【作者】 黄惠勇；

【导师】 章翔；

【作者基本信息】 第四军医大学 ， 外科学， 2013， 博士

【摘要】 人脑胶质细胞瘤是中枢神经系统(Central nervous system, CNS)中最常见的恶性肿瘤，其发生率占颅内肿瘤的40~50%。多形性胶质母细胞瘤（Glioblastoma multiforme,GBM, WHO IV）约占恶性胶质细胞瘤的60~70%，它能快速地增殖、浸润性生长至周围重要的神经结构，且对放、化疗不甚敏感。因而，具有病程短、死亡率高及预后很差等为其特点。虽然近年来包括手术技术的改良、立体定向放射外科、以细胞毒作用为基础的化疗、以及生物治疗等手段的快速发展，但是恶性胶质细胞瘤的病人预后并没有明显的提高。高级别的胶质细胞瘤病人初次手术后较快复发或是死亡，平均生存期不足1年。究其原因在于恶性胶质细胞瘤具有高度恶性的生物学特征，包括很高的增殖能力，较强的侵袭与迁移性，丰富的血管生成作用等。基于细胞毒作用为基础的替莫唑胺（Temozolomine, TMZ）化疗，是继神经导航手术治疗和立体定向放疗之后的、被广泛认可的一线治疗手段，一般认为能有效的延长病人的总生存时间，并提高病人的生活质量。然而，并不是所有恶性胶质细胞瘤病人都能受益于这一治疗方法，原因在于恶性胶质瘤细胞内DNA修复酶O6-甲基鸟嘌呤-DNA甲基转移酶（O6-methylguanine-DNA methyltransferase, MGMT）系统的高度活跃。近年来，随着分子靶向治疗的快速发展，在基因水平治疗恶性胶质细胞瘤已成为国内外学者研究的重点。因此，寻找治疗恶性胶质瘤细胞的关键分子靶点，并针对该分子进行实验性靶向治疗，同时联合TMZ的化疗，将具有较好的临床应用价值和广泛的社会意义。同源盒基因（Homeobox genes，HOXG）是含有共同183个核苷酸序列的转录因子家族，它具有高度保守性，且在胚胎发育、细胞分化过程中起主控基因的功能。在人类，共有39个I类同源盒（Homeobox，HOX）基因分别被发现分布在4个染色体基因簇（即：HOXA在7p15.3，HOXB在17q21.3，HOXC在12q13.3，HOXD在2q31）。每簇含有9~11个基因编排在同源的序列。HOX基因在正常胚胎发育过程中的时空变化起着非常重要的主控作用，同时它还具有发育后的监管职能。异常的HOX基因表达诱发不同人类疾病和癌症，包括白血病、淋巴瘤、乳腺癌、前列腺癌、结直肠癌、脑肿瘤等。多个HOX基因已被发现在原发性星形胶质细胞瘤和胶质母细胞瘤细胞系中过度表达。这些均提示HOX基因在胶质细胞瘤的发生、发展、复发和放、化疗耐受性方面具有很重要的作用。在人类39个HOX基因中，HOXA9是编码序列特异性转录调控因子，在胚胎的时空发育，细胞的分化、增殖与迁移，肿瘤的恶性演变和诱发凋亡都有很重要的作用。不正常的HOXA9激活和过表达被发现在各类肿瘤当中，其中包括星形胶质细胞瘤。然而，在星形胶质细胞瘤中，HOXA9基因异常激活的潜在作用机制和其对放、化疗药物的耐受机制仍不清楚。本课题重点研究HOXA9基因在人脑星形胶质细胞瘤中差异性表达和对细胞的定位，探讨其与胶质细胞瘤病理级别和胶质瘤病人预后的相关性；并初步分析HOXA9基因在不同表达水平对胶质细胞瘤恶性生物学行为的影响，以及它在胶质瘤细胞放、化疗耐受中的作用机制，为本类肿瘤的治疗提供实验依据。本课题主要包括以下4个方面的研究：1. HOXA9在人脑星形胶质细胞瘤中的差异性表达首先从mRNA和蛋白水平研究HOXA9基因在5株人脑星形胶质瘤细胞系（U87MG、U251、A172、T98G和BT325）中的表达。采用实时定量PCR（Quantitativereal time PCR, qRT-PCR）和Western blot的方法检测发现，HOXA9mRNA和蛋白在U87MG、U251、A172、T98G及BT325细胞中均呈高表达，以T98G细胞尤甚。这提示，HOXA9在恶性胶质瘤细胞系中的异常高表达可能与胶质细胞瘤恶性生物学行为之特征有关。本研究进一步采用免疫组织化学法（Immunohistochemistry, IHC）、qRT-PCR和Western blot方法检测了158例原发性星形胶质细胞瘤，50例复发多形性胶质母细胞瘤和6例非肿瘤脑组织标本，结果表明，HOXA9分子定位于星形胶质细胞瘤的胞核和胞浆，以胞核为主；HOXA9mRNA和蛋白在6例非肿瘤性脑组织标本中呈现低表达或是不表达，在各个病理级别的星形胶质细胞瘤组织中广泛表达，其表达量随着病理级别的升高而增高，且以复发性多形性胶质母细胞瘤最高。在158例原发性星形胶质细胞瘤、50例复发多形性胶质母细胞瘤和6例非肿瘤脑组织标本的免疫组化染色中，发现HOXA9蛋白表达在胶质细胞瘤中明显高于非肿瘤性脑组织标本（P<0.01），在高级别胶质细胞瘤标本中明显高于低级别胶质细胞瘤（P<0.01）。同时，经Spearman相关性分析发现，HOXA9的表达与Survivin表达（r=0.906,P<0.01）、肿瘤最大直径（r=0.507, P<0.01）、病理级别（r=0.683, P<0.01）、手术切除程度（r=0.152, P=0.029）和辅助治疗类型（r=0.366, P<0.01）呈正相关，而与病人的性别（P=0.624）、年龄（P=0.415）和肿瘤内坏死程度（P=0.351）无相关。基于HOXA9表达的免疫反应性评分（Immunoreactivity Score, IRS）结果，158例原发性星形胶质细胞瘤患者和50例复发多形性胶质母细胞瘤患者，根据临床随访资料，分为两组进行Kaplan-Meier生存分析，高表达HOXA9的病人总生存时间[Overall survival (OS);median,8months;95%confidence interval (CI),4-10months]和疾病无进展时间[Progression-free survival (PFS); median,7months; CI,3-8months]，均显著低于低表达HOXA9患者(OS; median,44months; CI,36-47months; P<0.01; PFS; median,43months; CI,34-46months; P<0.01）。经COX比例风险模型分析发现，病理级别(P=0.000)、KPS评分(P=0.000)、手术切除程度(P=0.015)、肿瘤最大直径(P=0.000)和HOXA9表达(P=0.007)，是胶质细胞瘤患者预后较差的危险因素。因此HOXA9的表达可作为判别星形胶质细胞瘤患者预后的一个潜在指标。本研究提示，HOXA9基因在人脑恶性胶质细胞瘤的发生及恶性演化过程中具有很重要的作用，这为我们下一步的研究奠定了基础。