HOXA9基因在人脑胶质细胞瘤对放化疗耐受性的作用机制研究
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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。
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 gliomas
The 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 vitro
According 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 pathways
In 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 pathways
In 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.
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。
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 gliomas
The 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 vitro
According 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 pathways
In 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 pathways
In 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.
引文
1. Ohgaki H. Epidemiology of brain tumors. Methods Mol Biol,2009,472:323-342.
2. Pytel P, Lukas RV. Update on diagnostic practice: tumors of the nervous system.Arch Pathol Lab Med,2009,133:1062-1077.
3. Kanu OO, Mehta A, Di C, et al. Glioblastoma multiforme: a review of therapeutictargets. Expert Opin Ther Targets,2009,13:701-718.
4. Puchner MJ, Herrmann HD, Berger J, et al. Surgery, tamoxifen, carboplatin, andradiotherapy in the treatment of newly diagnosed glioblastoma patients. Journal ofneuro-oncology,2000,49(2):147-155.
5. Clarke J, Butowski N, Chang S. Recent advances in therapy for glioblastoma.Archives of neurology,2004,67(3):279-283.
6. Chang JE, Khuntia D, Robins HI, et al. Radiotherapy and radiosensitizers in thetreatment of glioblastoma multiforme. Clin Adv Hematol Oncol,2007,5(11):894-902,7-15.
7. Keime-Guibert F, Chinot O, Taillandier L, et al. Radiotherapy for glioblastoma inthe elderly. The New England journal of medicine,2007,356(15):1527-1535.
8. Spiegl-Kreinecker S, Pirker C, Filipits M, et al. O6-Methylguanine DNAmethyltransferase protein expression in tumor cells predicts outcome oftemozolomide therapy in glioblastoma patients. Neuro Oncol,2010,12:28-36.
9. Friedman HS, McLendon RE, Kerby T, et al. DNA mismatch repair andO6-alkylguanine-DNA alkyltransferase analysis and response to Temodal in newlydiagnosed malignant glioma. J Clin Oncol,1998,16(12):3851-3857.
10. Gilbert MR, Friedman HS, Kuttesch JF, et al. A phase II study of temozolomide inpatients with newly diagnosed supratentorial malignant glioma before radiationtherapy. Neuro-oncology,2002,4(4):261-267.
11. Abdel-Fattah R, Xiao A, Bomgardner D, et al. Differential expression of HOXgenes in neoplastic and non-neoplastic human astrocytes. J Pathol,2006,209:15-24.
12. Akam M. The molecular basis for metameric pattern in the Drosophilaembryo.Development,1987,101:1-22.
13. Pearson JC, Lemons D, McGinnis W. Modulating Hox gene functions during animalbody patterning.Nat Rev Genet,2005,6:893-904.
14. Satokata I, Benson G, Maas R. Sexually dimorphic sterility phenotypes inHoxa10-deficient mice. Nature,1995,374:460-463.
15. Takahashi Y, Hamada J, Murakawa K, et al. Expression profiles of39HOX genes innormal human adult organs and anaplastic thyroid cancer cell lines by quantitativereal-time RT-PCR system. Exp Cell Res,2004,293:144-153.
16. Yamamoto M, Takai D, Yamamoto F, et al. Comprehensive expression profiling ofhighly homologous39Hox genes in26different human adult tissues by themodified systematic multiplex RT-PCR method reveals tissue-specific expressionpattern that suggests an important role of chromosomal structure in the regulation ofHox gene expression in adult tissues. Gene Expr,2003,11:199-210.
17. Neville SE, Baigent SM, Bicknell AB, et al. Hox gene expression in adult tissueswith particular reference to the adrenal gland. Endocr Res,2002,28:669-673.
18. Morgan R. Hox genes: a continuation of embryonic patterning? Trends Genet,2005,22:67-69.
19. Drabkin HA, Parsy C, Ferguson K, et al. Quantitative HOX expression inchromosomally defined subsets of acute myelogenous leukemia. Leukemia,2002,16(2):186-195.
20. Bijl J, Krosl, J, Lebert-Ghali, et al. Evidence for Hox and E2A-PBX1collaborationin mouse T-cell leukemia. Oncogene,2008,27:6356-6364.
21. Raman V, Martensen SA, Reisman D, et al. Compromised HOXA5function canlimit p53expression in human breast tumours. Nature,2000,405:974-978.
22. Ni ZY, Lou W, Leman ES, et al. Inhibition of constitutively activated STAT3signaling pathway suppresses growth of prostate cancer cell. Cancer Res,2000,60:1225.
23. Vider BZ, Zimber A, Hirsch D, et al. Human colorectal carcinogenesis is associatedwith deregulation of homeobox gene expression. Biochem Biophys Res Commun,1997,232(3):742-748.
24. Buccoliero AM, Castiglione F, Deglinnocenti DR, et al. Hox-D genes expression inpediatric low-grade gliomas: real-time-PCR study. Cell Mol Neurobiol,2008,29:1-6.
25. Tabuse M, Ohta S, Ohashi Y, et al. Functional analysis of HOXD9in humangliomas and glioma cancer stem cells. Mol Cancer,2011,10:60.
26. Gaspar N, Marshall L, Perryman L, et al. MGMT-independent temozolomideresistance in pediatric glioblastoma cells associated with a PI3-kinase-mediatedHOX/stem cell gene signature. Cancer Res,2010,70:9243-9252.
27. Murat A, Migliavacca E, Gorlia T, et al. Stem cell-related "self-renewal" signatureand high epidermal growth factor receptor expression associated with resistance toconcomitant chemoradiotherapy in glioblastoma. J Clin Oncol,2008,26:3015-3024.
28. Bodey B, Bodey B Jr, Siegel SE, et al. Immunocytochemical detection of thehomeobox B3, B4, and C6gene products in childhood medulloblastomas/primitiveneuroectodermal tumors. Anticancer Res,2000,20:1769-1780.
29. Louis DN, Ohgaki H, Wiestler OD, et a1. The2007WHO classification of tumoursof the central nervous system.Acta Neuropathol,2007,114:97-109．
30. Wirtz CR, Albert FK, Schwaderer M, Heuer C, Staubert A, Tronnier VM, Knauth M,Kunze S. The benefit of neuronavigation for neurosurgery analyzed by its impact onglioblastoma surgery.Neurological research,2000,22(4):354-60.
31. Helseth R, Helseth E, Johannesen TB, et al. Overall survival, prognostic factors, andrepeated surgery in a consecutive series of516patients with glioblastomamultiforme. Acta neurologica Scandinavica,2010,122(3):159-67.
32. Choudhari KA. Surgery for glioblastoma and influence of technology on outcomesbetween developed and developing parts of the world.Surgical neurology,2010,73(2): e16.
33. Stummer W, Stocker S, Wagner S, et a1. Intraoperative Detection of MalignantGliomas by5--Aminolevulinic Acid-induced Porphyrin Fluorescence．Neurosurgery,1998,42:518.
34. Stummer W, Pichlmeier U, Meinel T, et al. Fluorescence-guided surgery with5-aminolevulinic acid for resection of malignantglioma: a randomised controlledmuhicentre phase III trial. Lancet Oncology,2006,7:392．
35. Utsuki S，Oka H, Sato S, et al. Histological examination of false positive tissueresection using5-aminolevulinic acid-induced fluorescence guidance. NeurologiaMedico-Chirurgica,2007,47:210.
