PP4C在人脑胶质瘤中的表达、功能及其相关调控机制研究

发布时间：2018-04-23 00:16

本文选题：PP4C + 胶质瘤　；参考：《山东大学》2016年博士论文

【摘要】：研究背景及意义脑胶质瘤为中枢神经系统发病率最高的恶性肿瘤,约占颅内肿瘤的45%,占颅内恶性肿瘤的80%左右。脑胶质瘤起源于神经胶质细胞,生长迅速且具有较强的侵袭能力,给手术切除造成一定困难。根据2007年最新的WHO肿瘤分级标准,胶质瘤可分为Ⅰ-Ⅳ级,其中Ⅰ、Ⅱ级为低级别胶质瘤,Ⅲ、Ⅳ级为高级别胶质瘤。目前胶质瘤的标准治疗方案包括手术联合放疗和化疗,即使经过这些综合治疗,高级别胶质瘤的预后仍然不尽人意,尤其是恶性程度最高的多形性胶质母细胞瘤,5年总的生存率低于5%,平均生存期在14-15个月左右。胶质瘤的发病机制涉及到多基因、多因素的共同作用。许多相关基因的异常表达与胶质瘤的发生密切有关,但仍不能清楚的阐明胶质瘤发生、发展的全过程。因此,为了进一步揭示胶质瘤的发病机制,寻找胶质瘤的生物学标记,探求胶质瘤治疗的新靶点是当前亟待解决的难题。蛋白质的磷酸化是蛋白质翻译后修饰的重要组成部分,人体细胞中约有30%的蛋白质被磷酸化修饰,并且这种磷酸化修饰是调节细胞增殖、凋亡、分化和其他细胞功能的重要机制之一。蛋白质的磷酸化的水平由蛋白激酶和蛋白磷酸酶共同调节。蛋白磷酸酶曾被认为是伴随蛋白激酶左右的沉默伴侣,随机地逆转蛋白激酶的磷酸化效应,但越来越多的证据表明,蛋白磷酸酶组成了一个与蛋白激酶相平行的结构复杂多样的酶家族,能主动、特异地对特定底物去磷酸化。蛋白磷酸酶根据氨基酸序列同源性、结构及催化机制可分为三个家族。第一家族是丝/苏氨酸蛋白磷酸酶,包括磷酸蛋白磷酸酶(Phosphoprotein phosphatase, PPP)及镁或锰离子依赖性蛋白磷酸酶(Protein phosphatase Mg2+ or Mn2+ dependent, PPM).其中PPP作为其中种类最多并且最为经典的家族,又可分为PP1、PP2A、PP、PP5、PP6、PP7。酪氨酸蛋白磷酸酶及基于天冬氨酸伴有DXDXT/V催化的蛋白磷酸酶分别组成第二和第三家族。PP4 (Protein Phosphatase 4)独立于其他的磷酸酶,参与多种病理生理过程,越来越受到众多研究者的关注。PP4在结构上与PP2A相似,以催化亚基PP4C(Protein Phosphatase 4 catalytic subunit)及不同的调节亚基相结合,组成异源多聚体的形式存在并发挥功能。因此PP4C作为PP4的核心催化亚基,被认为是起重要作用的亚基。PP4C由ppp4c基因编码,定位于人染色体16p1 1.2区域,最初由Brewis ND等人从兔肝到cDNA文库中分离并鉴定,起初称之为PPX,1993年在哺乳动物中克隆出PP4C基因,然后在果蝇、线虫、酵母中也陆续发现PP4C的同源物。PP4C参与多种病理生理过程,如微管的形成、大脑皮质的发育、免疫调节、DNA损伤修复及多个细胞信号通路的调节。PP4C可以促进细胞增殖、抑制细胞凋亡,并调节NF-κB、JNK和m-TOR等肿瘤相关信号通路,提示PP4C参与肿瘤的发生、发展过程。近年来,有学者相继发现PP4C在乳腺癌、肺癌、胰腺导管癌及结直肠癌中高表达,并且高表达PP4C的患者与胰腺导管癌、结直肠癌患者的预后差密切相关。然而,目前国内外学者尚未有关于PP4C在胶质瘤中的报道。本研究共分为三部分。第一部分,应用Western Blot、Real-time PCR和免疫组织化学检测PP4C在胶质瘤中的表达,并分析PP4C的异常表达与胶质瘤患者临床病理特征及预后的相关性。第二部分,检测人胶质瘤细胞系A172、U251、U87口正常胶质细胞Astrocyte中PP4C的表达,并探讨沉默PP4C基因对人脑胶质瘤细胞系U251、U87增殖、迁移和侵袭的影响。第三部分,应用生物信息软件预测调控PP4C的潜在miRNA,即miR-128-3p,通过荧光素酶报告基因实验进一步验证其真实性；并用Western Blot及Real-time PCR检测miR-128-3p对PP4C蛋白及mRNA的影响;进一步在胶质瘤组织中检测miR-128-3p的表达,并分析其与PP4C的相关性。第一部分PP4C在人脑胶质细胞瘤组织中表达及其临床病理特征相关性研究研究目的：1.探讨PP4C蛋白和mRNA在人脑胶质细胞瘤组织和正常脑组织的表达情况。2.探讨PP4C异常表达与胶质瘤患者临床病理特征及预后的相关性。研究方法：1.采用Western Blot和Real-time PCR和检测PP4C蛋白和mRNA在新鲜胶质细胞瘤组织及正常脑组织中的表达。2.采用免疫组织化学方法检测120例脑胶质瘤组织及10例正常脑组织中PP4C蛋白表达情况及分析其表达与胶质瘤WHO病理级别的相关性。研究结果：1.PP4C蛋白和mRNA在脑胶质瘤组织中的表达显著高于正常脑组织。并且PP4C蛋白和mRNA表达随WHO病理级别增高有逐渐升高的趋势,与胶质瘤的恶性程度相关。2.PP4C阳性表达为细胞核和胞浆染成浅黄色、棕黄色或者黄褐色。PP4C蛋白在脑胶质瘤组织的高表达率为60%(72/120),在10例正常脑组织中PP4C均呈低表达。除此之外,PP4C蛋白在Ⅱ级、Ⅲ级、Ⅳ级胶质瘤的高表达率分别为21.43%(6/28) 、55.56%(20/36)、82.14%(46/56),差异具有统计学意义(P0.001)。Kaplan-Meier生存分析表明PP4C高表达的患者预后比PP4C低表达的患者预后差。Cox比例风险模型进行单因素、多因素分析显示PP4C蛋白高表达是影响胶质瘤患者预后的独立危险因素。结论：PP4C蛋白和mRNA在胶质瘤中的表达与正常脑组织相比显著增高,并与胶质瘤病理分级相关；预后分析表明PP4C高表达是胶质瘤患者的预后独立危险因素。第二部分PP4C在人胶质瘤细胞系中的表达及沉默PP4C基因对细胞增殖、迁移和侵袭的影响研究目的：1.探讨人胶质瘤细胞系A172、U251、U87及正常胶质细胞Astrocyte中PP4C的表达情况。2.探讨沉默PP4C基因对人脑胶质瘤细胞系U251、U87增殖、迁移和侵袭的影口向。研究方法：1. 采用Western Blot和Real-time PCR和检测PP4C蛋白和mRNA在人胶质瘤细胞系及正常胶质细胞中的表达情况。2. si-RNA的构建及转染：设计合成PP4C-siRNA干扰序列,并实验将U251和U87细胞株各分成两组,即实验组(转染PP4C-siRNA)、阴性对照组(转染非特异性siRNA),用转染试剂进行转染。3.PP4C基因沉默效率的检测：使用Real-time PCR和Western Blot检测PP4C-siRNA沉默效率。4. 采用CCK-8及EdU实验检测沉默PP4C基因后U251、U87细胞的增殖能力变化。5.采用划痕实验检测沉默PP4C基因后U251、U87细胞迁移能力的变化。6.采用Transwell实验检测沉默PP4C基因后U251、U87细胞侵袭能力的变化。