基因芯片对中段食管癌基因表达谱的研究

目的研究中段食管癌基因表达谱,筛选特异性基因。方法利用基因芯片技术对食管癌的基因表达进行分析,通过生物信息学手段研究其内在关系。结果筛选出在中段食管癌中显著差异表达的基因21个,其中11个基因表达水平增高,10个基因表达水平降低。结论利用基因芯片技术可以高通量地筛选出肿瘤组织差异表达基因,这些特异性基因的表达水平,可以区分不同的肿瘤组织。对将来基因诊断和治疗有一定价值。     

胶质瘤恶性进展相关基因的生物信息学分析

背景与目的:利用基因芯片技术和生物信息学分析基因在系统水平的调控机制,是当前功能基因组学的重要研究手段,它明显优于过去的单一基因研究模式,可以在整体的、基因组的水平对基因的表达调控网络机制,进行系统、全面地分析。本研究利用生物信息学技术对我们建立的胶质瘤恶性进展相关基因表达谱作系统的聚类分析,并对候选基因作初步的数据库检索,目的在于筛选与肿瘤恶性进展相关的基因,为进一步克隆其全长及研究其功能提供依据。方法:首先对基因表达谱做聚类分析,在数据分析时我们取双点之间的变异系数<0.33的数据进行分析,以便尽量减少实验误差。又取至少3组比值差异均在2倍以上、双点之间变异系数<0.33的基因,据此从16363个基因数据中筛选出368个做聚类分析。进而选取在3份肿瘤样品中基因表达呈现逐步升高的11条基因和逐步降低的6条基因,进一步做生物信息学数据库的检索,去分析它们对应的结构和功能。结果:得到了两大类基因。第一类都是从WHOⅡ级、WHOⅢ级、WHOIV级胶质瘤中表达逐步升高,有11条;另一大类则相反,是表达逐步递减的,有6条。生物信息学分析从中找到3条基因X55987(EDN,嗜酸细胞来源的神经毒素)、AB011097(ARTS-1,肿瘤坏死因子受体调节因子-1)和M85085(CStF,剪切刺激因子)可能是胶质瘤恶性     

胶质瘤细胞诱导分化相关基因在胶质瘤组织中的表达

目的 利用已经建立的胶质瘤细胞诱导分化相关基因表达谱，筛选胶质瘤恶性进展相关基因。方法 按分子生物学实验要求，收集经病理确诊的不同恶性程度胶质瘤手术标本，应用反向多点杂交技术制作分子探针，与96个相关基因组成的小芯片杂交检测在不同恶性级别胶质瘤手术标本中的表达情况。结果共有8个基因在各个恶性级别胶质瘤中均呈高表达；有14个基因随胶质瘤恶性程度升高而表达频率呈上升趋势；有6个基因随胶质瘤恶性程度升高表达频率呈下降趋势；筛选到可能与胶质瘤恶性进展有关的新基因有DIP1、RPS7、CLK2、WWOX／FOR、GRIM-19等基因。结论 本研究制作的胶质瘤细胞分化相关基因小芯片，在不同级别胶质瘤组织标本中检测到的表达情况，可进一步用于胶质瘤恶性进展的分子机制研究。     

基因芯片在肾癌相关基因研究中的应用

目的：通过研究人肾癌组织和癌旁正常组织中差异表达的基因，寻找肾癌相关基因以用于诊断和治疗。方法：以包含8000个cDNA基因表达谱芯片研究1组肾癌组织样本的基因表达谱。按一步法抽提肾癌和对照组癌旁正常组织的总RNA并纯化mRNA；将等量的对照组织和肾癌组织mRNA分别逆转录合成荧光分子掺入的cDNA-链做探针，混合后杂交上述基因芯片。经严格洗片后用ScanArray3000扫描仪扫描芯片荧光信号图像，计算分析后比较2种组织中差异表达的基因。结果：共筛选出差异表达基因95条，其中未知基因44条。结论：基因芯片在筛选肾癌相关基因的改变具有快速、高通量、灵敏的特点，肾癌在细胞代谢、信号转录、蛋白合成的相关基因方面有异常表达。     

