【作者】 陈强；

【导师】 张世忠；

【作者基本信息】 南方医科大学 ， 神经外科学， 2014， 博士

【摘要】 神经胶质瘤是临床上最常见恶性原发性脑肿瘤,其发病率约占所有颅内原发性脑肿瘤的35.26～60.96%(平均44.69%)。通常,神经胶质瘤中心是一个出血坏死灶,而周围是与正常脑组织边界不清的高致密肿瘤细胞区。神经胶质瘤细胞具有无限增殖能力和很强的侵袭性,很容易向周围正常脑组织进行侵袭。胶质瘤生长迅速且与周围正常脑组织边界不清,呈浸润性生长,因此,神经胶质瘤很难通过外科手术切除手段达到完全病理学肿瘤切除的目的。另外,由于神经胶质瘤对放疗不敏感和对化疗药物容易产生耐药性,也导致了其复发率高和预后差的结果。因而,尽管给予了多种综合治疗手段,神经胶质瘤治疗效果仍不理想,特别是恶性程度高的胶质母细胞瘤(GBM)患者生存期仅有12-15个月。恶性胶质瘤的难治性很大程度就在于其侵袭性强的生物学特性。因此,关于神经胶质瘤侵袭性方面的研究和寻找更为有效的治疗手段非常有必要。通常,肿瘤基本的侵袭过程是瘤细胞从原发瘤灶脱落,侵袭基底膜(BM)和细胞外基质(ECM)相互作用的复杂过程。肿瘤细胞首先黏附于基底膜和血管内皮细胞,然后通过分泌蛋白水解酶和促进血管生成发挥作用。而所有级别的恶性胶质瘤都会发生弥漫性单细胞侵袭。胶质瘤细胞往往沿有髓纤维束和在血管周围间隙沿血管进行迁移。因此,神经胶质瘤这种侵袭特性与基底膜和细胞外基质的组成成分密不可分。多种基底膜和细胞外基质的组成成分已证实了在人胶质瘤中过度表达,如纤维连接蛋白、层粘连蛋白、Ⅲ和Ⅳ型胶原蛋白、玻连蛋白和腱糖蛋白,这些物质都与胶质瘤细胞的迁移和侵袭有着密切的联系。层粘连蛋白(laminin,LN)是一组细胞外基底膜异源三聚体的糖蛋白大家族,主要由α、β、γ这三种链不同的组合而构成。层粘连蛋白在许多基本的生物过程中发挥着重要作用,包括细胞粘附和迁移、胚胎发育、肿瘤浸润、血管生成、组织分化和伤口愈合等。层粘连蛋白不仅是血管基底膜的主要功能成分,也是细胞外基质的主要功能成分。而且,层粘连蛋白还是正常脑组织微血管BMs的主要组成成分。胶质瘤细胞自身也能合成和分泌多种LN参与血管基底膜和血管基底膜构成,也表达大量的整合素受体。有研究发现在低级别和高级别的胶质瘤及胶质母细胞瘤(GBM)邻近的正常脑组织中过度表达含α4链的LN。一种含α4链的LN亚型层粘连蛋白-8主要是在多形性胶质母细胞瘤和邻近多形性胶质母细胞瘤的脑组织中的血管基底膜上表达,而另一个含α4链的层粘连蛋白-9则主要在低级别胶质瘤和正常组织血管基底膜表达。LN-8是表达于血管内皮细胞的主要亚型。LN-8已被证实与相应的受体整合素结合,对于骨髓功能和血-脑屏障的维持起着十分重要的作用。LN-8还可以通过与整合素α3β1和α6β1的相互作用促进胶质瘤细胞黏附和迁移。过表达LN-8的GBM患者肿瘤复发时间和病人存活期与过表达LN-9的GBM患者相比较都有所缩短。而神经胶质瘤一个显著特征在于血管内皮细胞增殖和显著的肿瘤血管生成,后者是胶质瘤的生长、侵袭和转移过程中的一个重要的基础条件。含α4链层粘连蛋白本身就能促进肿瘤血管生成。以上所诉说明了LN-8在胶质瘤的侵袭和肿瘤血管生成过程中发挥着重要作用,可以促进胶质瘤的复发。随着脑胶质瘤的进展,毛细管基底膜中含有α4链层粘连蛋白的表达会显著上调,并且含有β2链的LN-9(α4β2β1)会被含有β1链的LN-8(α4βγ1)所取代。LN-8和LN-9具有相似的结构,只是其中的β链不同。胶质瘤进展过程中含β2链LN-9转变为含β1链(LAMB1) LN-8潜在的调节因素还尚为人所知。因此,对于LN-8的基因调控机制的研究就显得非常有意义。MicroRNAs新近发现的一类广泛存在于真核生物体中内生的短链非编码RNA,长约22nt(nt是核苷酸nucleotide的缩写,lnt代表一个核苷酸单位)。MicroRNAs可以通过作用于相应靶基因调控细胞增殖、凋亡、分化、发育、侵袭和转移,甚至还可以调节干细胞活化及新生血管生成。MicroRNAs基因通常位于在癌基因组的染色体脆性位点,被认为是一类新型的致癌基因和抑癌基因。在大多数实体瘤和血液系统恶性肿瘤中都发现MicroRNAs表达水平的异常。这些异常表达的MicroRNAs还可以抑制肿瘤细胞增殖、诱导肿瘤细胞凋亡、抑制肿瘤细胞的迁移性、下调多种靶基因的表达等等。脑部肿瘤MicroRNAs表达谱的一系列的研究也发现一些MicroRNAs的表达异常。这些表达异常的MicroRNAs基因位点通常位于典型的脑肿瘤的上游或下游信号通路,能调节脑肿瘤细胞的致癌表型。MicroRNA-124在各种哺乳动物的胚胎和成年皮质中大量表达,是一类脑组织中表达最为丰富特有的microRNA,约占脑组织microRNAs总量的25%～48%。MicroRNA-124对于维持正常的神经发育和功能有着重要的作用。对神经胶质瘤不同的表达谱的研究却发现miR-124在恶性程度较高的间变性星形细胞瘤(Ⅲ级)和多形性胶质母细胞瘤(Ⅳ级)中的表达量是显著下调的,提示miR-124的下调在神经胶质瘤形成和进展过程中发挥着关键作用。miR-124在神经胶质瘤中表达的恢复抑制了各种靶基因的表达,如lamininγ1和整合素β1,也抑制了肿瘤的转移和侵袭,从而延缓肿瘤的进展。miR-124-3p(miR-124或者miR-124a)和miR-124-5p(miR-124*)都是miR-124的两种成熟的形态(miR-124-5p在LAMB1的3’UTR一个公认的结合位点是由鸟嘌呤取代胞嘧啶而发生突变[星号标记])。miR-124-3p已证实在高级别胶质瘤中的表达是下调的,而且与胶质瘤患者预后相关。但是miR-124-5p在神经胶质瘤中的表达是否和niR-124-3p的结果一致还是未知数。因此,筛选出miR-124在胶质瘤发展调控网络中确切的靶标基因,对于胶质瘤的诊疗非常有意义。基于我们先前研究观察到GBM细胞系和组织中lamininβ1蛋白的高表达和miR-124-5p的低表达与胶质瘤进展关系,我们设想这两者之间在胶质瘤进展中是否存在相关关系。因此,本研究就针对miR-124-5p对lamininβ1的潜在调节机制进行探讨。我们首先通过Q-PCR和Western Blot方法检测GBM细胞系(U87和U251)及不同级别临床胶质瘤标本中miR-124-5p与LAMB1之间的表达水平,利用双荧光素酶检测系统验证miR-124-5p对LAMB1(蛋白质)表达的调控作用。通过体内、外研究对胶质瘤细胞系和组织中miR-124-5p的高表达和LAMB1的敲减对于细胞增殖、克隆形成、肿瘤生长和血管生成所产生的影响进行评估,验证miR-124-5p对LAMB1潜在的调节作用。