2.构建HOXA9RNAi慢病毒载体和稳转细胞系，观察RNAi后胶质瘤细胞生物学行为的变化本实验以HOXA9表达较高的人脑胶质瘤细胞系T98G为研究对象，设计构建特异性RNAi片段，将其克隆成经测序正确的pLKO.1-HOXA9shRNA-1和pLKO.1-HOXA9shRNA-2，通过慢病毒包装感染目的细胞，经嘌呤霉素筛选，建立了稳定转染的细胞系T98G-S1（T98G shRNA-1）、T98G-S2（T98G shRNA-2）和T98G-NC（阴性对照组）。以qRT-PCR与Western blot验证两组细胞的HOXA9mRNA和蛋白表达，见有明显下调，因而成功地获得了能够稳定下调HOXA9基因表达的恶性胶质细胞瘤细胞系。然后根据已经建立的稳定转染细胞系（T98G-S1、T98G-S2和T98G-NC）进行实验，观察HOXA9基因对恶性胶质瘤细胞的生长、增殖、分化、细胞周期和细胞凋亡、以及对侵袭性与迁移能力的影响。结果表明：HOXA9RNAi后，恶性胶质瘤细胞生长明显减缓，增殖亦有显著降低，分化大为减弱，细胞周期G0/G1期阻滞，细胞凋亡增加以及侵袭与迁移能力下降。本研究证明，HOXA9基因RNAi后对恶性胶质瘤细胞具有明显的抑制作用。3. HOXA9RNAi增强恶性胶质瘤细胞对TMZ敏感性作用的分子机制对158例原发性星形胶质细胞瘤、50例复发多形性胶质母细胞瘤和5株人脑星形胶质瘤细胞系（U87MG、U251、A172、T98G和BT325）的qRT-PCR及Western blot方法进行检测分析，发现在部分胶质细胞瘤病人的标本和胶质瘤细胞系BT325中呈低表达或是不表达MGMT mRNA和蛋白，仍然对TMZ具有耐药性。因此，我们推测这可能存在一条独立于MGMT甲基化地位TMZ耐药的作用机制。我们以胶质瘤细胞系T98G和BT325为研究对象，观察HOXA9RNAi后T98G和BT325细胞对TMZ的敏感性，探讨其可能的分子机制。结果表明，T98G和BT325的HOXA9基因经RNAi后对TMZ的敏感性明显增加，其中BT325细胞的生长增殖明显受到抑制（P<0.01），凋亡明显增加（P<0.01）；PI3K (Phosphoinositide-3-kinase)通路特异性抑制剂LY294002作用于T98G和BT325后，能达到与HOXA9RNAi相同的结果（P>0.05）；无论是在RNAi或是LY294002的作用之后，两株细胞系均出现NF-kappaBp65蛋白表达明显增多。因此我们认为，HOXA9基因的过度表达，一方面通过PI3K信号通路直接诱导TMZ耐药，此机制独立于MGMT甲基化地位；另一方面通过NF-kappaB信号通路上调MGMT转录水平，从而间接地诱导TMZ耐药。4. HOXA9RNAi增强恶性胶质瘤细胞对放疗敏感性作用的分子机制在158例原发性星形胶质细胞瘤患者和50例复发多形性胶质母细胞瘤患者的临床资料和随访记录中，发现高级别胶质细胞瘤患者对放射治疗（Radiotherary, RT）敏感性较差。有研究发现，NF-kappaB信号通路在肿瘤的发生与发展中扮演了很重要的作用，对于抑制NF-kappaB信号级联反应可能增加肿瘤对RT的敏感性，推测HOXA9过度表达可能通过NF-kappaB信号通路来增强胶质瘤细胞对RT的耐受性。我们以胶质瘤细胞系T98G和BT325为研究对象，观察HOXA9RNAi后胶质瘤细胞系T98G和BT325对RT的敏感性，探讨其可能的分子机制。本研究表明， T98G和BT325的HOXA9基因经RNAi后对RT的敏感性明显增加，细胞的生长与增殖受到明显抑制（P<0.01），凋亡显著增加（P<0.01），尤其是BT325细胞（P<0.05）；同时观察到单独给予RT上调了细胞内NF-kappaB信号通路中的NF-kappaB p65和IκB蛋白的表达，没有影响抑制性蛋白κB-ras1，而RT联合HOXA9RNAi却能阻滞整个NF-kappaB信号通路的激活。这说明恶性胶质瘤细胞通过上调HOXA9基因激活NF-kappaB信号通路来逃避RT的损伤，增强对RT的耐受性。本研究认为，HOXA9基因过表达是通过NF-kappaB信号通路，并参与了RT耐受性的作用机制。综上所述，本研究证实HOXA9在人脑星形胶质细胞瘤的生长、发展、侵袭、迁移及放、化疗中发挥了重要作用。以HOXA9基因为作用靶点进行恶性胶质细胞瘤的治疗具有较好的临床应用价值，并为星形胶质细胞瘤的进一步治疗提供了新的思路。更多还原

【Abstract】 Gliomas are the most common adult primary central nervous system tumours, whichaccount for approximately40~50%of brain tumours. Glioblastoma multiforme (GBMWHO IV) is the most common gliomas and accounts for approximately60~70%ofmalignant gliomas with the worst prognosis. GBM is characterised by a short course andhigh mortality of disease, which prognosis is far worse than that of other tumours becauseof its rapid growth and invasion of the central nervous system and resistance toconventional therapies. Imaging-guided neurosurgery, stereotactic radiation therapy andcytotoxic chemotherapy have been unable to fundamentally cure GBM. GBM patientsusually die from tumour recurrence within six months of the initial surgery. Difficulties ontreatment are closely associated with the malignant biology phenotype of gliomas.Although temozolomide (TMZ)-based chemotherapy effectively prolongs overall survivaland enhances the quality of life of