36. Eljamel MS, Goodman C, Moseley H. AIA and Photofrin(R) fluorescence-guidedresection and repetitive PDT in glioblastoma multiforme：a single centre Phase IIIrandomised controlled trial. Lasers in Medical Science,2008,23:361.
37. Valdes PA, Fan XY, Ji SB, et al. Estimation of Brain Deformation for VolumetricImage Updating in Protoporphyrin IX F1uorescence Guided Resection. Stereotacticand Functional Neurosurgery,2010,88:1.
38. Feigl GC, Ritz R, Moraes M, et al. Resection ofmalignant brain tumors in eloquentcortical areas: a new multimodal approach combining5-aminolevulinic acid andintraoperative monitoring Clinical article. Joumal of Neurosurgery,2010,113:352.
39. Utsuki S, Oka H, Miyajima Y, et al. Auditory alert system for fluorescence-guidedresection of gliomas. Neurologia Medico-Chirurgica,2008,48:95.
40. Hall EJ. Radiobiology for radiologist.4th edition. Philadelphia: JB Lippincott,1994,p1-13.
41. Walker MD, Strike TA, Sheline GE. An analysis of dose-effect relationship in theradiotherapy of malignant gliomas.Int J Radiat Oncol Biol Phys1979,5:1725-1731.
42. Kristiansen K, Hagen S, Kollevold T, et al. Combined modality therapy of operatedastrocytomas grade III and IV. Confirmation of the value of postoperative irradiationand lack of potentiation of bleomycin on survival time: a prospective multicentertrial of the Scandinavian Glioblastoma Study Group. Cancer1981,47:649-652.
43. Wallner KE, Galicich JH, Krol G, Arbit E, Malkin MG. Patterns of failure followingtreatment for glioblastoma multiforme and anaplastic astrocytoma. Internationaljournal of radiation oncology, biology, physics,1989,16(6):1405-1409.
44. Hochberg FH, Pruitt A. Assumptions in the radiotherapy of glioblastoma. Neurology,1980,30(9):907-911.
45. Shapiro WR, Green SB, Burger PC, et al, Strike TA, et al. Randomized trial of threechemotherapy regimens and two radiotherapy regimens and two radiotherapyregimens in postoperative treatment of malignant glioma.Brain Tumor CooperativeGroup Trial8001. Journal of neurosurgery,1989,71(1):1-9.
46. Cardinale R, Won M, Choucair A, et al. A phase II trial of accelerated radiotherapyusing weekly stereotactic conformal boost for supratentorial glioblastomamultiforme: RTOG0023. International journal of radiation oncology, biology,physics,2006,65(5):1422-1428.
47. Beauchesne P, Bernier V, Carnin C, et al. Prolonged survival for patients with newlydiagnosed, inoperable glioblastoma with3-times daily ultrafractionated radiationtherapy. Neuro Oncol,2010,12(6):595-602.
48. Beauchesne PD, Taillandier L, Bernier V, et al. Concurrent radiotherapy:fotemustine combination for newly diagnosed malignant glioma patients, a phase IIstudy. Cancer Chemother Pharmacol,2009,64(1):171-175.
49. Nwokedi EC, DiBiase SJ, Jabbour S, et al. Gamma knife stereotactic radiosurgeryfor patients with glioblastoma multiforme. Neurosurgery,2002,50:41-46
50. Souhami L, Seiferheld W, Brachman D, et al. Randomized comparison ofstereotactic radiosurgery followed by conventional radiotherapy with carmustine toconventional radiotherapy with carmustine for patient with glioblastoma multiforme:report of Radiation Therapy Oncology Group93-05protocol. Int J Radiat OncolBiol Phys,2004,60(3):853-860.
51. Kong DS, Lee JI, Park K, et al. Efficacy of stereotactic radiosurgery as a salvagetreatment for recurrent malignant gliomas. Cancer,2008,112:2046-2051.
52. Graf R, Hildebrandt B, Tilly W, et al. Dose-escalated conformal radiotherapy ofglioblastomas--results of a retrospective comparison applying radiation doses of60and70Gy. Onkologie,2005,28(6-7):325-330.
53. Corn BW, Wang M, Fox S, et al. Health related quality of life and cognitive status inpatients with glioblastoma multiforme receiving escalating doses of conformal threedimensional radiation on RTOG98-03. J Neurooncol,2009,95(2):247-257.
54. Piroth MD, Pinkawa M, Holy R, et al. Integrated-boost IMRT or3-D-CRT usingFET-PET based auto-contoured target volume delineation for glioblastomamultiforme--a dosimetric comparison. Radiat Oncol,2009,23(4):57.
55. Panet-Raymond V, Ansbacher W, Zavgorodni S, et al. Coplanar versus noncoplanarintensity-modulated radiation therapy (IMRT) and volumetric-modulated arctherapy (VMAT) treatment planning for fronto-temporal high-grade glioma. J ApplClin Med Phys,2012,13(4):3826.
56. Davidson MT, Masucci GL, Follwell M, et al. Single arc volumetric modulated arctherapy for complex brain gliomas: is there an advantage as compared to intensitymodulated radiotherapy or by adding a partial arc? Technol Cancer Res Treat,2012,11(3):211-220.
57. Stupp R, Mason WP, van den Bent MJ, et al. Radiotherapy plus concomitant andadjuvant temozolomide for glioblastoma. N Engl J Med,2005,352:987-996.
58. van Nifterik KA, van den Berg J, Stalpers LJ, et al. Differential radiosensitizingpotential of temozolomide in MGMT promoter methylated glioblastoma multiformecell lines. Int J Radiat Oncol Biol Phys,2007,69:1246-1253.
59. Nelson DF, Diener-West M, Weinstein AS, et al. A randomized comparison ofmisonidazole sensitized radiotherapy plus BCNU and radiotherapy plus BCNU fortreatment of malignant glioma after surgery: final report of an RTOG study. Int JRadiat Oncol Biol Phys,1986,12:1793-1800.
60. Prados MD, Seiferheld W, Sandler HM, et al. Phase III randomized study ofradiotherapy plus procarbazine, lomustine, and vincristine with or without BUdRfor treatment of anaplastic astrocytoma: final report of RTOG9404. Int J RadiatOncol Biol Phys,2004,58:1147-1152.
61. Anderson B, Anderson B Jr. Necrotizing uveitis incident to perfusion of intractraialmalignancies with nitrogen mustard or related compounds. Trans Am OphthalmolSoc,1960,58:95-104.
62. Galanis E, Buekner JC, Burch PA, et al. PhaseⅡtrial of nitrogen mustard,vincristine and procarbazine in patients with recurrent glioma: North Central CancerTreatment Group results. J Clin Oncol,1998,16:2953-2958.
63. Stewart LA. Chemotherapy in adult high-grade glioma: a systematic review andmeta-analysis of individual patient data from12randomised trials. Lancet,2002,359(9311):1011-1018.
64. Chang CH, Horton J, Schoenfeld D, et al. Comparison of postoperative radiotherapyand combined postoperative radiotherapy and chemotherapy in the multidisciplinarymanagement of malignant gliomas, A joint Radiation Therapy Oncology Group andEastern Cooperative Oncology Group study. Cancer,1983,52:997-1007.
65. Walker MD, Alexander E Jr, Hunt WE, et al. Evaluation of BCNU and/orradiotherapy in the treatment of anaplastic gliomas. A cooperative clinical trial.JNeurosurg,1978,49:333-343.
