Metadherin在弥漫大B细胞淋巴瘤侵袭性中的作用及调控机制

发布时间：2018-06-28 15:25

本文选题：Metadherin + 上皮间质转化　；参考：《山东大学》2016年博士论文

【摘要】：弥漫大B细胞淋巴瘤(Diffuse large B-cell lymphoma, DLBC L)是一组高度侵袭性的淋巴瘤,在成人非霍奇金淋巴瘤(non-Hodgkin lymphoma, NH L)中约占30-40%,是NHL中最常见的亚型。DLBCL可原发于淋巴结或结外组织器官,也可由慢性淋巴细胞白血病/小淋巴细胞淋巴瘤、边缘区淋巴瘤和滤泡性淋巴瘤(follicular lymphoma, FL)等低度恶性淋巴瘤转化而来。作为高度侵袭性的淋巴瘤,DLBCL在细胞遗传学、分子生物学、组织形态学、临床表现及预后等方面均存在很大的异质性。尽管由美罗华联合环磷酰胺、阿霉素、长春新碱和泼尼松(R-CHOP)的一线方案,显著提高了DLBCL患者的治疗效果,但仍有约40%的患者难治、复发导致死亡。因此,深入研究DLBCL的生物学异质性和侵袭机制,对于开发特异性治疗靶点、优化一线治疗方案的选择、提高完全缓解率、改善患者预后具有重要意义。异黏蛋白(metadherin, MTDH)是近年来发现的一个新的癌基因,又称为星状细胞上调基因-1(Astrocyte elevated gene-1, AEG-1)。最早是在研究人类免疫缺陷病毒(Human Immunodeficiency Virus-1, HIV-1)感染相关脑病时发现的新基因,命名为AEG-1。后来从侵袭性较高的乳腺癌细胞株中克隆出该蛋白,并且同时发现该蛋白能够促进乳腺癌细胞向肺部转移,从而命名该蛋白为MTDH。人类的MTDH基因位于染色体8q22,基因全长3611bp,编码产物是一种跨膜蛋白,包含582个氨基酸,分子量64KD。目前,已有大量的研究表明,MTDH在全身多种肿瘤中高表达,如乳腺癌、黑色素瘤、肝癌、肺癌、前列腺癌、肾癌、结肠癌、神经胶质瘤、胆囊癌、卵巢癌、淋巴瘤等。且进一步的研究证实,MTDH能够通过调节PI3K/AKT、NF-κB、Wnt/β-catenin等多个信号传导通路及其相关基因的表达水平,来调控肿瘤的生长增殖、血管生成、侵袭转移及耐药等。同时,近年来发现,上皮-间质转化(epithelial-mesenchymal transition, EMT)与肿瘤细胞的局部浸润和远处转移密切相关。EMT是指上皮细胞在特定生理或病理情况下向间质细胞转化,并伴随明显基因和表型改变的一个多阶段过程。肿瘤细胞发生EMT时,细胞的形态、极性、迁移侵袭能力等形态学和生物学行为会出现变化,同时伴有上皮标志物E-cadherin等表达减低和功能缺失,间质细胞标志物β-catenin、vimentin等过量表达,以及某些转录因子Snail、Twist和ZEB1等表达和/或功能上调。在乳腺癌、肺癌、肝细胞癌、头颈鳞癌、骨肉瘤、结肠癌及子宫颈癌等多种恶性肿瘤中已证实,MTDH可以通过调节肿瘤细胞的EMT来促进肿瘤的侵袭转移。MTD H与EMT标志物的表达和肿瘤分化、临床分期、治疗预后密切相关。MTDH促进肿瘤细胞发生EMT,是通过调控多种信号通路来实现的,如：PI3K/AKT、Wnt/p-catenin、NF-κB和MAPK等信号通路,从而来调控肿瘤的侵袭性。其中Wnt/β-catenin是 MTDH发挥作用的一条重要信号通路,该通路在调控胚胎发育,维持成体组织细胞自我更新平衡中起重要作用,其异常激活与肿瘤的发生发展有关。该信号通路中的β-catenin在EMT和激活Wnt信号通路方面起到双重作用。DLBCL为高度侵袭性淋巴瘤,易出现结外侵犯,DLBCL中是否存在EMT现象、MTDH与EMT在DLBCL侵袭性中的作用及调控机制如何,目前尚不清楚。本研究应用免疫组织化学、细胞生物学及基因沉默等技术,阐明了MTDH和EMT在DLBCL中的表达情况及二者的调控关系,探讨了MTDH表达变化对DLBCL细胞侵袭性的影响和调控机制,为开发新的DLBCL分子标志物和治疗靶点提供了依据。第一部分MTDH和EMT标志物在弥漫大B细胞淋巴瘤中的表达及临床意义目的：DLBCL为高度侵袭性淋巴瘤,存在很大的异质性,但对其侵袭机制的研究尚不清楚。MTDH是近年来发现的一个新的癌基因。大量研究表明,MTDH在全身多种肿瘤中高表达,并通过调节PI3K/AKT、NF-κB、Wnt/β-catenin等多个信号传导通路及其相关基因的表达水平,来调控肿瘤的生长增殖、血管生成、侵袭转移及耐药等。EMT与肿瘤细胞的局部浸润和远处转移密切相关。肿瘤细胞发生EMT时,其生物学行为和分子标志物会出现改变。MTDH可以通过调节肿瘤细胞的EMT来促进肿瘤的侵袭转移。为了研究MTDH口EMT在DLBCL中的表达情况,我们以人DLBCL细胞株LY1和LY8为研究对象,首先采用蛋白印记分析技术检测MTDH和EMT标志物vimentin、ZEB1蛋白在DLBCL细胞株中的表达情况。并收集病理诊断明确的DLBCL和淋巴结反应性增生的组织标本,应用免疫组织化学技术对MTDH在两组织中的表达情况进行检测,并与患者的临床资料进行相关性分析,探讨它们在DLBCL侵袭性中的作用。材料与方法：1.标本收集和分离外周血单个核细胞2.LY1和LY8细胞株培养3.蛋白提取与蛋白印迹分析4.标本和临床病例资料收集5.免疫组织化学分析6.统计学分析结果：1. Western blot结果显示,MTDH、vimentin和 ZEB1蛋白在DLBCL细胞株中均高表达,而在对照正常人外周血单个核细胞中几乎不表达。MTDH的目的条带位于75kD处,vimentin的目的条带位于54kD处,ZEB1的分子量最大,目的条带位于124kD处。2.30例DLBC L组织标本中只有1例评分为0,40.0%(12/30例)的DLBCL组织标本中MTDH为低表达,60.0%(18/30例)的DLBCL组织标本中MTDH为高表达,表达的MTDH染色多见于细胞浆,也可少量见于细胞核中。而MTDH在淋巴结反应性增生组织中几乎不表达。3. MTDH的表达与DLBCL患者临床特征的相关性分析结果显示,MTDH的高表达与DLBCL患者的年龄(P=0.458)、性别(P=0.284)、LDH (P=0.249)无相关性,而与临床分期(P=0.04)、有B症状(P=0.004)、危险度分层(P=0.033)呈正相关,P值均0.05,有统计学意义。结论：1.本研究通过Western blot技术的检测,发现MTDH、vimentin和 ZEB1蛋白在DLBCL细胞株中均高表达。2.IHC结果显示,60.0%(18/30例)的DLBCL组织中MTDH为高表达,表达的MTDH多位于细胞浆中,少量见于细胞核中。3. MTDH的高表达与DLBCL患者的临床分期、有B症状和危险度分层呈正相关。第二部分MTDH表达变化对弥漫大B细胞淋巴瘤侵袭性的影响及其调控机制目的：在多种恶性肿瘤中证实,MTDH可以通过调节肿瘤细胞的EMT来促进肿瘤的侵袭转移。MTDH与EMT标志物的表达和肿瘤分化、临床分期、治疗预后密切相关。MTDH是通过调控PI3K/AKT, NF-κB. Wnt/β-catenin和MAPK等多种信号通路来促进肿瘤细胞EMT的发生,从而调控肿瘤的侵袭性。本研究以人DLBCL细胞株LY8为研究对象,应用慢病毒载体介导的RNAi沉默MTDH基因,然后检测EMT标志物vimentin和ZEB1蛋白的表达变化和DLBCL细胞侵袭能力的改变。进一步研究在DLBCL中是否存在EMT现象,以及MTDH表达变化对DLBCL细胞侵袭性的影响和调控机制。材料与方法：1.LY8细胞株培养2.慢病毒载体介导的RNAi靶向沉默LY8细胞株的MTDH基因3.流式细胞术检测细胞转染率4.蛋白提取及蛋白印迹分析5.侵袭小室实验6.统计学分析结果：1.慢病毒载体介导的MTDH RNAi可以抑制DLBCL细胞中MTDH的表达。2.应用慢病毒颗粒沉默MTDH基因后,DLBCL细胞中Vimentin和ZEB1蛋白的表达水平明显降低(P0.05)3. Transwell侵袭小室实验结果显示,慢病毒载体介导的MTDH基因沉默可以降低DLBCL细胞株的迁移力和侵袭力(P0.05)。结论：1. DLBCL中存在类EMT现象。2. MTDH基因沉默可以降低EMT标志物Vimentin 和 ZEB1蛋白在DLBCL细胞株中的表达。3.抑制MTDH的表达可以降低DLBCL细胞株的迁移力和侵袭力。4.MTDH可能是通过Wnt/β-catenin信号通路调节DLBCL的EMT。