研究结果：1.PP4C在脑胶质瘤细胞系中的表达应用Western Blot及Real-time PCR检测胶质瘤细胞系A172、U251、U87及正常胶质细胞Astrocyte结果显示,PP4C蛋白及mRNA在4种细胞系中均存在表达,在U251、U87细胞中相对表达量较高,A172细胞次之,Astrocyte中相对表达量最低,因此筛选出U251、U87优势表达细胞系进行下一步实验。2. 检测PP4C-siRNA的沉默效率使用Real-time PCR和Western Blot检测PP4C-siRNA沉默效率。Real-timePCR及Western Blot结果显示,转染48小时后,相对于阴性对照组,PP4C-siRNA明显降低胶质瘤细胞系U251、U87中PP4C mRNA及蛋白的表达(P均0.01)。3.PP4C基因沉默对U251、U87细胞系增殖能力的影响CCK-8增殖实验结果表明,与阴性对照组(NC)相比,P4C-siRNA实验组U251、U87细胞增值率显著降低,分别降低了54%、50%,差异均具有统计学意义(P0.01)。 EdU增殖实验结果表明,与阴性对照组(NC)相比,PP4C-siRNA实验组细胞增殖率显著降低,U251、U87细胞分别降低了51%、64%,差异具有统计学意义(P0.01)。这些结果表明,PP4C可以促进胶质瘤细胞的体外增殖能力,沉默PP4C后胶质瘤细胞的增殖明显降低。4.PP4C基因沉默对U251、U87细胞系迁移能力的影响划痕实验结果显示,相对于阴性对照组,PP4C-siRNA组细胞迁移数明显下降,其中U251细胞减少了56%,U87细胞减少了66%,差异具有统计学意义(P0.01)。5.PP4C基因沉默对U251、U87细胞系侵袭能力的影响Transwell侵袭实验结果表明,与阴性对照组(NC)相比,PP4C-siRNA实验组穿过小室结晶紫染色细胞显著减少,其中U251细胞减少了52%,U87细胞减少了39%,差异具有统计学意义(P0.01)。结论：1.PP4C在胶质瘤细胞株中高表达,并筛选出U251、U87优势细胞株进行后续生物学实验。2. PP4C siRNA可以明显沉默PP4C的表达,并可以抑制U251、U87细胞的增殖、迁移及侵袭能力。表明PP4C可以促进胶质瘤细胞的增殖、迁移和侵袭能力。第三部分miR-128-3p与PP4C基因的靶向调控关系研究研究目的：1.预测可能调控PP4C的潜在miRNA。2.验证miR-128-3p是否转录后水平调控PP4C的表达。3.检测miR-128-3p在胶质瘤组织中的表达,并分析miR-128-3p与PP4C表达相关性。研究方法：1.采用靶基因预测算法对调控PP4C的潜在miRNA进行生物信息学预测,分析PP4C的3,-UTR区域存在miR-128-3p的结合位点。2.利用荧光素酶报告基因实验在293T细胞中验证miR-128-3p与PP4C是否存在直接相互作用,进一步在胶质瘤细胞系U251、U87中验证miR-128-3p与PP4C是否存在直接相互作用。3. miR-128-3p mimics、miR-NC mimics转染U251、U87细胞,分别利用VWestern Blot和Real-time PCR检测PP4C的蛋白和mRNA水平。4. Real-time PCR检测miR-128-3p在脑胶质瘤组织中的表达,并分析miR-128-3p与PP4C表达相关性。研究结果：1. 生物信息学预测软件显示在PP4C的3,-UTR区域存在miR-128-3p的结合位点。2.首先在293T细胞中,miR-128-3p能直接与PP4C 3'-UTR上的结合位点结合从而抑制荧光素酶表达。其次,miR-128-3p可以在U251和U87细胞中可以与PP4C 3'-UTR结合从而抑制荧光素酶活性,从而验证了miR-128-3p与PP4C的直接相互作用,PP4C是miR-128-3p的直接靶基因。3.48小时后,转染miR-128-3p过表达miR-128-3p U251、U87细胞中PP4C蛋白水平相比与转染NC的U251、U87细胞是显著降低的,而胶质瘤U251和U87细胞中过表达miR-128-3p后,PP4C mRNA水平没有显著变化。预示PP4C作为miR-128-3p的直接靶基因,并被其负调控。4. miR-128-3p在胶质瘤中的表达水平显著低于正常脑组织。并且发现miR-128-3p的表达水平随着胶质瘤病理级别的增高逐渐降低。在胶质瘤组织中,miR-128-3p的表达水平与PP4C蛋白表达水平呈显著负相关。结论：1. miR-128-3p对胶质瘤细胞系U251、U87中PP4C mRNA的表达无调节作用,对U251、U87细胞中PP4C蛋白的转录后表达具有负调控作用,提示PP4C是miR-128-3p直接的靶基因,并被其负调控。2. miR-128-3p在胶质瘤组织中低表达,PP4C与miR-128-3p在胶质瘤组织中的表达呈负相关。
[Abstract]:The background and significance of brain glioma is the highest incidence of the central nervous system, accounting for about 45% of the intracranial tumors, accounting for about 80% of the intracranial malignant tumors. Glioma originated from glial cells, which grew rapidly and had strong invasion ability, which made it difficult to excision. According to the latest WHO tumor classification in 2007 Standard, glioma can be divided into grade I - IV, of which grade I, grade II is low grade glioma, grade III and grade IV is high grade glioma. The standard treatment for glioma currently includes surgery combined with radiotherapy and chemotherapy. Even through these comprehensive treatment, the prognosis of high grade gliomas is still unsatisfactory, especially the highest malignant polyglia. The total survival rate of 5 years is less than 5% and the average survival time is about 14-15 months. The pathogenesis of glioma involves multiple genes and multiple factors. The abnormal expression of many