66. Ushio Y, Hayakawa T, Hasegawa H, et al. Chemotherapy of malignant gliomas.Neumsurg Rev,1984,7:3-12.
67. Komblith PL. Walker M. Chemotherapy for malignant gliomas. J Neurosurg,1988,68:1-17.
68. Lassman LP, Pearce GW, Gang J. Sensitivity of intraeranial gliomas to vincristinesulphate.Lancet,1965,1:296-298.
69. Kaneko S, Abe H, Aida T, et al. Evaluation of radiation immunochemotherapy in thetreatment of malignant glioma. Combined use of ACNU,VCR and PS-K. HokkaidoIgaku Zasshi,1983,58:622-630.
70. Levin VA, Uhm JH, Jaeckle KA, et al. Phase III randomized study ofpostradiotherapy chemotherapy with alpha-difluoromethylornithine-procarbazine,N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosurea, vincristine (DFMO-PCV) versusPCV for glioblastoma multiforme. Clin Cancer Res,2000,6(10):3878-3884.
71. Levin VA, Lamborn K, Wara W, et al. Phase II study of6-thioguanine, procarbazine,dibromodulcitol, lomustine, and vincristine chemotherapy with radiotherapy fortreating malignant glioma in children. Neuro-oncology,2000,2(1):22-28.
72. Hildebrand JG. Chemotherapy of malignant glioma. Results of studies of theEORTC group of brain tumors. Rev Neurol (Paris),1992,148:435-440.
73. Jeremic B, Jovanovic D, Djuric LJ, et al. Advantage of post radiotherapychemotherapy with CCNU, procarbazine and vineristine(mPCV) over chemotherapywith VM-26and CCNU for malignant gliomas. J Chemother,1992,4:123-126.
74. Ushio Y, Abe H, Suzuki J, Tanaka R, et al. Evaluation of ACNU alone andcombined with tegafur as additions to radiotherapy of the treatment of malignantgliomas-a cooperative clinical trial. No To Shinkei,1985,37:999-1006.
75. Shapiro WR, Green SB, Burger PC, et al. A randomized comparison of intra-arterialversus intravenous BCNU,with or without intravenous5-fluomuracil,for newlydiagnosed patients with malignant glioma. J Neurosurg,1992,76:772-781.
76. Menei P, Boisdron-Celle M, Croue A, et al. Effect of stereotactic implantation ofbiodegradable5-fluorouracil-loaded microspheres in healthy and C6glioma-bearingrats. Neurosurgery,1996,39:117-123．
77. Stewart DJ, Richard MT, Benoit B, et al. Cisplatin plus cytosine arabinoside inadults with malignant gliomas. J Neurooncol,1984,2:29-34.
78. Stevens MF, Hickman JA, Stone R, et al. Antitumor imidazotetrazines.1. Synthesisand chemistry of8-carbamoyl-3-(2-chloroethy1)imidazo[5,1-d]-1,2,3,5-tetrazin-4(3H)-one，a novel broad-spectrum antitumor agent. J Med Chem,1984,27:196-201.
79. van Rijn J, Heimans JJ, van den Berg J, et al. Survival of human glioma cells treatedwith various combination of temozolomide and X-rays. International journal ofradiation oncology, biology, physics,2000,47(3):779-784.
80. Wedge SR, Porteous JK, Glaser MG, et al. In vitro evaluation of temozolomidecombined with X-irradiation. Anti-cancer drugs,1997,8(1):92-97.
81.0’Reilly SM, Newlands ES, Glaser MG, et al. Temozolomide: a new oral cytotoxicchemotherapeutic agent with promising activity against primary brain tumours. EurJ Cancer,1993,29A:940-942.
82. Stupp R, Mason WP, van den Bent MJ, et al. Radiotherapy plus concomitant andadjuvant temozolomide for glioblastoma. The New England journal of medicine,2005,352(10):987-996.
83. Ali-Osman F, Brunner JM, Kutluk TM, et al. Prognostic signifi cance ofglutathione S-transferase pi expression and subcellular localization in humangliomas. Clin Cancer Res,1997,3:2253-2261.
84. Goto S, Kamada K, Soh Y, Ihara Y, et al. Significance of nuclear glutathioneS-transferase pi in resistance to anti-cancer drugs. Jpn J Cancer Res,2002,93:1047-1056.
85. Batchelor T. Temozolomide for malignant brain tumours. Lancet,2000,355(9210):1115-6.
86. MacConnachie AM. Temozolomide (Temodal) for treatment of primary braintumours. Intensive Crit Care Nurs,2000,16(1):59-60.
87. Becker K, Gregel C, Fricke C, et al. DNA repair protein MGMT protects againstN-methyl-N-nitrosourea-induced conversion of benign into malignant tumors.Carcinogenesis,2003,24(3):541-546.
88. Omar AI, Mason WP. Temozolomide: The evidence for its therapeutic efficacy inmalignant astrocytomas. Core evidence,2010,4:93-111.
89. Hegi ME, Diserens AC, Gorlia T, et al. MGMT gene silencing and benefit fromtemozolomide in glioblastoma. N Engl J Med,2005,352:997-1003.
90. Fukushima T, Takeshima H, Kataoka H. Anti-glioma therapy with temozolomideand status of the DNA-repair gene MGMT．Anticancer Res,2009,29:4845-4854.
91. Lai A, Tran A, Nghiemphu PL, et al. Phase II study of bevacizumab plustemozolomide during and after radiation therapy for patients with newly diagnosedglioblastoma multiforme. J Clin Oncol,2011,29(2):142-148.
92. Vredenburghj J,Desjardins A，Herndon J End, et al. Phase II trial of bevacizumabandirinotecan inrecurrent malignant glioma. Clin Cancer Res,2007,13:1253-1259.
93. Shah N, Sukumar S. The Hox genes and their roles in oncogenesis.Nat Rev Cancer,2010,10(5):361-371.
94. McGinnis W, Garber RL, Wirz J, et al.A homologous protein-coding sequence inDrosophila homeotic genes and its conservation in other metazoans. Cell,1984,37(2):403-408.
95. Soubeyran P, Haglund K, Garcia S, et al. Homeobox gene Cdx1regulates Ras,Rhoand PI3kinase pathways leading to transformation and tumorigenesis of intestinalepithelial cells. Oncogene,2001,20(31):4180-4187.
96. Ford, H L.Homeobox genes: a link between development, cell cycle, and cancer?Cell Biol Int,1998,22(6):397-400.
97. Maroulakou IG, Spyropoulos DD. The study of HOX gene function inhematopoietic, breast and lung carcinogenesis.Anticancer Res,2003,23(3A):2101-2110.
98. Fanti L, Perrini B, Piacentini L, et al. The trithorax group and Pc group proteins aredifferentially involved in heterochromatin formation in Drosophila. Chromosoma,2008,117:25-39.
99. Hanson RD, Hess JL, Yu BD, et al. Mammalian Trithorax and polycomb-grouphomologues are antagonistic regulators of homeotic development. Proc. Natl Acad.Sci,1999,96:14372-14377.
100. Weiss FU, Marques IJ, Woltering JM, et al. Retinoic acid receptor (RAR)antagonists inhibit miR-10a expression and block metastatic be haviour ofpancreatic cancer. Gastroenterology,2009,137:2136-2145.
101. Popovic R, Riesbeck LE, Velu CS, et al. Regulation of mir-196b by MLL and itsoverexpression by MLL fusions contributes to immortalization. Blood,2009,113:3314-3322.