第一部分各级别神经胶质瘤层粘连蛋白-8和miR-124-5p的表达关系目的：检测层粘连蛋白-8LAMB1mRNA和蛋白,miR-124-5p在各级别神经胶质瘤中的表达水平,并分析它们与神经胶质瘤进展之间是否存在关联。方法：人U87和U251胶质瘤细胞系进行培养。于珠江医院神经外科和病理科各级别胶质瘤组织标本进行采集及处理。采用Q-PCR和Western blots检测非肿瘤性脑组织、Ⅰ/Ⅱ级胶质瘤、间变性星形细胞瘤(AAs, WHO Ⅲ级),多形性胶质瘤母细胞瘤(GBMWHOⅣ级)和U87、U251胶质瘤细胞系中的LAMB1mRNA和蛋白、miR-124-5p的表达水平。结果：LAMB1mRNA和蛋白在AAs和GBM组织中的表达水平相比较于正常脑组织和Ⅰ/Ⅱ级神经胶质瘤表现出显著上调,胶质母细胞瘤组织中的LAMB1表达量最高。MiR-124-5p在间变性星形细胞瘤(AAs, WHO Ⅲ级)和多形性胶质母细胞瘤(GBM WHO Ⅳ级)的表达量与正常脑组织和Ⅰ/Ⅱ级胶质瘤相比显著下调。对miR-124-5P表达水平与LAMB1mRNA和蛋白表达水平进行秩相关分析：miR-124-5p表达水平与LAMB1mRNA和蛋白表达水平呈负相关关系。结论：LAMB1mRNA和蛋白的表达水平随着神经胶质瘤级别和严重程度的增加而提高,而miR-124-5p表达水平随着神经胶质瘤级别和严重程度而下降,因此,miR-124-5p和LN-8可以作为神经胶质瘤的生物标志物。下调的miR-124-5p和上调的LAMB1与胶质瘤进展相关,两者之间呈负相关关系。第二部分Has-miR-124-5p调控LAMB1基因的表达目的：验证miR-124-5p是否对LAMB1转录后水平具有调控作用。方法：采用MicroCosm Targets Version5microRNA靶基因预测软件in silico分析miR-124-5p在LAMB13’UTR端结合位点。MiR-124-5p minic、NC (miRNA minic negative control)、miR-124-5p inhibitor和miRNA NC inhibitor脂质体转染U87和U251胶质瘤细胞,分别利用Q-PCR和Western blots检测U87和U251胶质瘤细胞中LAMB1mRNA和蛋白水平。我们还利用双荧光素酶检测系统鉴定miR-124-5p对LAMB1(蛋白质)表达的调控作用。结果：MicroRNA靶基因预测软件in silico分析显示miR-124-5p是LAMB13’UTR端结合位点。72小时后,转染miR-124-5p minic过表达miR-124-5pU87和U251细胞的LAMB1蛋白水平相比于转染NC的U87和U251细胞是下调的。转染miR-124-5p inhibitor的U87和U251细胞的LAMB1蛋白水平相比于转染NC inhibitor的U87和U251细胞是上调的。同时,LAMB1mRNA水平却不受过表达或敲低miR-124-5p的影响。miR-124-5p对LAMB1基因的翻译产生抑制作用。结论：miR-124-5p对U87和U251胶质瘤细胞LAMB1mRNA的表达无调节作用,对U87和U251胶质瘤细胞LAMB1(蛋白)的转录后水平表达有直接的负调控作用。LAMB1是：miR-124-5p直接的靶基因。第三部分MiR-124-5p表达恢复对异种移植胶质瘤肿瘤生长和微血管密度的抑制作用目的：评估miR-124-5p表达的恢复对于胶质瘤细胞增殖、细胞周期和迁移以及肿瘤生长和血管生成产生的影响。方法：1.体外实验：miR-124-5p minic和miRNA minic negative control (NC)脂质体转染U87胶质瘤细胞。利用MTT实验和Transwell细胞迁移实验对转染miR-124-5p minic和NC (miRNA minic negative control)的U87胶质瘤细胞的细胞增殖能力、细胞周期和细胞侵袭能力进行分析。2.体内实验：建立免疫缺陷nu/nu裸鼠U87和U251皮下胶质瘤动物模型。当肿瘤体积达到150-200mm3,将小鼠随机分配为2个处理组：miR-124-5p minic组和NC组(每组n=6),分别给予miR-124-5p minic和NC原位注射处理,监测3个星期的肿瘤体积变化情况。利用Q-PCR, Western blots和免疫组化技术检测各组肿瘤组织中LAMB1mRNA和蛋白水平变化；通过对肿瘤标本的肿瘤血管内皮标记物CD31行免疫组化分析肿瘤微血管密度的变化。结果：miR-124-5p对U87胶质瘤细胞的细胞增殖能力、细胞周期和细胞侵袭能力无调控作用。miR-124-5p minic组的肿瘤生长与NC组相比是受到抑制的。miR-124-5p minic组的LAMB1蛋白水平与NC组相比较是下调的,而niR-124-5p minic组的LAMB1mRNA水平与NC组相比较无统计学意义。miR-124-5p minic组的肿瘤微血管密度与NC组相比较明显下降。结论：miR-124表达的恢复通过抑制LAMB1基因的蛋白表达导致胶质瘤肿瘤血管生成和肿瘤生长受到抑制。更多还原

【Abstract】 Gliomas is the most common malignant brain tumors, the incidence is about35.26～60.96%(average44.69%) of all intracranial primary brain tumors. Generally, the center of gliomas is a hemorrhagic necrosis, while the surrounding is a high density of tumor cell area. Glioma cells are characterized by unlimited proliferation and strong aggression. Glioma is easy to invade into the surrounding normal brain tissue. Gliomas grow rapidly and with a characteristic of invasive growth, therefore, glioma is difficult to be completely removed by surgery. In addition, glioma is insensitive to radiotherapy and chemotherapy, so glioma has a high recurrence rate and poor