related genes is closely related to the occurrence of glioma. However, the whole process of glioma and the whole process of glioma can not be clearly elucidated. To show the pathogenesis of glioma, to find the biological markers of glioma and to explore new targets for the treatment of glioma, the phosphorylation of protein is an important part of post-translational modification of protein, and about 30% of the proteins in human cells are phosphorylated, and this phosphorylation modification is the regulation of cell proliferation. One of the important mechanisms of apoptosis, differentiation, and other cell functions. The level of phosphorylation of proteins is regulated by protein kinase and protein phosphatase. Protein phosphatase has been considered a silent companion with protein kinase, which reverses the phosphorylation effect of protein kinase at random, but more and more evidence suggests that the protein phosphatase group is a protein phosphatase group. A complex and diverse family of enzymes, parallel to protein kinase, can actively and specifically dephosphorylate specific substrates. Protein phosphatase can be divided into three families according to amino acid sequence homology, structure and catalytic mechanism. The first family is silk / threonine protein phosphatase, including phosphate phosphatase (Phosphoprotein phosph). ATase, PPP) and magnesium or manganese ion dependent protein phosphatase (Protein phosphatase Mg2+ or Mn2+ dependent, PPM), in which PPP is the largest and most classic family, and can be divided into PP1, PP2A, tyrosine phosphatase and protein phosphatase based on aspartate catalyzed by aspartic acid, respectively. The two and third family.PP4 (Protein Phosphatase 4) is independent of other phosphatase and participates in a variety of pathophysiological processes. More and more researchers are concerned that.PP4 is structurally similar to PP2A to catalyze the combination of subunit PP4C (Protein Phosphatase 4 catalytic subunit) and different regulatory subunits to form a heterogeneous polymer. PP4C, as the core catalytic subunit of PP4, is considered to be an important subunit of the subunit.PP4C, encoded by the ppp4c gene, located in the human chromosome 16p1 1.2 region, originally isolated and identified by Brewis ND and others from rabbit liver to cDNA library, originally called PPX. In 1993, the PP4C gene was cloned in mammals, and then in the mammalian In Drosophila, nematodes, and yeast, PP4C's homologous.PP4C is also found to be involved in a variety of pathophysiological processes, such as microtubule formation, cerebral cortex development, immune regulation, DNA damage repair and multiple cell signaling pathways that promote cell proliferation, inhibit cell withering, and regulate the tumor related signaling pathways such as NF- kappa B, JNK and m-TOR. It is suggested that PP4C is involved in the occurrence and development of tumor. In recent years, some scholars have discovered that PP4C is highly expressed in breast cancer, lung cancer, pancreatic