Smad4静默前后PanIN细胞基因芯片检测及表达谱的研究

背景与目的:由己建立的小鼠基因打靶模型证实,K-ras突变启动了胰腺癌前病变即胰腺导管上皮内瘤变(pancreatic intraepithelial neoplasia,PanIN),Smad4基因失活可促使PanIN细胞恶性转化.PanIN细胞中Smad4基因静默后其下游基因表达将如何改变及促使PanIN细胞恶性转化,目前尚不清楚.基于此目的,我们在己成功分离建立K-ras突变启动的PanIN细胞,并应用siRNA干扰技术静默PanIN细胞株中内源性Smad4表达的基础上,应用全基因表达谱芯片研究Smad4基因静默前PanIN细胞与其静默后PanIN-S细胞基因表达谱的变化.方法:应用包含41174个小鼠基因转录本Agi lent小鼠4×44K全基因芯片检测Smad4基因静默前后PanIN细胞基因表达谱变化,分析差异表达基因,并选择其中有差异表达的部分基因进行实时荧光定量多聚酶链反应(Real-time PCR)验证.结果:基因芯片筛选出差异倍数2倍以上的差异表达基因,如Fut9、CXCR4、MMP2、PIK3C2G、Cplx4、Lemdl、TIMP4、Reln和PITX2等.Real-time PCR验证差异表达基因CXCR4、MMP2、PIK3C2G、TIMP4、PITX2与基因芯片结果一致.结论:Smad4基因静默前后PanIN细胞中CXCR4、MMP2、PIK3C2G、TIMP4、PITX2基因表达存在显著差异,其中CXCR4、PIK3C2G基因可能与胰腺肿瘤新生血管形成相关,MMP2、TIMP4基因可能与胰腺肿瘤细胞黏附、浸润、迁移相关,PITX2表达高低可能与胰腺肿瘤预后相关.     

应用基因芯片技术筛选肝癌相关基因

目的:本研究利用基因芯片技术筛查肝癌组织中特异表达的基因,为深入研究肝癌的发生、发展提供线索,为肝癌的药物治疗提供指导.方法:选取5例人肝癌组织标本和3例人正常肝组织标本,Trizol法提取组织总RNA,RNeasy Mini kit进行纯化.使用One-Cycle cDNA Synthesis Kjt反转录成cDNA,Sample Cleanup Module kit纯化双链cDNA,VT Labeling Kit体外转录合成并纯化生物素标记cRNA,与人基因组U133+2.0基因芯片杂交.Scanner 3000 7G扫描仪获取基因表达的荧光信号强度值,用预先选定的内参照基因进行均衡和修正,Microarray SuiteVersion5.0软件分析杂交信号的强度和比值,应用SAM统计分析结果,按照常规标准q-value(%)＜5%,fold change≥2或≤0.5筛选出表达差异明显的基因.结果:与正常肝组织相比,肝癌组织存在967个共同差异表达的基因,包括562个上调基因,405个下调基因,其中有58个基因变化倍数大于2倍以上,包括35个上调基因和23个下调基因,与肝癌的发病与进展具有一定相关性,其中包括凋亡相关基因、细胞周期调节相关基因、信号传导相关基因、转录调控相关基因等发生异常改变.结论:基因表达谱芯片可以快速筛选与肝癌发生和进展相关的多个环节上起重要作用的基因,对于肝癌的基础研究和药物治疗具有指导作用,为肝癌的临床诊断、预防、易感性预测和治疗提供分子标记和靶基因,为建立个体化治疗方案和预后评估提供帮助.     