prognosis. Despite given a variety of treatments, the effect of treatment is not satisfactory. Survival of glioblastoma patients is only12-15months. Malignant glioma is not easily being cured largely depended by its strong invasion. Therefore, it is necessary to investigate invasiveness of glioma and find more effective treatments.Commonly, the process of tumor invasion is complex. Tumor cells shed from a primary tumor foci and invade into basement membrane, then have an interaction with extracellular matrix (ECM). Firstly, tumor cells adhere to endothelial cells and basement membrane, and secrete proteolytic enzymes and promote angiogenesis. All malignant glioma cells will invade into the surrounding tissue. Glioma cells often invade into the surrounding tissue along with myelinated fibers and blood vessels. Therefore, invasion of glioma is related to compositions of the basement membrane and extracellular matrix. Compositions of the basement membrane and extracellular matrix have been confirmed overexpressed in human gliomas, such as fibronectin, laminin, III and IV collagen, vitronectin and tenascin. These substances are closely linked to migration and invasion of glioma cell.Laminin (laminin, LN) is a large family of extracellular heterologous glycoprotein trimers and mainly constituted by three different α, β, γ chain. Laminin plays an important role in many fundamental biological processes, including cell adhesion and migration, embryonic development, tumor invasion, angiogenesis, tissue differentiation and wound healing. Laminin is a major functional component of the vascular basement membrane extracellular matrix (ECM). Moreover, Laminin is main component of microvascular BMs of normal brain tissue. Glioma cells can synthesize and secrete a variety of LN involved in the vascular basement membrane and vascular basement membrane. A large number of integrin receptors are expressed on the glioma cell surfaces. Studies have found that overexpression of α4chain laminin is observed in the low-level or high-level glioma and normal brain tissue adjacent to GBM. The majority of GBMs had increased expression of laminin-8(α4β1γ1) chains in blood vessel walls, whereas low-grade tumors overexpressed laminin-9(α4β2γ1) chains.Laminin-8(a4b1g1) is the predominant isoform expressed in vascular endothelial cell BMs. Along with its receptor integrin a6β1, laminin-8has been shown to be important for BM function and the maintenance of the blood-brain barrier. Laminin-8also promote adhesion and migration of glioma cell through interactions with α3β1and α6β1integrin. GBMs that predominantly expressed laminin-8had a shorter time to recurrence than did GBMs that predominantly expressed laminin-9.GBM is characterized by endothelial cell proliferation and prominent vascularization arising from a combination of blood vessel co-option and