARPP-19与患者的无病生存率(RFS)和总生存率(OS)之间的相关性。结果在转染了ARPP-19的si RNA后A172细胞中,ARPP-19的m RNA表达明显下降。ARPP-19被si RNA敲低以后五天之内的细胞生长曲线显示A172细胞的生长数目明显降低,软琼脂克隆形成率明显降低,转移能力和侵袭能力也有了很大程度的降低划痕创伤愈合能力也被大大削弱。关于下游机制,Western Blot实验显示当A172细胞的ARPP-19的表达水平被敲低以后,cyclin D1和c-myc的表达水平也相应降低。81例胶质瘤患者中,高级别胶质瘤43例,低级别38例。临床病理资料显示ARPP-19的表达水平与神经胶质瘤的肿瘤高分级具有显著相关性,而与年龄、性别、KPS评分的相关性不显著。ARPP-19在高级别神经胶质瘤中的表达水平明显高于低级别神经胶质瘤(ARPP-19 P=0.001),ARPP-19高表达的神经胶质瘤患者的无病生存率(RFS)以及整体生存率(OS)明显低于ARPP-19低表达患者(RPF P=0.002;OS P=0.001)。结论ARPP-19能够促进神经胶质瘤的增殖和转移,可能是是通过正向调节cyclin D1和c-myc的表达而发挥作用。ARPP-19的蛋白表达水平与神经胶质瘤患者的预后具有显著正相关性,即高表达患者预后差,低表达患者预后好。提示ARPP-19可能作为神经胶质瘤患者疾病进展和生存预测的生物指标。第二部分:LC3B,p62在神经胶质瘤中的表达和意义目的细胞自噬autophagy是真核生物中进化保守的、依赖溶酶体途径的、对胞质蛋白和细胞器进行降解、对细胞内物质进行周转的重要过程。自噬是维持细胞稳定性和代谢平衡的一个基本生理代谢机制;同时,细胞自噬在促进多种肿瘤的发生、发展以及抗肿瘤治疗耐受方面也发挥着重要作用。近年来在多种肿瘤中发现自噬的增强或减弱:一方面,细胞自噬可能抑制肿瘤的发生,自噬水平的下降也导致肿瘤的恶化和进展,因此恶性肿瘤则表现为自噬降低。另一方面,自噬使肿瘤细胞迅速降解过多错误表达的蛋白质以适应恶性生长的需要、或者免受饥饿、化疗、放疗所导致的损伤而持续生存。导致自噬促进肿瘤细胞对恶性环境的适应,表现为自噬增高。文献显示,细胞自噬一般参与了神经胶质瘤的恶性发展过程。文献报道,在细胞自噬生理过程中自噬小体的形成是关键的步骤,而微管蛋白1轻链3(microtubleassociated protein 1 light chain3,MAP1LC3,简称LC3)是编码自噬相关蛋白的重要基因,其编码形成自噬小体膜的重要蛋白,因此,在细胞自噬的研究过程中,人们常将LC3蛋白作为一个标志物来衡量细胞自噬的程度、频度等。但LC3蛋白在其中发挥的具体作用我们还知之甚少,即LC3参与了增强自噬的过程还是降低自噬的过程。LC3蛋白包含三个亚基(LC3A,LC3B,LC3C),一般通过检测LC3B亚基来衡量整个LC3表达水平。同时根据LC3B的表达水平观察其形成的自噬小体在胶质瘤中的作用。在细胞自噬中还有一种蛋白发挥着关键作用,它就是p62。Sequestosome 1(p62/SQSTM1)是一种由应激诱导产生的细胞内蛋白并作用于选择性自噬。p62包含多种蛋白作用域,可作为将要被自噬作用降解的小泡的受体,也可作为要被清除的泛素化蛋白聚集物的受体。p62蛋白可结合泛素,也可与LC3结合,从而靶向自噬体并促进泛素化蛋白的清除。p62作为细胞自噬生理过程中一些酶作用过程中不可或缺的底物,在信号转导过程中发挥重要作用。现有文献已知P62在恶性肿瘤中的高表达与肿瘤的发生、发展、恶性行为及病人预后差高度相关。本研究拟检测神经胶质瘤标本LC3B和p62蛋白表达水平,并分析它们与各临床病理参数以及病人的生存率之间的相关性。了解LC3B在胶质瘤中的表达特点;同时了解作为参与自噬过程中自噬小体组建的LC3B和参与自噬降解过程中的底物的p62在神经胶质瘤中的相关性。方法收集安医大二附院2009-2015年间81例经胶质瘤的石蜡固标本,利用免疫组织化学技术检测LC3B,p62蛋白的表达水平;通过电话随访和门诊随访的方式统计上述病人的无病生存和总生存情况。使用皮尔森卡方检验,卡普兰-迈耶曲线(Kaplan-Meier curves)分析它们与各临床病理参数以及与病人的无病生存率(RFS)和总生存率(OS)之间的相关性。结果81例胶质瘤患者中,高级别胶质瘤43例,低级别38例。LC3B和p62的表达水平与神经胶质瘤的肿瘤高分级具有显著相关性,而与年龄、性别、KPS评分的相关性不显著。相对于低级别胶质瘤,LC3B和p62在高级别的神经胶质瘤组织中为高表达(LC3B P=0.015,p62 P=0.002)。LC3B蛋白表达水平较高的病人,与LC3B蛋白表达水平较低的病人相比表现出更低的3年无病生存率(RFS)(P=0.044)和3年整体生存率(OS)(P=0.022)。LC3B与p62蛋白二者之间皮尔森相关性系数是0.274。结论在神经胶质瘤中,LC3B参与的细胞自噬促进了肿瘤的恶性发展过程。同时LC3B和p62在神经胶质瘤中的表达水平和患者预后具有正相关性,提示LC3B和p62可能在神经胶质瘤中协同发挥作用,共同促进神经胶质瘤的肿瘤发生、发展和恶性进程。更多还原

【Abstract】 Part One: The roles of ARPP-19 in gliomaObjectiveGlioma is the most common and aggressive type of human primary brain tumor with a poor outcome.The molecular mechanisms understanding glioma development and progression are still poorly understood.c AMP-regulated phosphoprotein 19(ARPP-19),belongs to the family of alpha-endosulfine(ENSA),and is identified in the mammalian brain as a substrate for protein kinase A(PKA).ARPP-19 plays an important regulatory role in the central nervous system,including the signal transduction of many neural factors,the expression regulation of the genes related to nervous system.ARPP-19 was an important regulator to modify the growth and physiological status of synapses.On the other hand,ARPP-19 also plays a role in the pathologic process of neuronal system dieases.For example,Kim et al revealed that decreased levels of ARPP-19 may contribute to the progression of Down syndrome and Alzheimer’s disease.In addition,the studies have also demonstrated a novel role of ARPP-19 in human cancer development and progression.Recent studies have demonstrated a novel role of ARPP-19 in regulation of cell mitosis and cancer progression.Therefore,ARPP-19 can be considered a promoter of oncogenes.However,no study has been conducted to determine the role of ARPP-19 in human glioma cells,and analyzed the expression and clinical significance of ARPP-19 in human glioma.In this study,we investigate the role of ARPP-19 in human glioma cell line A172 using immunohistochemical staining and protein immunoblotting,and to further explore the role of ARPP-19 in the genesis and development of human glioma.A retrospective study was performed to examine the