[Abstract]:Diffuse large B cell lymphoma (Diffuse large B-cell lymphoma, DLBC L) is a highly invasive group of lymphoma, which accounts for 30-40% in adult non Hodgkin's lymphoma (non-Hodgkin lymphoma, NH L). It is the most common subtype in lymph node or extranodal organs, or chronic lymphocytic leukemia / lymphatic lymph nodes. Low degree malignant lymphoma such as cell lymphoma, marginal zone lymphoma and follicular lymphoma (follicular lymphoma, FL). As a highly invasive lymphoma, DLBCL has great heterogeneity in cytogenetics, molecular biology, histomorphology, clinical manifestation, and prognosis. The first-line scheme of amines, adriamycin, vincristine and prednisone (R-CHOP) significantly improves the therapeutic effect of DLBCL patients, but there are still about 40% of the patients' refractory and recurrent deaths. Therefore, the study of the biological heterogeneity and invasion mechanism of DLBCL, the development of specific therapeutic targets, the optimization of the selection of first-line treatment options, and the improvement of the complete delay. The rate of solution is important to improve the prognosis of patients. Metadherin (MTDH) is a new oncogene found in recent years, also known as -1 (Astrocyte elevated gene-1, AEG-1). It was first found in the study of human immunodeficiency virus (Human Immunodeficiency Virus-1, HIV-1) infection related encephalopathy. The new gene, named AEG-1., was later cloned from the aggressive breast cancer cell strain and found that the protein could promote the metastasis of breast cancer cells to the lungs, which named the MTDH gene of MTDH. human being on chromosome 8q22, the full 3611bp of the gene, and the encoding product of a transmembrane protein containing 582 ammonia. At present, basic acid, molecular weight 64KD., a large number of studies have shown that MTDH is highly expressed in various tumors of the whole body, such as breast cancer, melanoma, liver cancer, lung cancer, prostate cancer, kidney cancer, colon cancer, glioma, gallbladder cancer, ovarian cancer, lymphoma and so on. Further research has confirmed that MTDH can be used to regulate PI3K/AKT, NF- kappa B, Wnt/ beta -catenin and so on. The expression level of signal transduction pathway and its related genes to regulate tumor growth, angiogenesis, invasion and metastasis and resistance. Meanwhile, in recent years, epithelial-mesenchymal transition (EMT) is closely related to the local infiltration and distant metastasis of tumor cells..EMT refers to the epithelial cells in a specific life. Changes in morphological and biological behavior such as cell morphology, polarity, migration and invasion ability and other changes in cell morphology, polarity, migration and invasion, as well as epithelial markers such as E-cadherin expression reduction and functional deletion, and interstitial cells marked by EMT. The expression and / or function of some transcriptional factors, such as beta -catenin, vimentin, and some transcription factors, such as Snail, Twist and ZEB1, are up-regulated in many malignant tumors, such as breast cancer, lung cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, osteosarcoma, colon and cervical cancer, and MTDH can promote tumor invasion and metastasis by regulating the EMT of tumor cells. The expression of D H and the expression of EMT markers, tumor differentiation, clinical stage and treatment prognosis are closely related to.MTDH to promote EMT in tumor cells. It is realized by regulating a variety of signal pathways, such as PI3K/AKT, Wnt/p-catenin, NF- kappa B and MAPK, to regulate the invasiveness of the tumor. This pathway plays an important role in regulating embryo development and maintaining the self renewal balance of adult tissue cells. Its abnormal activation is related to the occurrence and development of tumor. The beta -catenin in this signal pathway plays a dual role in EMT and activation of Wnt signaling pathway,.DLBCL is highly invasive lymphoma and is prone to extranodal invasion. It is not clear whether there is a phenomenon of EMT in DLBCL, the role of MTDH and EMT in DLBCL invasiveness and its regulatory mechanism. This study uses immunohistochemistry, cell biology and gene silencing techniques to elucidate the expression of MTDH and EMT in DLBCL and the regulation relationship between the two and the MTDH expression changes to DLBCL cells. Invasive effects and regulatory mechanisms provide a basis for the development of new DLBCL markers and therapeutic targets. Part 1 the expression and clinical significance of MTDH and EMT markers in diffuse large B cell lymphoma: DLBCL is a highly invasive lymphoma with great heterogeneity, but the study of its invasion mechanism is not clear.MTD H is a new oncogene found in recent years. A large number of studies have shown that MTDH is highly expressed in many kinds of tumors and regulates the growth and proliferation of tumor, blood Guan Shengcheng, invasion and resistance and other.EMT and tumor cells by regulating the expression of multiple signal transduction pathways, such as PI3K/AKT, NF- kappa B, Wnt/ beta -catenin, and other related genes. Local invasion is closely related to distant metastasis. When the tumor cells occur EMT, the biological behavior and molecular markers will change.MTDH to promote the invasion and metastasis of the tumor by regulating the EMT of the tumor cells. In order to study the expression of MTDH mouth EMT in DLBCL, we take the human DLBCL cell strain LY1 and LY8 as the research object, first of all. The expression of MTDH and EMT markers vimentin, ZEB1 protein in DLBCL cell lines was detected with protein imprint analysis technique. The pathological diagnosis of DLBCL and lymphoid reactive hyperplasia tissue specimens were collected, and the expression of MTDH in two tissues was detected by immunohistochemistry and the clinical data of the patients were carried out. Correlation analysis, their role in DLBCL invasiveness. Materials and methods: 1. specimens collected and separated from peripheral blood mononuclear cells 2.LY1 and LY8 cell lines, 3. protein extraction and Western blot analysis 4. specimens and clinical case data collection 5. immunohistochemical analysis 6. statistical analysis results: 1. Western blot results showed that MTDH, vimentin and ZEB1 protein were highly expressed in DLBCL cell lines, but the target band of almost non expression.MTDH in peripheral blood mononuclear cells of normal human was located at 