tumor angiogenesis, the latter being a fundamental aspect of tumor growth, invasion, and metastasis. a4chain laminin itself can promote tumor angiogenesis. Above illustrate that laminin-8plays an important role in the invasion and angiogenesis of gliomas to promote tumor recurrence. During progression of human gliomas, the expression of capillary basement membrane(BM) laminins containing a4chain switched from the predominant laminin-9(α4β2γ1) to laminin-8(α4β1γ1). Laminin-8is distinguished to laminin-9by having αβ1subunit. The factors regulating the switch to laminin β1expression in gliomas are still unknown. Therefore, it is very meaningful to study the mechanisms of gene regulation about laminin-8.MicroRNAs are an abundant class of endogenous small RNA molecules,20-25nucleotides in length. The role of miRNAs as key regulatory molecules that control a wide variety of fundamental cellular processes, such as proliferation, death, differentiation, motility, invasiveness, etc. Because miRNA genes are frequently located at the chromosomal fragile sites of cancer genomes, miRNAs have been considered as novel classes of oncogenes and tumor suppressors.Abnormal expression levels of microRNAs were found in most solid tumors and hematologic malignancies. These abnormal expression of microRNAs can also inhibit tumor cell proliferation and induce tumor cell apoptosis and inhibit tumor cell migration, reduced expression of multiple target genes, and so on.A series of microRNAs expression profiling about brain tumors also found a number of abnormal microRNAs expression. The genes of these abnormal expression microRNAs usually located upstream or downstream signaling pathways of brain tumors to regulate oncogenic phenotype of brain tumor cells. The microRNA (miRNA) miR-124is abundantly expressed in the embryonic and adult cortex in various mammalian species, suggesting an essential role in normal functioning of the brain. Indeed, expression profiling of various human brain tumors has shown that miR-124is among the most dramatically downregulated miRNAs in anaplastic astrocytoma (Ⅲ grade) and glioblastoma (IV grade), suggesting reduction of miR-124plays a key role in formation and progression of glioma. Restoring miR-124expression in gliomas may slow tumor progression by repressing various miR-124targets such as laminingl and integrin β1. MiR-124-3p (known as miR-124or miR-124a) and miR-124-5p (known as miR-124*) are both mature forms of miR-124(A putative miR-124-5p binding site in the3’UTR of LAMB1was mutated by replacing the cytosine with guanine [marked by an asterisk]. MiR-124-3p is significantly downregulated in AA and GBM tumors, which is correlated with poor prognosis in glioma patients However, whether the expression of miR-124-5p has a similar result as miR-124-3p in human gliomas is still unknown.Therefore, it is