胶质母细胞瘤复发相关基因的筛选、表达和临床预后分析

背景与目的:脑胶质瘤是常见的中枢神经系统原发性恶性脑肿瘤之一,胶质母细胞瘤恶性程度高、侵袭性强、容易复发,复发后患者的预后极差。筛选胶质母细胞瘤复发相关的基因,并分析其在胶质瘤中的表达、临床病理学参数和预后的关系。方法:通过对GEO数据库中胶质母细胞瘤相关数据集进行挖掘,筛选包含胶质母细胞瘤原发和复发病例的相关数据集,并分析胶质母细胞瘤原发病例和复发病例样本间的差异表达基因(differentially expressed gene,DEG)。对DEG进行基因本体功能和信号富集分析,构架蛋白质相互作用(protein-protein interaction,PPI)网络,筛选Hub基因。通过PPI网络和Venn图筛选关键基因,而后研究使用基因表达谱数据动态分析(Gene Expression Profiling Interactive Analysis,GEPIA)和中国脑胶质瘤基因组图谱(Chinese Glioma Genome Atlas,CGGA)数据库进行关键基因的生存分析和表达分析,分析其表达与胶质瘤临床病理学参数的关系。结果:GSE62153数据集筛选到40个DEG,上调...     

基于生物信息学方法分析甲酰肽受体1在胶质瘤中的表达及临床意义

目的：分析胶质瘤与正常组织表达差异的基因,筛选和胶质瘤预后相关的关键基因。方法：通过下载GEO（Gene Expression Omnibus）数据库GSE15824原始数据,利用R语言分析正常组织与胶质瘤组织中差异表达的基因,通过生物信息学分析工具（DAVID、String、Cytoscape、oncomine和GEPIA）对差异表达的基因进行生物学功能、蛋白互作网络（PPI）分析及筛选胶质瘤的预后标志物。结果：共筛选出648个差异表达的mRNAs。差异表达mRNAs的生物学功能主要富集于T细胞的激活,调节白细胞的黏附和细胞的迁移。KEGG信号通路分析显示差异基因主要富集于PI3K/Akt,MAPK和钙离子信号通路。FPR1在胶质瘤中明显高表达,其表达水平与胶质瘤的预后呈负相关（Log-rank P < 0.01）。结论：通过基因芯片数据库的分析,FPR1在胶质瘤中高表达,且与患者生存预后相关,为进一步分子机制的研究提供了新思路。     

非小细胞肺癌mRNA的表达谱分析

目的 检测非小细胞肺癌（NSCLC）组织和正常肺组织中mRNA的表达差异,并进行生物信息学分析。方法 采用基因芯片技术对3例NSCLC组织和3例正常肺组织的mRNA进行检测,筛选差异表达基因,进一步对差异表达基因进行Gene Ontoloty（GO）分析和信号通路分析。结果 在20 716条目的基因中共发现两种组织差异表达基因896条,其中593条基因表达增加,303条基因表达降低;生物信息学分析表明上述差异性表达的mRNA参与了NSCLC重要的生物学调节功能和通路。结论 基因芯片技术可以筛选出NSCLC组织和正常肺组织间差异表达的基因,mRNA在NSCLC组织和正常肺组织间的表达水平有明显差异,差异性表达的mRNA参与了NSCLC重要的生物学功能。     

基因芯片分析RNA干涉MSP58后细胞周期相关基因表达的变化

目的:采用基因芯片技术,观察RNA干涉法抑制人脑胶质瘤U251细胞MSP58基因表达后细胞周期相关基因的变化,从而了解MSP58在细胞周期相关基因通路中的可能定位.方法:将特异性的MSP58干涉表达载体pSilencer3.1-MSP58转染人脑胶质瘤细胞系U251,同时构建相应的阴性对照载体pSilencer3.1-NC以及空载体pSilencer3.1-H1neo.运用半定量RT-PCR及蛋白质印迹法,检测稳定转染和未转染干涉载体的胶质瘤细胞MSP58的mRNA和蛋白表达水平.运用基因芯片技术分析pSilencer3.1-MSP58转染后细胞周期相关基因的变化.结果:成功建立了具有稳定下调MSP58基因表达的U251-S细胞、阴性对照U251-NC细胞以及含有空载体的U251-H1 neo细胞.干涉组细胞MSP58的表达无论在mRNA水平还是在蛋白质水平均较阴性对照组及空载体组细胞显著降低,P＜0.01.基因芯片技术共筛选细胞周期相关基因128个,其中上调及下调>2倍的共有33个基因.结论:干涉MSP58基因表达后,胶质瘤细胞增殖的抑制与细胞周期相关基因表达的变化有关.     