75kD. The target band of vimentin was located at 54kD, and the molecular weight of ZEB1 was the largest. The target band was located at 124kD.2.30 case of DLBC tissue specimens, only 1 cases were scored ( The expression of MTDH in DLBCL tissue specimens of 12/30 cases was low, and MTDH was highly expressed in DLBCL tissue specimens of 60% (18/30). The expression of MTDH staining was mostly found in cytoplasm and in the nucleus, but MTDH in the reactive hyperplasia tissues of lymph nodes almost did not express the correlation analysis between the expression of.3. MTDH and the clinical characteristics of DLBCL patients. The high expression of MTDH was not related to the age of DLBCL patients (P=0.458), sex (P=0.284) and LDH (P=0.249), but it was positively correlated with the clinical stage (P=0.04), B symptoms (P=0.004), risk stratification (P=0.033), and P values were 0.05, and there were statistical significance. The high expression of.2.IHC in DLBCL cell lines showed that MTDH was highly expressed in 60% (18/30) DLBCL tissues, and the MTDH expressed in the cytoplasm was mostly located in the cytoplasm. The high expression of.3. MTDH in the nucleus was in positive correlation with the clinical stages of DLBCL patients. There was a positive correlation between the B symptoms and risk stratification of B. The second part MTDH expression changed to diffuse large B. The influence and regulatory mechanism of cell lymphoma invasiveness: in a variety of malignant tumors, MTDH can promote tumor invasion and metastasis by regulating the EMT of tumor cells to transfer the expression of.MTDH and EMT markers and tumor differentiation. Clinical staging, the prognosis closely related to the prognosis of.MTDH is through the regulation of PI3K/AKT, NF- kappa B. Wnt/ beta -catenin and MAPK. A variety of signal pathways are used to promote the occurrence of tumor cell EMT and regulate tumor invasiveness. In this study, the human DLBCL cell line LY8 was used as the research object. The RNAi silencing MTDH gene mediated by the lentivirus vector was used to detect the changes in the expression of the EMT markers vimentin and ZEB1 protein and the change of the invasive ability of DLBCL cells. Whether there is EMT in DLBCL, and the effect of MTDH expression on the invasiveness of DLBCL cells and its regulatory mechanism. Materials and methods: 1.LY8 cell line culture 2. lentivirus vector mediated RNAi targeted silent LY8 cell line, MTDH gene 3. flow cytometry, cell transfection rate 4. protein extraction and Western blot analysis 5. invasive chamber experiment 6 The results of statistical analysis: 1. MTDH RNAi mediated by lentivirus vector can inhibit the expression of MTDH in DLBCL cells,.2. application of lentivirus particles silencing MTDH gene, the expression level of Vimentin and ZEB1 protein in DLBCL cells decreased significantly (P0.05) 3. Transwell invasive laboratory results showed that the lentivirus vector mediated MTDH gene silencing could To reduce the mobility and invasiveness of DLBCL cell lines (P0.05). Conclusion: the existence of EMT phenomenon.2. MTDH gene silencing in 1. DLBCL can reduce the expression of EMT markers Vimentin and ZEB1 protein in DLBCL cell lines. The signal pathway regulates the EMT. of DLBCL
【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】：R733.1

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