102. Chopra VS, Mishra RK.“Mir”acles in hox gene regulation. Bioessays,2006,28:445-448.
103. Han L, Witmer PD, Casey E, et al. DNA methylation regulates MicroRNAexpression. Cancer Biol Ther,2007,6:1284-1288.
104. Sessa L, Breiling A, Lavorgna G, et al. Noncoding RNA synthesis and loss ofPolycomb group repression accompanies the collinear activation of the humanHOXA cluster. RNA,2007,13:223-239.
105. Petruk S, Sedkov Y, Brock HW, et al. A model for initiation of mosaic HOX geneexpression patterns by non-coding RNAs in early embryos. RNA Biol,2007,4:1-6.
106. Rinn JL, Kertesz M, Wang JK, et al. Functional demarcation of active and silentchromatin domains in human HOX loci by noncoding RNAs. Cell,2007,129:1311-1323.
107. Nieto M A, Gilardi-Hebenstreit P, Charnay P, et al. A receptor protein tyrosinekinase implicated in the segmental patterning of the hindbrain and mesoderm.Development,1992,116(4):1137-1150.
108. Riddle R D, Johnson R L, Laufer E, et al. Sonic hedgehog mediates the polarizingactivity of the ZPA.Cell,1993,75(7):1401-1416.
109. Trivedi CM, Patel RC&Patel CV. Homeobox gene HOXA9inhibits nuclearfactor-κB dependent activation of endothelium. Atherosclerosis,2007,195: e50-e60.
110. Trivedi CM, Patel RC&Patel CV. Differential regulation of HOXA9expression bynuclear factor kappaB (NF-κB) and HOXA9. Gene,2008,408:187-195.
111. Manohar CF, Salwen HR, Furtado MR, et al. Upregulation of HOXC6, HOXD1,and HOXD8homeobox gene expression in human neuroblastoma cells followingchemical induction of differentiation. Tumour Biol,1996,17:34-47.
112. Zhang X, Hamada J, Nishimoto A, et al. HOXC6and HOXC11increasetranscription of S100beta gene in BrdU-induced in vitro differentiation of GOTOneuroblastoma cells into Schwannian cells. J Cell Mol Med,2007,11:299-306.
113. Jung C, Kim RS, Lee SJ, et al. HOXB13homeodomain protein suppresses thegrowth of prostate cancer cells by the negative regulation of T-cell factor4. CancerRes,2004,64:3046-3051.
114. Jung C, Kim RS, Zhang HJ, et al. HOXB13induces growth suppression of prostatecancer cells as a repressor of hormone activated androgen receptor signaling. CancerRes,2004,64:9185-9192.
115. Cheng W, Liu J, Yoshida H, et al. Lineage infidelity of epithelial ovarian cancers iscontrolled by HOX genes that specify regional identity in the reproductive tract.Nature Med,2005,11:531-537.
116. Dubeau L. The cell of origin of ovarian epithelialtumors and the ovarian surfaceepithelium dogma: does the emperor have no clothes? Gynecol Oncol,1999,72:437-442.
117. Lawrence HJ, Sauvageau G, Humphries RK, et al. The role of HOX homeoboxgenes in normal and leukemic hematopoiesis. Stem Cells,1996,14:281-291.
118. Bijl J, van Oostveen JW, Kreike M, et al. Expression of HOXC4, HOXC5, andHOXC6in human lymphoid cell lines, leukemias, and benign and malignantlymphoid tissue. Blood,1996,87:1737-1745.
119. Wu X, Chen H, Parker B, et al. HOXB7, a homeodomain protein, is overexpressed inbreast cancer and confers epithelialmesenchymal transition. Cancer Res,2006,66:9527-9534.
120. Rubin E, Wu X, Zhu T, Cheung JC, et al. A role for the HOXB7homeodomainprotein in DNA repair. Cancer Res,2007,67:1527-1535.
121. Chirnomas D, Taniguchi T, de la Vega M, et al. Chemosensitization to cisplatinbyinhibitors of the Fanconi anemia/BRCA pathway. Mol Cancer Ther,2006,5:952-961.
122. Raman V, Martensen SA, Reisman D, et al. Compromised HOXA5function canlimit p53expression in human breast tumours. Nature,2000,405:974-978.
123. Chen H, Chung S, Sukumar S. HOXA5-induced apoptosis in breast cancer cells ismediated by caspases2and8. Mol Cell Biol,2004,24:924-935.
124. Chu MC, Selam FB, Taylor HS. HOXA10regulates p53expression and matrigelinvasion in human breast cancer cells. Cancer Biol Ther,2004,3:568-572.
125. Morgan R, Pirard PM, Shears L, et al. Antagonism of HOX/PBX dimer formationblocks the in vivo proliferation of melanoma. Cancer Res,2007,67:5806-5813.
126. Plowright L, Harrington KJ, Pandha HS, et al. HOX transcription factors arepotential therapeutic targets in non-small-cell lung cancer (targeting HOX genes inlung cancer). Br J Cancer,2009,100:470-475.
127. Lawrence HJ and Largman C. Homeobox genes in normal hematopoiesis andleukemia. Blood,1992,80:2445-2453.
128. Argiropoulos B and Humphries RK. Hox genes in hematopoiesis andleukemogenesis. Oncogene,2007,26:6766-6776.
129. Calvo KR, Sykes DB, Pasillas MP, et al. Nup98-HoxA9immortalizes myeloidprogenitors, enforces expression of Hoxa9, Hoxa7and Meis1, and alterscytokine-specific responses in a manner similar to that induced by retroviralco-expression of Hoxa9and Meis1. Oncogene,2002,21:4247-4256.
130. Hu YL, Fong S, Ferrell C, et al. HOXA9modulates its oncogenic partner Meis1toinfluence normal hematopoiesis. Mol Cell Biol,2009,29:5181-5192.
131. Whelan JT, Ludwig DL, Bertrand FE. HoxA9induces insulin-like growth factor-1receptor expression in B-lineage acute lymphoblastic leukemia. Leukemia,2008,22:1161-1169
132. Costa BM, Smith JS, Chen Y, et al. Reversing HOXA9oncogene activation by PI3Kinhibition: epigenetic mechanism and prognostic significance in humanglioblastoma. Cancer Res,2010,70(2):453-62.
133. Cha TL, Zhou BP, Xia W, et al. Akt-mediated phosphorylation of EZH2suppressesmethylation of lysine27in histone H3. Science,2005,310:306-10.
134. Rinn JL, Kertesz M, Wang JK, et al. Functional demarcation of activeand silentchromatin domains in human HOX loci by noncodingRNAs. Cell,2007,129:1311-1323.
135. Lin H, Wang Y, Zhang X, et al: Prognostic significance of kappaB-Ras1expressionin gliomas. Med Oncol,2012,29(2):1272-1296.
136. Lin H, Xiong W, Zhang X, et al. Notch-1activation-dependent p53restorationcontributes to resveratrol-induced apoptosis in glioblastoma cells. Oncol Rep,2011,26(4):925-930.
137. Huang H, Lin H, Zhang X, Li J. Resveratrol reverses temozolomide resistance bydownregulation of MGMT in T98G glioblastoma cells by the NF-κB-dependentpathway. Oncol Rep,2012,27(6):2050-2056.
138. Yan T, Skaftnesmo KO, Leiss L, et al. Neuronal markers are expressed in humangliomas and NSE knockdown sensitizes glioblastoma cells to radiotherapy andtemozolomide. BMC Cancer,2011,11:524.
139. Ghosh S, Karin M. Missing pieces in the NF-kB puzzle. Cell,2002;109: S81-96.
140. Aggarwal BB. Nuclear factor-kB: the enemy within. Cancer Cell,2004,6(3):203-208.
141. Lin Y, Bai L, Chen W, Xu S. The NF-kB activation pathways, emerging moleculartargets for cancer prevention and therapy.Ther Targets,2010,14(1):45-55.