patients, however, not all GBM patients can be benefitfrom TMZ-based chemotherapy because of the active GSTs system and abundant MGMTcontent of GBM cells. Therefore, it is important to find new ways to treat and curegliomas. Molecular targeted therapy is one of the most promising potential treatments. New markers to predict clinical outcome, tumour recurrence and resistance to therapiesoften determine the diagnosis and therapy of some cancer subtypes.Homeobox genes encode transcription factors important for anteroposteriorpatterning during embryogenesis and are divided into two classes: the class I clusteredhomeobox genes (HOX), and the class II dispersed non-HOX genes (PAX, EMX, MSX,and so on). In humans, there are39class I homeobox (HOX) genes found in four genomicclusters (HOXA at7p15.3, HOXB at17q21.3, HOXC at12q13.3, and HOXD at2q31).Their spatial and temporal expression patterns are critical for body patterning duringnormal development, and HOX genes also have critical postdevelopmental regulatoryfunctions. Aberrant HOX gene expression initiates different types of human diseases andcancers including leukaemia, breast cancer and brain tumours. Multiple HOX genes havebeen shown to be overexpressed in primary astrocytomas and GBM cell lines, whichsuggests an important role for these genes in gliomagenesis, glioma recurrence and drugresistance. However, the potential mechanisms underlying HOX activation, in addition totheir functional relevance in GBM cells, have not been determined. Of the39HOX genesin humans, the HOXA9genes encode critical transcriptional regulators of embryonicdevelopment and postdevelopmental stages that have been implicated in gliomas.However, the potential mechanisms with HOXA9activation and gliomas’ recurrent,TMZ-resistance and radiotherary (RT)-resistance also have not been explored.Therefore, to further understand the relationship between HOXA9and brain gliomasand the role of HOXA9in malignant gliomas’ proliferation and invasion, gliomas’recurrent, TMZ-resistance and RT-resistance, we performed four experiments as follows.1. The significance of HOXA9expression in human gliomasThe expression of HOXA9mRNA and protein were first investigated in five humanglioma cell lines (U87MG、U251、A172、T98G and BT325) by quantitative real time PCR(qRT-PCR) and western blot respectively. The results showed that the HOXA9was widelyexpressed in five human glioma cell lines, and the most were T98G and BT325cells. Andthen, we performed immunohistochemistry (IHC), qRT-PCR and western blot to detect theexpression of HOXA9on158primary glioma specimens,50recurrent glioma specimens and6non-neoplastic brain parenchyma specimens. In the158primary glioma specimens,50recurrent glioma specimens and6non-neoplastic brain parenchyma specimens by IHC,we found that HOXA9expression was significantly higher in gliomas than in normal