very meaningful to screen out exact target genes of miR-124in the development of regulatory networks in glioma.Based on the reduced miR-124-5p expression and high levels of laminin β1protein that were observed in GBM cell lines and tissues, the present work investigated the potential regulation of laminin β1by miR-124-5p.Therefore, the potential regulatory mechanisms of miR-124-5p this were discussed. The expression levels of LAMB1and miR-124-5p were examined in glioma cell lines (U87and U251) and GBM tissue samples by quantitative PCR and Western blotting. Regulation of miR-124-5p on LAMB1(protein) expression is defined by dual luciferase assay system. The potential regulation of LAMB1by miR-124-5p was investigated by assessing the effects of restored miR-124-5p expression on cell proliferation, colony formation, and tumor growth and angiogenesis. Chapter I Relationships between laminin8and miR-124-5p expression in gliomasObjective:Detection of laminin-8mRNA and protein, miR-124-5p expression levels in all grade gliomas, and to analyze whether there is an association between laminin-8and miR-124-5p in glioma progression.Methods:U87and U251human glioma cell lines were cultured. Glioma tissue samples were obtained and proscessed through the Department of Neurosurgery1andDepartment of Pathologyof Zhujiang Hospita. The expression levels of LAMB1and miR-124-5p were examined in glioma cell lines (U87and U251) and GBM tissue samples by quantitative PCR and Western blotting.Results:AA and GBM tissues showed significantly higher levels of LAMB1mRNA and protein relative to normal brain tissue, and grade I/II gliomas, and GBM tissues had the highest level of LAMB1gene expression. A significant decrease in miR-124-5p expression was observed in AAs and GBMs compared with normal brains and grade Ⅰ/Ⅱ gliomas. Rank correlations calculated between miR-124-5P expression level and LAMB1mRNA or protein levels indicated that elevated LAMB1mRNA or protein expression occurred concomitantly with reduced miR-124-5p expression.Conclusions:Progressive increases in the levels of LAMB1mRNA and protein were observed with increasing glioma severity. miR-124-5p expression was significantly downregulated during glioma progression. miR-124-5p and laminin-8can act as biomarkers of glioma. Reduction of miR-124-5p and overexpression of LAMB1is associated with progression of gliomas. There is a negative correlation between miR-124-5p and LAMB1. Chapter Ⅱ MiR-124-5p Regulates LAMB1Expression at the Posttranscriptional LevelObjective:To verify regulation of LAMB1expression at the posttranscriptional level by miR-124-5p.Methods:An in silico analysis using MicroCosm Targets Version5test a potential miR-124-5p binding site in the3’UTR of LAMB1. Lipofectamine2000transfecte U87and U251glioma cells by miR-124-5p minic, NC, miR-124-5p inhibitor and miRNA NC