ductal carcinoma and colorectal cancer, and the patients with high expression of PP4C are closely related to pancreatic ductal carcinoma, and the prognosis of colorectal cancer patients is closely related. However, scholars at home and abroad have not yet found that PP4C is in glioma. This study is divided into three parts. In the first part, the expression of PP4C in glioma was detected by Western Blot, Real-time PCR and immunohistochemistry, and the correlation between the abnormal expression of PP4C and the clinicopathological features and prognosis of the patients with glioma was analyzed. The second part, the human glioma cell line A172, U251, and U87 normal glial cells Ast were detected. The expression of PP4C in rocyte and the effect of silent PP4C gene on the proliferation, migration and invasion of human glioma cell line U251, U87. The third part, using bioinformatics software to predict the potential miRNA of PP4C, namely miR-128-3p, further verify its authenticity by luciferase reporter gene experiment, and use Western Blot and Real-time PCR. To detect the effect of miR-128-3p on PP4C protein and mRNA; further detect the expression of miR-128-3p in glioma tissue and analyze its correlation with PP4C. Part 1 PP4C in human glioblastoma tissue and the correlation of clinicopathological features: 1. to explore the expression of PP4C protein and mRNA in human glioma tissue The expression of normal brain tissue and.2. study the correlation between abnormal expression of PP4C and the clinicopathological features and prognosis of patients with glioma. Methods: 1. using Western Blot and Real-time PCR and detecting the expression of PP4C protein and mRNA in fresh glioma tissue and normal brain tissue, the immunohistochemical method was used to detect 120 cases. The expression of PP4C protein in glioma tissues and 10 normal brain tissues and the correlation between the expression of WHO and the pathological grade of glioma. The results showed that the expression of 1.PP4C protein and mRNA in glioma tissues was significantly higher than that of normal brain tissue, and the expression of PP4C protein and mRNA was gradually increased with the increase of the pathological grade of WHO. The positive expression of.2.PP4C in the malignant degree of glioma was light yellow in nucleus and cytoplasm, and the high expression rate of brown or yellowish brown.PP4C protein in glioma tissues was 60% (72/120). The expression of PP4C in the normal brain tissues was low. In addition, the high expression rate of PP4C protein in grade II, grade III and grade IV glioma was respectively For 21.43% (6/28), 55.56% (20/36), 82.14% (46/56), the difference was statistically significant (P0.001).Kaplan-Meier survival analysis showed that the prognosis of the patients with high expression of PP4C was more than that of the.Cox proportional risk model with the poor prognosis of PP4C, and the multifactor analysis showed that the high expression of PP4C protein was an independent risk factor affecting the prognosis of glioma