2. Pytel P, Lukas RV. Update on diagnostic practice: tumors of the nervous system.Arch Pathol Lab Med,2009,133:1062-1077.
3. Kanu OO, Mehta A, Di C, et al. Glioblastoma multiforme: a review of therapeutictargets. Expert Opin Ther Targets,2009,13:701-718.
4. Puchner MJ, Herrmann HD, Berger J, et al. Surgery, tamoxifen, carboplatin, andradiotherapy in the treatment of newly diagnosed glioblastoma patients. Journal ofneuro-oncology,2000,49(2):147-155.
5. Clarke J, Butowski N, Chang S. Recent advances in therapy for glioblastoma.Archives of neurology,2004,67(3):279-283.
6. Chang JE, Khuntia D, Robins HI, et al. Radiotherapy and radiosensitizers in thetreatment of glioblastoma multiforme. Clin Adv Hematol Oncol,2007,5(11):894-902,7-15.
7. Keime-Guibert F, Chinot O, Taillandier L, et al. Radiotherapy for glioblastoma inthe elderly. The New England journal of medicine,2007,356(15):1527-1535.
8. Spiegl-Kreinecker S, Pirker C, Filipits M, et al. O6-Methylguanine DNAmethyltransferase protein expression in tumor cells predicts outcome oftemozolomide therapy in glioblastoma patients. Neuro Oncol,2010,12:28-36.
9. Friedman HS, McLendon RE, Kerby T, et al. DNA mismatch repair andO6-alkylguanine-DNA alkyltransferase analysis and response to Temodal in newlydiagnosed malignant glioma. J Clin Oncol,1998,16(12):3851-3857.
10. Gilbert MR, Friedman HS, Kuttesch JF, et al. A phase II study of temozolomide inpatients with newly diagnosed supratentorial malignant glioma before radiationtherapy. Neuro-oncology,2002,4(4):261-267.
11. Abdel-Fattah R, Xiao A, Bomgardner D, et al. Differential expression of HOXgenes in neoplastic and non-neoplastic human astrocytes. J Pathol,2006,209:15-24.
12. Akam M. The molecular basis for metameric pattern in the Drosophilaembryo.Development,1987,101:1-22.
13. Pearson JC, Lemons D, McGinnis W. Modulating Hox gene functions during animalbody patterning.Nat Rev Genet,2005,6:893-904.
14. Satokata I, Benson G, Maas R. Sexually dimorphic sterility phenotypes inHoxa10-deficient mice. Nature,1995,374:460-463.
15. Takahashi Y, Hamada J, Murakawa K, et al. Expression profiles of39HOX genes innormal human adult organs and anaplastic thyroid cancer cell lines by quantitativereal-time RT-PCR system. Exp Cell Res,2004,293:144-153.
16. Yamamoto M, Takai D, Yamamoto F, et al. Comprehensive expression profiling ofhighly homologous39Hox genes in26different human adult tissues by themodified systematic multiplex RT-PCR method reveals tissue-specific expressionpattern that suggests an important role of chromosomal structure in the regulation ofHox gene expression in adult tissues. Gene Expr,2003,11:199-210.
17. Neville SE, Baigent SM, Bicknell AB, et al. Hox gene expression in adult tissueswith particular reference to the adrenal gland. Endocr Res,2002,28:669-673.
18. Morgan R. Hox genes: a continuation of embryonic patterning? Trends Genet,2005,22:67-69.
19. Drabkin HA, Parsy C, Ferguson K, et al. Quantitative HOX expression inchromosomally defined subsets of acute myelogenous leukemia. Leukemia,2002,16(2):186-195.
20. Bijl J, Krosl, J, Lebert-Ghali, et al. Evidence for Hox and E2A-PBX1collaborationin mouse T-cell leukemia. Oncogene,2008,27:6356-6364.
21. Raman V, Martensen SA, Reisman D, et al. Compromised HOXA5function canlimit p53expression in human breast tumours. Nature,2000,405:974-978.
22. Ni ZY, Lou W, Leman ES, et al. Inhibition of constitutively activated STAT3signaling pathway suppresses growth of prostate cancer cell. Cancer Res,2000,60:1225.
23. Vider BZ, Zimber A, Hirsch D, et al. Human colorectal carcinogenesis is associatedwith deregulation of homeobox gene expression. Biochem Biophys Res Commun,1997,232(3):742-748.
24. Buccoliero AM, Castiglione F, Deglinnocenti DR, et al. Hox-D genes expression inpediatric low-grade gliomas: real-time-PCR study. Cell Mol Neurobiol,2008,29:1-6.
25. Tabuse M, Ohta S, Ohashi Y, et al. Functional analysis of HOXD9in humangliomas and glioma cancer stem cells. Mol Cancer,2011,10:60.
26. Gaspar N, Marshall L, Perryman L, et al. MGMT-independent temozolomideresistance in pediatric glioblastoma cells associated with a PI3-kinase-mediatedHOX/stem cell gene signature. Cancer Res,2010,70:9243-9252.
27. Murat A, Migliavacca E, Gorlia T, et al. Stem cell-related "self-renewal" signatureand high epidermal growth factor receptor expression associated with resistance toconcomitant chemoradiotherapy in glioblastoma. J Clin Oncol,2008,26:3015-3024.
28. Bodey B, Bodey B Jr, Siegel SE, et al. Immunocytochemical detection of thehomeobox B3, B4, and C6gene products in childhood medulloblastomas/primitiveneuroectodermal tumors. Anticancer Res,2000,20:1769-1780.
29. Louis DN, Ohgaki H, Wiestler OD, et a1. The2007WHO classification of tumoursof the central nervous system.Acta Neuropathol,2007,114:97-109．
30. Wirtz CR, Albert FK, Schwaderer M, Heuer C, Staubert A, Tronnier VM, Knauth M,Kunze S. The benefit of neuronavigation for neurosurgery analyzed by its impact onglioblastoma surgery.Neurological research,2000,22(4):354-60.
31. Helseth R, Helseth E, Johannesen TB, et al. Overall survival, prognostic factors, andrepeated surgery in a consecutive series of516patients with glioblastomamultiforme. Acta neurologica Scandinavica,2010,122(3):159-67.
32. Choudhari KA. Surgery for glioblastoma and influence of technology on outcomesbetween developed and developing parts of the world.Surgical neurology,2010,73(2): e16.
33. Stummer W, Stocker S, Wagner S, et a1. Intraoperative Detection of MalignantGliomas by5--Aminolevulinic Acid-induced Porphyrin Fluorescence．Neurosurgery,1998,42:518.
34. Stummer W, Pichlmeier U, Meinel T, et al. Fluorescence-guided surgery with5-aminolevulinic acid for resection of malignantglioma: a randomised controlledmuhicentre phase III trial. Lancet Oncology,2006,7:392．
35. Utsuki S，Oka H, Sato S, et al. Histological examination of false positive tissueresection using5-aminolevulinic acid-induced fluorescence guidance. NeurologiaMedico-Chirurgica,2007,47:210.
36. Eljamel MS, Goodman C, Moseley H. AIA and Photofrin(R) fluorescence-guidedresection and repetitive PDT in glioblastoma multiforme：a single centre Phase IIIrandomised controlled trial. Lasers in Medical Science,2008,23:361.