brainparenchyma and was much more lower in more malignant gliomas than in less malignantgliomas (P<0.01). HOXA9expression was inversely correlated with survivin expression(r=0.906, P<0.01), large tumor diameter (r=0.507, P<0.01), pathological grade (r=0.683,P<0.01), extent of resection (r=0.152, P=0.029) and type of adjuvant treatment (r=0.366,P<0.01) in gliomas. Spearman’s rank correlation analysis did not show a statisticallysignificant correlation between HOXA9and patients’ gender (P=0.624), age (P=0.415)and frequent intra-tumorous necrosis (P=0.351). Based on IRS results, we divided theglioma specimens into two categories for the Kaplan-Meier survival analysis. HOXA9expression was associated with OS and PFS in all the glioma patients. Patients whosetumors high expressed HOXA9had a significantly shorter overall survival [OS; median,8months;95%confidence interval (CI),4-10months] compared with those whose tumorslow expression of HOXA9(median,44months; CI,36-47months; P<0.01). Similarly,patients whose tumors high expressed HOXA9had a significantly shorter progression-freesurvival (PFS; median,7months; CI,3-8months) compared with those whose tumors lowexpression of HOXA9(median,43months; CI,34-46months; P<0.01). Cox stepwiseproportional hazards model involving the IRS score of HOXA9and survivin expressionand eight clinical parameters (age, gender, pathologic grade, Kamofsky performance sore,largest tumor diameter, extent of resection, type of adjuvant treatment, intra-tumornecrosis) identified five prognostic variables including pathologic grade (P=0.000), KPS(P=0.000), extent of resection (P=0.015), largest tumor diameter (P=0.000) and HOXA9expression (P=0.007). Therefore, HOXA9activation is a novel, independent, and negativeprognostic marker in human gliomas. The results showed that the HOXA9locates innucleus and cytoplasm of glioma cells, especially in nucleus; A weak to strong range ofHOXA9staining with increasing pathologic grade of gliomas at the mRNA and proteinlevels, especially recurrent GBM.2. Construct HOXA9RNAi expression lentivirus to infect T98G cells, and effects of HOXA9down-regulation on T98G cells proliferation, apoptosis and invasion in vitroAccording to HOXA9cDNA sequence, the specific RNAi fragments targetingHOXA9gene were designed and synthesized, which were cloned into pLKO.1-HOXA9vector of HOXA9shRNA was constructed. After lentiviral packaging, HOXA9RNAiexpression lentivirus infected T98G cells. pLKO.1-HOXA9shRNA-1, pLKO.1-HOXA9shRNA-2and pLKO.1-NC were transfected into T98G cells. After puromycin selection,stably transfected cell lines including T98G-S1(T98G shRNA-1), T98G-S2(T98GshRNA-2) and T98G-NC (negative control group) were established. To detect expressionof HOXA9mRNA and protein in T98G-S1, T98G-S2and T98G-NC using qRT-PCR andWestern blot. The result showed that expressions of HOXA9in