patients. Conclusion: the expression of PP4C protein and mRNA in glioma is significantly higher than that of normal brain tissue, and is related to the pathological classification of glioma. The prognosis analysis shows that the high expression of PP4C is an independent risk factor for the patients with glioma. Second part of PP4C in human glioma cell lines and the silence of PP4C gene for cell proliferation and migration Objective: 1. to investigate the expression of PP4C in human glioma cell lines A172, U251, U87 and normal glial cell Astrocyte.2. to explore the shadow of the silent PP4C gene on the human glioma cell line U251, U87 proliferation, migration and invasion. Research methods: 1. Western Blot and Real-time, and the detection of PP4C The expression of A in human glioma cell line and normal glial cell line.2. si-RNA construction and transfection: design and synthesize PP4C-siRNA interference sequence and divide U251 and U87 cell lines into two groups, that is, experimental group (transfected PP4C-siRNA), negative control group (transfected non-specific siRNA), and transfection reagents to transfect.3.PP4C gene silencing effect Detection of rate: using Real-time PCR and Western Blot to detect the PP4C-siRNA silencing efficiency.4., CCK-8 and EdU tests were used to detect the U251 of the silent PP4C gene and the proliferation ability of the U87 cells. Changes in the invasiveness of U87 cells. Results: the expression of 1.PP4C in glioma cell lines using Western Blot and Real-time PCR to detect glioma cell lines A172, U251, U87 and normal glia Astrocyte showed that PP4C protein and mRNA were expressed in 4 cell lines. The 2 cell was the next, the relative expression in Astrocyte was the lowest, so U251 was screened out. The U87 superiority expressed the cell line for the next experiment of.2. detection of the PP4C-siRNA silence efficiency using Real-time PCR and Western Blot detection PP4C-siRNA silencing efficiency.Real-timePCR and Western results showed, after 48 hours transfection, relative to negative control group. SiRNA significantly reduced the expression of PP4C mRNA and protein in the glioma cell line, PP4C mRNA and protein (P 0.01).3.PP4C gene silencing on the proliferation ability of U251 and U87 cell lines, the proliferation test of CCK-8 showed that, compared with the negative control group (NC), the P4C-siRNA experimental group decreased significantly, and decreased by 54%, 50%, respectively. Study significance (P0.01). The results of EdU proliferation test showed that compared with negative control group (NC), the cell proliferation rate of PP4C-siRNA experimental group decreased significantly, U251, U87 cells decreased by 51%, 64%, respectively, and the difference was statistically significant (P0.01). These results showed that PP4C could promote the proliferation ability of glioma cells in vitro and silenced the glioma cells after PP4C. The effect of.4.PP4C gene silencing on the migration ability of U251 and U87 cell lines was significantly reduced. The results of the scratch