37. Valdes PA, Fan XY, Ji SB, et al. Estimation of Brain Deformation for VolumetricImage Updating in Protoporphyrin IX F1uorescence Guided Resection. Stereotacticand Functional Neurosurgery,2010,88:1.
38. Feigl GC, Ritz R, Moraes M, et al. Resection ofmalignant brain tumors in eloquentcortical areas: a new multimodal approach combining5-aminolevulinic acid andintraoperative monitoring Clinical article. Joumal of Neurosurgery,2010,113:352.
39. Utsuki S, Oka H, Miyajima Y, et al. Auditory alert system for fluorescence-guidedresection of gliomas. Neurologia Medico-Chirurgica,2008,48:95.
40. Hall EJ. Radiobiology for radiologist.4th edition. Philadelphia: JB Lippincott,1994,p1-13.
41. Walker MD, Strike TA, Sheline GE. An analysis of dose-effect relationship in theradiotherapy of malignant gliomas.Int J Radiat Oncol Biol Phys1979,5:1725-1731.
42. Kristiansen K, Hagen S, Kollevold T, et al. Combined modality therapy of operatedastrocytomas grade III and IV. Confirmation of the value of postoperative irradiationand lack of potentiation of bleomycin on survival time: a prospective multicentertrial of the Scandinavian Glioblastoma Study Group. Cancer1981,47:649-652.
43. Wallner KE, Galicich JH, Krol G, Arbit E, Malkin MG. Patterns of failure followingtreatment for glioblastoma multiforme and anaplastic astrocytoma. Internationaljournal of radiation oncology, biology, physics,1989,16(6):1405-1409.
44. Hochberg FH, Pruitt A. Assumptions in the radiotherapy of glioblastoma. Neurology,1980,30(9):907-911.
45. Shapiro WR, Green SB, Burger PC, et al, Strike TA, et al. Randomized trial of threechemotherapy regimens and two radiotherapy regimens and two radiotherapyregimens in postoperative treatment of malignant glioma.Brain Tumor CooperativeGroup Trial8001. Journal of neurosurgery,1989,71(1):1-9.
46. Cardinale R, Won M, Choucair A, et al. A phase II trial of accelerated radiotherapyusing weekly stereotactic conformal boost for supratentorial glioblastomamultiforme: RTOG0023. International journal of radiation oncology, biology,physics,2006,65(5):1422-1428.
47. Beauchesne P, Bernier V, Carnin C, et al. Prolonged survival for patients with newlydiagnosed, inoperable glioblastoma with3-times daily ultrafractionated radiationtherapy. Neuro Oncol,2010,12(6):595-602.
48. Beauchesne PD, Taillandier L, Bernier V, et al. Concurrent radiotherapy:fotemustine combination for newly diagnosed malignant glioma patients, a phase IIstudy. Cancer Chemother Pharmacol,2009,64(1):171-175.
49. Nwokedi EC, DiBiase SJ, Jabbour S, et al. Gamma knife stereotactic radiosurgeryfor patients with glioblastoma multiforme. Neurosurgery,2002,50:41-46
50. Souhami L, Seiferheld W, Brachman D, et al. Randomized comparison ofstereotactic radiosurgery followed by conventional radiotherapy with carmustine toconventional radiotherapy with carmustine for patient with glioblastoma multiforme:report of Radiation Therapy Oncology Group93-05protocol. Int J Radiat OncolBiol Phys,2004,60(3):853-860.
51. Kong DS, Lee JI, Park K, et al. Efficacy of stereotactic radiosurgery as a salvagetreatment for recurrent malignant gliomas. Cancer,2008,112:2046-2051.
52. Graf R, Hildebrandt B, Tilly W, et al. Dose-escalated conformal radiotherapy ofglioblastomas--results of a retrospective comparison applying radiation doses of60and70Gy. Onkologie,2005,28(6-7):325-330.
53. Corn BW, Wang M, Fox S, et al. Health related quality of life and cognitive status inpatients with glioblastoma multiforme receiving escalating doses of conformal threedimensional radiation on RTOG98-03. J Neurooncol,2009,95(2):247-257.
54. Piroth MD, Pinkawa M, Holy R, et al. Integrated-boost IMRT or3-D-CRT usingFET-PET based auto-contoured target volume delineation for glioblastomamultiforme--a dosimetric comparison. Radiat Oncol,2009,23(4):57.
55. Panet-Raymond V, Ansbacher W, Zavgorodni S, et al. Coplanar versus noncoplanarintensity-modulated radiation therapy (IMRT) and volumetric-modulated arctherapy (VMAT) treatment planning for fronto-temporal high-grade glioma. J ApplClin Med Phys,2012,13(4):3826.
56. Davidson MT, Masucci GL, Follwell M, et al. Single arc volumetric modulated arctherapy for complex brain gliomas: is there an advantage as compared to intensitymodulated radiotherapy or by adding a partial arc? Technol Cancer Res Treat,2012,11(3):211-220.
57. Stupp R, Mason WP, van den Bent MJ, et al. Radiotherapy plus concomitant andadjuvant temozolomide for glioblastoma. N Engl J Med,2005,352:987-996.
58. van Nifterik KA, van den Berg J, Stalpers LJ, et al. Differential radiosensitizingpotential of temozolomide in MGMT promoter methylated glioblastoma multiformecell lines. Int J Radiat Oncol Biol Phys,2007,69:1246-1253.
59. Nelson DF, Diener-West M, Weinstein AS, et al. A randomized comparison ofmisonidazole sensitized radiotherapy plus BCNU and radiotherapy plus BCNU fortreatment of malignant glioma after surgery: final report of an RTOG study. Int JRadiat Oncol Biol Phys,1986,12:1793-1800.
60. Prados MD, Seiferheld W, Sandler HM, et al. Phase III randomized study ofradiotherapy plus procarbazine, lomustine, and vincristine with or without BUdRfor treatment of anaplastic astrocytoma: final report of RTOG9404. Int J RadiatOncol Biol Phys,2004,58:1147-1152.
61. Anderson B, Anderson B Jr. Necrotizing uveitis incident to perfusion of intractraialmalignancies with nitrogen mustard or related compounds. Trans Am OphthalmolSoc,1960,58:95-104.
62. Galanis E, Buekner JC, Burch PA, et al. PhaseⅡtrial of nitrogen mustard,vincristine and procarbazine in patients with recurrent glioma: North Central CancerTreatment Group results. J Clin Oncol,1998,16:2953-2958.
63. Stewart LA. Chemotherapy in adult high-grade glioma: a systematic review andmeta-analysis of individual patient data from12randomised trials. Lancet,2002,359(9311):1011-1018.
64. Chang CH, Horton J, Schoenfeld D, et al. Comparison of postoperative radiotherapyand combined postoperative radiotherapy and chemotherapy in the multidisciplinarymanagement of malignant gliomas, A joint Radiation Therapy Oncology Group andEastern Cooperative Oncology Group study. Cancer,1983,52:997-1007.
65. Walker MD, Alexander E Jr, Hunt WE, et al. Evaluation of BCNU and/orradiotherapy in the treatment of anaplastic gliomas. A cooperative clinical trial.JNeurosurg,1978,49:333-343.
66. Ushio Y, Hayakawa T, Hasegawa H, et al. Chemotherapy of malignant gliomas.Neumsurg Rev,1984,7:3-12.
67. Komblith PL. Walker M. Chemotherapy for malignant gliomas. J Neurosurg,1988,68:1-17.
68. Lassman LP, Pearce GW, Gang J. Sensitivity of intraeranial gliomas to vincristinesulphate.Lancet,1965,1:296-298.
69. Kaneko S, Abe H, Aida T, et al. Evaluation of radiation immunochemotherapy in thetreatment of malignant glioma. Combined use of ACNU,VCR and PS-K. HokkaidoIgaku Zasshi,1983,58:622-630.