T98G cells weresignificantly inhibited. Based on T98G-S1, T98G-S2, and T98G-NC cells, we found thatthe downregulation of HOXA9gene expression significantly inhibited proliferation,migration and invasion of glioma cells.3. Overexpression of HOXA9is partially associated with MGMT-independentTMZ-resistance in glioblastoma cells via phosphoinositide-3-kinase pathways andNF-kappaB pathwaysIn the158primary glioma specimens,50recurrent glioma specimens and5gliomas’cells (U87MG, U251, A172, T98G and BT325) by qRT-PCR and western blot, we foundthat the expressions of MGMT mRNA and protein were very lower or hardly detected insome gliomas’ specimens and BT325cell, however, they were TMZ resistanced. Giventhat HOXA9plays an important role on BT325cells with TMZ-resistance through PI3Kpathways, we investigated whether knockdown of HOXA9or block of PI3K pathways canenhance BT325cells to TMZ-sensitivity and subsequently reverse TMZ-resistancethrough repressed HOXA9expression in T98G and BT325cells. The results showed thatknockdown of HOXA9was done with siRNA that resulted in at least70%inhibition ofHOXA9mRNA and protein expression72h posttransfection in T98G and BT325cells asassessed by PI3K signaling specific inhibitor LY294002, and LY294002also enhanced theprotein expression of NF-kappaB p65. Therefore, overexpression of HOXA9is associatedwith MGMT-independent TMZ-resistance in glioblastoma cells via PI3K pathways and NF-kappaB pathways.4. Overexpression of HOXA9is associated with RT-resistance in glioblastoma cells viaNF-kappaB pathwaysIn the158primary glioma and50recurrent glioma patients’ clinical follow-upparameters, we found that the patients with high-grade gliomas were higher RT-resistance.Studies showed that the NF-kappaB signaling pathway plays an important role in tumordevelopment and progression, and results in unsatisfactory treatment outcome. Inhibitionof the NF-kappaB signaling cascade may sensitize the resistant cancer cells toradiotherapy. Given it was an important role to RT-resistance in GBM by NF-kappaBsignaling pathways, knockdown of HOXA9by siRNA could be enhanced radiosensitive.When glioblastoma T98G and BT325cells upon radiated with4Gy treatment, we foundthat the mRNA and protein expression of NF-kappaB p65increased as well andknockdown of HOXA9was done with siRNA that resulted in at least70%inhibition ofNF-kappaB p65and I B mRNA and protein expression48h posttransfection in T98Gand BT325cells upon the same radiated treatment. These results support thatoverexpression of HOXA9is partially associated with RT-resistance in glioblastoma cellsvia NF-kappaB pathways.In sum, HOXA9gene may play an important role in the pathogenesis andproliferation of human gliomas. HOXA9is not only a negative prognostic biomarker ofbrain gliomas, but also a potential candidate target for gene therapy in malignant gliomas.更多还原