expression of ARPP-19 protein in 81 cases of human glioma tissue,and to explore the correlation ARPP-19,and to analyze the relationship between the pathological parameters and the survival rate of patients.Our aims are to clarify the clinical significance of the ARPP-19 in glioma patients.We hoped that ARPP-19 can be used as biological markers to analyze the survival rate of high-grade glioma,and to find a new treatment target and strategy for high-grade glioma.MethodsIn this study,the human glioma cell line(A172)was used in the cell experiment research in vitro,and to explore the role of ARPP-19 genes in A172 cell line.After the transfection of synthetic ARPP-19 si RNA,the expression of ARPP-19 was detected in A172 glioma cell line,and the proliferation,non-anchorage-dependent growth and malignant behavior of the transfected A172 cell line were examined by cell soft agar cloning assay.Trans-well chamber and cell scratch wound healing experiments were performed to examine the migration and invasion of A172 cells after the transfection with ARPP-19 si RNA.Western Blot was used to detect the expression of cyclin D1 and c-myc proteins in A172 cells transfected with ARPP-19 si RNA and control group cells(β-actin as the internal reference gene).At the same time,81 cases of human glioma clinical tissue samples was collected.The protein expression of ARPP-19 was detected by immunochemistry.The overall survival of these patients was recorded by telephone and outpatient follow-up.The correlation between the clinical pathological parameters and patient’s disease-free survival(RFS)and overall survival(OS)were analyzed using Pearson chi-square,Kaplan Meier curve(Kaplan Meier curves).ResultsAfter the transfection with ARPP-19 si RNA in A172 cells,ARPP-19 m RNA expression was significantly decreased.The cell growth curve of A172 cells 5 days after the transfection with ARPP-19 si RNA was detected,and the results showed that the number of cell growth was significantly decreased.Consistent with the results of cell growth,the formation of soft agar clones in A172 cells after the transfection of ARPP-19 si RNA was also significantly reduced.In addition,the ability of metastasis and cell invasion have a significant degree of reduction in the A172 cell line after the si RNA transfection,which due to the reduced expression level of ARPP-19.Concomitantly,when the ARPP-19 expression level was knocked down in the A172 cells,the ability of scratched wound healing was greatly weakened.Moreover,we found that the expression level of cyclin D1 and c-myc decreased after knockdown of ARPP-19 in A172 cells.ConclusionOur results suggested that ARPP-19 had the effect of promoting the proliferation and metastasis of human glioma.These effects of ARPP-19 may be related to significantly up-regulate the expression of cyclin D1 and c-myc genes.The protein expression level of ARPP-19 was significantly correlated in malignant degree of glioma.High expression of ARPP-19 in gliomas was generally related to high-grade malignancy of the tumor,including high-grade tumors and poor prognosis.Part Two:The roles of LC3 B,p62 expression in gliomaObjectiveCell autophagy is a basic physiological metabolic mechanism to maintain cell stability and metabolic balance,and by which cells degrade their unwanted molecules and components.Simultaneously autophagy also play a important role in the development,progression and anti-cancer therapy resistance of many types of human cancers.As previously reported,the formation of autophagosomes is a key step in the process of autophagy.LC3 is associated with the membrane of autophagosomes,and involved in the autophagy.Therefore,LC3 is widely used as an autophagy marker in the study of autophagy.In the other way,with the progress of biotechnology,the detection of LC3 by immunohistochemistry,immunoblotting and other methods using specific antibodies was more common.Many