70. Levin VA, Uhm JH, Jaeckle KA, et al. Phase III randomized study ofpostradiotherapy chemotherapy with alpha-difluoromethylornithine-procarbazine,N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosurea, vincristine (DFMO-PCV) versusPCV for glioblastoma multiforme. Clin Cancer Res,2000,6(10):3878-3884.
71. Levin VA, Lamborn K, Wara W, et al. Phase II study of6-thioguanine, procarbazine,dibromodulcitol, lomustine, and vincristine chemotherapy with radiotherapy fortreating malignant glioma in children. Neuro-oncology,2000,2(1):22-28.
72. Hildebrand JG. Chemotherapy of malignant glioma. Results of studies of theEORTC group of brain tumors. Rev Neurol (Paris),1992,148:435-440.
73. Jeremic B, Jovanovic D, Djuric LJ, et al. Advantage of post radiotherapychemotherapy with CCNU, procarbazine and vineristine(mPCV) over chemotherapywith VM-26and CCNU for malignant gliomas. J Chemother,1992,4:123-126.
74. Ushio Y, Abe H, Suzuki J, Tanaka R, et al. Evaluation of ACNU alone andcombined with tegafur as additions to radiotherapy of the treatment of malignantgliomas-a cooperative clinical trial. No To Shinkei,1985,37:999-1006.
75. Shapiro WR, Green SB, Burger PC, et al. A randomized comparison of intra-arterialversus intravenous BCNU,with or without intravenous5-fluomuracil,for newlydiagnosed patients with malignant glioma. J Neurosurg,1992,76:772-781.
76. Menei P, Boisdron-Celle M, Croue A, et al. Effect of stereotactic implantation ofbiodegradable5-fluorouracil-loaded microspheres in healthy and C6glioma-bearingrats. Neurosurgery,1996,39:117-123．
77. Stewart DJ, Richard MT, Benoit B, et al. Cisplatin plus cytosine arabinoside inadults with malignant gliomas. J Neurooncol,1984,2:29-34.
78. Stevens MF, Hickman JA, Stone R, et al. Antitumor imidazotetrazines.1. Synthesisand chemistry of8-carbamoyl-3-(2-chloroethy1)imidazo[5,1-d]-1,2,3,5-tetrazin-4(3H)-one，a novel broad-spectrum antitumor agent. J Med Chem,1984,27:196-201.
79. van Rijn J, Heimans JJ, van den Berg J, et al. Survival of human glioma cells treatedwith various combination of temozolomide and X-rays. International journal ofradiation oncology, biology, physics,2000,47(3):779-784.
80. Wedge SR, Porteous JK, Glaser MG, et al. In vitro evaluation of temozolomidecombined with X-irradiation. Anti-cancer drugs,1997,8(1):92-97.
81.0’Reilly SM, Newlands ES, Glaser MG, et al. Temozolomide: a new oral cytotoxicchemotherapeutic agent with promising activity against primary brain tumours. EurJ Cancer,1993,29A:940-942.
82. Stupp R, Mason WP, van den Bent MJ, et al. Radiotherapy plus concomitant andadjuvant temozolomide for glioblastoma. The New England journal of medicine,2005,352(10):987-996.
83. Ali-Osman F, Brunner JM, Kutluk TM, et al. Prognostic signifi cance ofglutathione S-transferase pi expression and subcellular localization in humangliomas. Clin Cancer Res,1997,3:2253-2261.
84. Goto S, Kamada K, Soh Y, Ihara Y, et al. Significance of nuclear glutathioneS-transferase pi in resistance to anti-cancer drugs. Jpn J Cancer Res,2002,93:1047-1056.
85. Batchelor T. Temozolomide for malignant brain tumours. Lancet,2000,355(9210):1115-6.
86. MacConnachie AM. Temozolomide (Temodal) for treatment of primary braintumours. Intensive Crit Care Nurs,2000,16(1):59-60.
87. Becker K, Gregel C, Fricke C, et al. DNA repair protein MGMT protects againstN-methyl-N-nitrosourea-induced conversion of benign into malignant tumors.Carcinogenesis,2003,24(3):541-546.
88. Omar AI, Mason WP. Temozolomide: The evidence for its therapeutic efficacy inmalignant astrocytomas. Core evidence,2010,4:93-111.
89. Hegi ME, Diserens AC, Gorlia T, et al. MGMT gene silencing and benefit fromtemozolomide in glioblastoma. N Engl J Med,2005,352:997-1003.
90. Fukushima T, Takeshima H, Kataoka H. Anti-glioma therapy with temozolomideand status of the DNA-repair gene MGMT．Anticancer Res,2009,29:4845-4854.
91. Lai A, Tran A, Nghiemphu PL, et al. Phase II study of bevacizumab plustemozolomide during and after radiation therapy for patients with newly diagnosedglioblastoma multiforme. J Clin Oncol,2011,29(2):142-148.
92. Vredenburghj J,Desjardins A，Herndon J End, et al. Phase II trial of bevacizumabandirinotecan inrecurrent malignant glioma. Clin Cancer Res,2007,13:1253-1259.
93. Shah N, Sukumar S. The Hox genes and their roles in oncogenesis.Nat Rev Cancer,2010,10(5):361-371.
94. McGinnis W, Garber RL, Wirz J, et al.A homologous protein-coding sequence inDrosophila homeotic genes and its conservation in other metazoans. Cell,1984,37(2):403-408.
95. Soubeyran P, Haglund K, Garcia S, et al. Homeobox gene Cdx1regulates Ras,Rhoand PI3kinase pathways leading to transformation and tumorigenesis of intestinalepithelial cells. Oncogene,2001,20(31):4180-4187.
96. Ford, H L.Homeobox genes: a link between development, cell cycle, and cancer?Cell Biol Int,1998,22(6):397-400.
97. Maroulakou IG, Spyropoulos DD. The study of HOX gene function inhematopoietic, breast and lung carcinogenesis.Anticancer Res,2003,23(3A):2101-2110.
98. Fanti L, Perrini B, Piacentini L, et al. The trithorax group and Pc group proteins aredifferentially involved in heterochromatin formation in Drosophila. Chromosoma,2008,117:25-39.
99. Hanson RD, Hess JL, Yu BD, et al. Mammalian Trithorax and polycomb-grouphomologues are antagonistic regulators of homeotic development. Proc. Natl Acad.Sci,1999,96:14372-14377.
100. Weiss FU, Marques IJ, Woltering JM, et al. Retinoic acid receptor (RAR)antagonists inhibit miR-10a expression and block metastatic be haviour ofpancreatic cancer. Gastroenterology,2009,137:2136-2145.
101. Popovic R, Riesbeck LE, Velu CS, et al. Regulation of mir-196b by MLL and itsoverexpression by MLL fusions contributes to immortalization. Blood,2009,113:3314-3322.
102. Chopra VS, Mishra RK.“Mir”acles in hox gene regulation. Bioessays,2006,28:445-448.
103. Han L, Witmer PD, Casey E, et al. DNA methylation regulates MicroRNAexpression. Cancer Biol Ther,2007,6:1284-1288.
104. Sessa L, Breiling A, Lavorgna G, et al. Noncoding RNA synthesis and loss ofPolycomb group repression accompanies the collinear activation of the humanHOXA cluster. RNA,2007,13:223-239.