inhibitor. The expression levels of LAMB1were examined in glioma cell lines (U87and U251) by quantitative PCR and Western blotting. Regulation of miR-124-5p on LAMB1(protein) expression is defined by dual luciferase assay system.Results:An in silico analysis using MicroCosm Targets Version5revealed a potential miR-124-5p binding site in the3’UTR of LAMB1. After72hours, LAMB1protein expression was reduced in cells overexpressing miR-124-5p compared with NC-transfected cells. Furthermore, suppression of miR-124-5p resulted in higher levels of LAMB1protein compared with cells transfected with NC inhibitor. Significantly, the levels of LAMB1mRNA were unaffected by miR-124-5p overexpression or inhibition. LAMB1(protein) expression is directly and negatively regulated by miR-124-5p.Conclusions:MiR-124-5p has no effect on expression LAMB1mRNA in U87and U251cells, but a direct negative regulation of LAMB1(protein) at the posttranscriptional level. LAMB1is a direct target gene of MiR-124-5p.Chapter Ⅲ Restoration of miR-124-5p expression inhibits tumor growth and microvessel density in glioma xenograftsObjective:To assess the effects of restored miR-124-5p expression on cell proliferation, cell cycle, cell migration as well as tumor growth and angiogenesis.Methods:1.In vitro:miR-124-5p minic and miRNA minic negative control (NC) transfect U87glioma cells by Lipofectamine2000.Cell proliferation, cell cycle and cell invasion of U87glioma cell transfected miR-124-5p minic and miRNA minic negative control were analyzed by MTT assay and Trans well cell migration assay.2. In vivo:Glioblastoma xenografts immunocompromised nu/nu mice models of U87and U251were established. When tumor volume reached150-200mm3, the mice were randomly assigned to two treatment groups:miR-124-5p minic and NC groups (n=6), the mice were respectively given miR-124-5p minic and NC. We monitored tumor volume changes for three weeks. The expression levels of LAMB1in glioma tissues were examined by Q-PCR, Western blotting and immunohistochemistry. Tumor microvessel density changes of tumor specimens is determined by immunohistochemical analysis of endothelial marker CD31.Results:miR-124-5p did not significantly inhibit U87cell proliferation, cell cycle and cell invasion. A significant growth inhibition of U87and U251xenograft tumors injected with miR-124-5p minic was observed, compared with tumors injected with the NC.LAMB1protein levels of MiR-124-5p minic group were reduced, compared with the NC group. LAMB1mRNA levels of MiR-124-5p minic group were not statistically significant to NC group. Injection of the miR-124-5p minic to restore miR-124-5p expression led to a dramatic reduction in microvessel density in U87and U251xenografts.Conclusions:Restoration of miR-124-5p expression can inhibits tumor growth and microvessel density in glioma xenografts.更多还原