test showed that the number of cell migration decreased significantly in the PP4C-siRNA group compared with the negative control group, in which the U251 cells decreased by 56%, and the U87 cells decreased by 66%. The difference was statistically significant (P0.01).5.PP4C gene silencing of U251, U87 cell lines invaded. The influence of the attack ability on the Transwell invasion test showed that compared with the negative control group (NC), the PP4C-siRNA experimental group passed through the chamber crystal violet staining cells significantly decreased, of which U251 cells decreased by 52%, U87 cells decreased by 39%, and the difference was statistically significant (P0.01). Conclusion: 1.PP4C was highly expressed in glioma cell lines, and U251, U was screened. 87 dominant cell lines in subsequent biological experiments.2. PP4C siRNA can clearly silence the expression of PP4C, and can inhibit the proliferation, migration and invasion of U251, U87 cells. It shows that PP4C can promote the proliferation, migration and invasion of glioma cells. The target regulation relationship between the third part of miR-128-3p and the PP4C gene is studied: 1. Prediction of potential miRNA.2. validation of PP4C, miR-128-3p post transcriptional level, PP4C expression,.3. detection of miR-128-3p expression in glioma tissue, and analysis of the correlation between miR-128-3p and PP4C expression. Research methods: 1. use target gene prediction algorithm to predict the potential miRNA for PP4C and analyze PP4C 3, the binding site of miR-128-3p in the -UTR region.2. uses luciferase reporter gene test to verify whether there is a direct interaction between miR-128-3p and PP4C in 293T cells, and further in the glioma cell line U251 and U87 to verify whether there is a direct interaction between miR-128-3p and PP4C. VWestern Blot and Real-time PCR were used to detect the expression of PP4C protein and mRNA level.4. Real-time PCR detection miR-128-3p in brain glioma tissue, and the correlation between miR-128-3p and PP4C expression was analyzed. Results: 1. bioinformatics prediction software showed the 3 of the binding site. In 293T cells, miR-128-3p can bind to the binding site on PP4C 3'-UTR to inhibit luciferase expression. Secondly, miR-128-3p can inhibit the activity of luciferase by binding to PP4C 3'-UTR in U251 and U87 cells, thus verifying the direct interaction between miR-128-3p and PP4C. PP4C is a direct target gene. After 48 hours, transfected miR-128-3p overexpressed miR-128-3p U251, and the level of PP4C protein in U87 cells was significantly lower than that of NC transfected U251, but there was no significant change in the PP4C levels after the expression of miR-128-3p in the glioma U251 and U87 cells. The expression level of miR-128-3p in glioma was significantly lower than that in normal brain tissue, and the expression level of glioma was increased with pathological grade of glioma.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R739.41

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