studies revealed high LC3 expression associates with poor prognosis in diverse cancer.In the process of development of human malignancies,cell autophagy can promote tumor cells to adapt to the malignant environment,and thereby contributing to the malignant behavior of malignant tumors.Therefore,the high-degree malignancy tumor with poor prognosis is often accompanied by high LC3 expression.Many studies revealed high LC3 expression associates with poor prognosis in diverse cancer patients through autophagy.In malignant gliomas,a few studies documented the association of autophagy with cancer aggravation.But there was no one studying systematically about the relation between LC3 and clinical behaviors of glioma patients.LC3 consists of three isoforms: LC3 A,LC3B,and LC3 C.In immunohistochemical testing,LC3 A shows three patterns: diffuse cytoplasmic,cytoplasmic /juxtanuclear,and stone-like pattern,and which pattern represented the functional LC3 A involved in autophagy remains confusing.In all tissues,LC3 C is much lower than that of LC3 A and LC3 B.So LC3 B was selected to study the relation between LC3 and clinical behavior of glioma patients here,which has a high expression level and has a defined punctate pattern by immunohistochemical testing participating in autophagy.p62 plays a key role in cell autophagy,which is involved in the process of cell autophagy as an essential substrate for some of the enzymes.Therefore,p62 is commonly used to study the cell autophagy.In order to understand the correlation between P62 and LC3 B,we also detected the P62.In the study,we performed a retrospective study to examine the expression of LC3 B and p62 protein in 81 cases of human glioma tissue,and the correlation between LC3 B and p62 was analyzed in 81 cases.In addition,the correlation between the clinical pathologic parameters and the survival rate of patients was illustrated.Our aims is to clarify the clinical significance of the above proteins in glioma patients,and hoped that LC3 B and p62 can be used as a biological index to measure the survival rate of high-grade glioma,and to find a new treatment target and strategy for high-grade glioma.MethodsWe collected 81 cases of human glioma clinical tissue samples from Department of Pathology of the Second Affiliated Hospital of Anhui Medical University from 2009 to 2015.The expression of LC3 B and p62 protein was detected using immunohistochemical technique in human gliomas tissues from 81 patients.The correlation between the expression level of LC3 B and p62 protein and the clinical pathological parameters and patient’s disease-free survival(RFS)and overall survival(OS)were analyzed using Pearson chi-square,Kaplan Meier curve(Kaplan Meier curves).ResultsIn this study,we detected the expression of two autophagic protein LC3 B and p62 in 81 glioma tissues by Immunohistochemistry analysis.LC3 B and p62 expressed high in high-grade glioma tissues,and expressed low in low-grade glioma tissues.High LC3 B and p62 protein level were also associated with advanced tumor stages,worse relapse-free survival(RFS)and overall survival(OS)in glioma patients,but not with patients’ age,gender or KPS.Furthermore,we found a significant positive correlation between LC3 B and p62 expression in gliomas,suggesting that LC3 B and p62 may play a role in gliomas and contribute to tumorigenesis and progression of gliomas malignant deterioration.ConclusionOur results suggested that the expression of LC3 B and p62 protein was closely related to autophagy,which can promote the development of malignant glioma and the prognosis of patients with poor prognosis.Highly expressed cell autophagy-related proteins,LC3 B and p62,are predictive of high malignancy and poor prognosis of gliomas.Therefore,therapies based on autophagy,or drugs targeting to the LC3 B and p62 genes,are a potential can be used to treat the advanced glioblastoma.更多还原