105. Petruk S, Sedkov Y, Brock HW, et al. A model for initiation of mosaic HOX geneexpression patterns by non-coding RNAs in early embryos. RNA Biol,2007,4:1-6.
106. Rinn JL, Kertesz M, Wang JK, et al. Functional demarcation of active and silentchromatin domains in human HOX loci by noncoding RNAs. Cell,2007,129:1311-1323.
107. Nieto M A, Gilardi-Hebenstreit P, Charnay P, et al. A receptor protein tyrosinekinase implicated in the segmental patterning of the hindbrain and mesoderm.Development,1992,116(4):1137-1150.
108. Riddle R D, Johnson R L, Laufer E, et al. Sonic hedgehog mediates the polarizingactivity of the ZPA.Cell,1993,75(7):1401-1416.
109. Trivedi CM, Patel RC&Patel CV. Homeobox gene HOXA9inhibits nuclearfactor-κB dependent activation of endothelium. Atherosclerosis,2007,195: e50-e60.
110. Trivedi CM, Patel RC&Patel CV. Differential regulation of HOXA9expression bynuclear factor kappaB (NF-κB) and HOXA9. Gene,2008,408:187-195.
111. Manohar CF, Salwen HR, Furtado MR, et al. Upregulation of HOXC6, HOXD1,and HOXD8homeobox gene expression in human neuroblastoma cells followingchemical induction of differentiation. Tumour Biol,1996,17:34-47.
112. Zhang X, Hamada J, Nishimoto A, et al. HOXC6and HOXC11increasetranscription of S100beta gene in BrdU-induced in vitro differentiation of GOTOneuroblastoma cells into Schwannian cells. J Cell Mol Med,2007,11:299-306.
113. Jung C, Kim RS, Lee SJ, et al. HOXB13homeodomain protein suppresses thegrowth of prostate cancer cells by the negative regulation of T-cell factor4. CancerRes,2004,64:3046-3051.
114. Jung C, Kim RS, Zhang HJ, et al. HOXB13induces growth suppression of prostatecancer cells as a repressor of hormone activated androgen receptor signaling. CancerRes,2004,64:9185-9192.
115. Cheng W, Liu J, Yoshida H, et al. Lineage infidelity of epithelial ovarian cancers iscontrolled by HOX genes that specify regional identity in the reproductive tract.Nature Med,2005,11:531-537.
116. Dubeau L. The cell of origin of ovarian epithelialtumors and the ovarian surfaceepithelium dogma: does the emperor have no clothes? Gynecol Oncol,1999,72:437-442.
117. Lawrence HJ, Sauvageau G, Humphries RK, et al. The role of HOX homeoboxgenes in normal and leukemic hematopoiesis. Stem Cells,1996,14:281-291.
118. Bijl J, van Oostveen JW, Kreike M, et al. Expression of HOXC4, HOXC5, andHOXC6in human lymphoid cell lines, leukemias, and benign and malignantlymphoid tissue. Blood,1996,87:1737-1745.
119. Wu X, Chen H, Parker B, et al. HOXB7, a homeodomain protein, is overexpressed inbreast cancer and confers epithelialmesenchymal transition. Cancer Res,2006,66:9527-9534.
120. Rubin E, Wu X, Zhu T, Cheung JC, et al. A role for the HOXB7homeodomainprotein in DNA repair. Cancer Res,2007,67:1527-1535.
121. Chirnomas D, Taniguchi T, de la Vega M, et al. Chemosensitization to cisplatinbyinhibitors of the Fanconi anemia/BRCA pathway. Mol Cancer Ther,2006,5:952-961.
122. Raman V, Martensen SA, Reisman D, et al. Compromised HOXA5function canlimit p53expression in human breast tumours. Nature,2000,405:974-978.
123. Chen H, Chung S, Sukumar S. HOXA5-induced apoptosis in breast cancer cells ismediated by caspases2and8. Mol Cell Biol,2004,24:924-935.
124. Chu MC, Selam FB, Taylor HS. HOXA10regulates p53expression and matrigelinvasion in human breast cancer cells. Cancer Biol Ther,2004,3:568-572.
125. Morgan R, Pirard PM, Shears L, et al. Antagonism of HOX/PBX dimer formationblocks the in vivo proliferation of melanoma. Cancer Res,2007,67:5806-5813.
126. Plowright L, Harrington KJ, Pandha HS, et al. HOX transcription factors arepotential therapeutic targets in non-small-cell lung cancer (targeting HOX genes inlung cancer). Br J Cancer,2009,100:470-475.
127. Lawrence HJ and Largman C. Homeobox genes in normal hematopoiesis andleukemia. Blood,1992,80:2445-2453.
128. Argiropoulos B and Humphries RK. Hox genes in hematopoiesis andleukemogenesis. Oncogene,2007,26:6766-6776.
129. Calvo KR, Sykes DB, Pasillas MP, et al. Nup98-HoxA9immortalizes myeloidprogenitors, enforces expression of Hoxa9, Hoxa7and Meis1, and alterscytokine-specific responses in a manner similar to that induced by retroviralco-expression of Hoxa9and Meis1. Oncogene,2002,21:4247-4256.
130. Hu YL, Fong S, Ferrell C, et al. HOXA9modulates its oncogenic partner Meis1toinfluence normal hematopoiesis. Mol Cell Biol,2009,29:5181-5192.
131. Whelan JT, Ludwig DL, Bertrand FE. HoxA9induces insulin-like growth factor-1receptor expression in B-lineage acute lymphoblastic leukemia. Leukemia,2008,22:1161-1169
132. Costa BM, Smith JS, Chen Y, et al. Reversing HOXA9oncogene activation by PI3Kinhibition: epigenetic mechanism and prognostic significance in humanglioblastoma. Cancer Res,2010,70(2):453-62.
133. Cha TL, Zhou BP, Xia W, et al. Akt-mediated phosphorylation of EZH2suppressesmethylation of lysine27in histone H3. Science,2005,310:306-10.
134. Rinn JL, Kertesz M, Wang JK, et al. Functional demarcation of activeand silentchromatin domains in human HOX loci by noncodingRNAs. Cell,2007,129:1311-1323.
135. Lin H, Wang Y, Zhang X, et al: Prognostic significance of kappaB-Ras1expressionin gliomas. Med Oncol,2012,29(2):1272-1296.
136. Lin H, Xiong W, Zhang X, et al. Notch-1activation-dependent p53restorationcontributes to resveratrol-induced apoptosis in glioblastoma cells. Oncol Rep,2011,26(4):925-930.
137. Huang H, Lin H, Zhang X, Li J. Resveratrol reverses temozolomide resistance bydownregulation of MGMT in T98G glioblastoma cells by the NF-κB-dependentpathway. Oncol Rep,2012,27(6):2050-2056.
138. Yan T, Skaftnesmo KO, Leiss L, et al. Neuronal markers are expressed in humangliomas and NSE knockdown sensitizes glioblastoma cells to radiotherapy andtemozolomide. BMC Cancer,2011,11:524.
139. Ghosh S, Karin M. Missing pieces in the NF-kB puzzle. Cell,2002;109: S81-96.
140. Aggarwal BB. Nuclear factor-kB: the enemy within. Cancer Cell,2004,6(3):203-208.
141. Lin Y, Bai L, Chen W, Xu S. The NF-kB activation pathways, emerging moleculartargets for cancer prevention and therapy.Ther Targets,2010,14(1):45-55.