Detection of gene amplification and deletion in high-grade gliomas using a genome DNA microarray (GenoSensor Array 300)

Glioblastoma is a rapidly growing tumor that accounts for more than 50% of all primary gliomas. Amplification of oncogenes and deletion of tumor suppressor genes frequently affects tumor progression. Thus, the goal of this study was to conduct a comprehensive analysis of gene aberrations of individual glioblastomas. A genome DNA microarray (GenoSensor Array 300), spotted with 287 target genes, was used to analyze resected tissue from 11 different high-grade gliomas. The average number of gene aberrations was 9.0 per case (WHO grade III) and 13.3 per case (WHO grade IV).EGFR was the most frequent amplified gene in this series (4 of 11 cases), and high-level amplification was also detected forEGFR, SAS/CDK4, andAKT1. A high frequency of deleted genes was observed in 6 of 11 cases (54.5%), includingFGFR2, MTAP, andDMBT1. The detected gene aberrations were matched to the classical primary glioblastoma pathway in five of nine cases. We conclude that the GenoSensor Array 300 genomic DNA microarray is a useful method for the comprehensive identification of amplified and deleted genes in glioblastoma.     

Distinct transcription profiles of primary and secondary glioblastoma subgroups

Glioblastomas are invasive and aggressive tumors of the brain, generally considered to arise from glial cells. A subset of these cancers develops from lower-grade gliomas and can thus be clinically classified as "secondary," whereas some glioblastomas occur with no prior evidence of a lower-grade tumor and can be clinically classified as "primary." Substantial genetic differences between these groups of glioblastomas have been identified previously. We used large-scale expression analyses to identify glioblastoma-associated genes (GAG) that are associated with a more malignant phenotype via comparison with lower-grade astrocytomas. We have further defined gene expression differences that distinguish primary and secondary glioblastomas. GAGs distinct to primary or secondary tumors provided information on the heterogeneous properties and apparently distinct oncogenic mechanisms of these tumors. Secondary GAGs primarily include mitotic cell cycle components, suggesting the loss of function in prominent cell cycle regulators, whereas primary GAGs highlight genes typical of a stromal response, suggesting the importance of extracellular signaling. Immunohistochemical staining of glioblastoma tissue arrays confirmed expression differences. These data highlight that the development of gene pathway-targeted therapies may need to be specifically tailored to each subtype of glioblastoma.     

Integrated genomic analyses identify ERRFI1 and TACC3 as glioblastoma-targeted genes

The glioblastoma genome displays remarkable chromosomal aberrations, which harbor critical glioblastoma-specific genes contributing to several oncogenetic pathways. To identify glioblastoma-targeted genes, we completed a multifaceted genome-wide analysis to characterize the most significant aberrations of DNA content occurring in glioblastomas. We performed copy number analysis of 111 glioblastomas by Digital Karyotyping and Illumina BeadChip assays and validated our findings using data from the TCGA (The Cancer Genome Atlas) glioblastoma project. From this study, we identified recurrent focal copy number alterations in 1p36.23 and 4p16.3. Expression analyses of genes located in the two regions revealed genes which are dysregulated in glioblastomas. Specifically, we identify EGFR negative regulator, ERRFI1, within the minimal region of deletion in 1p36.23. In glioblastoma cells with a focal deletion of the ERRFI1 locus, restoration of ERRFI1 expression slowed cell migration. Furthermore, we demonstrate that TACC3, an Aurora-A kinase substrate, on 4p16.3, displays gain of copy number, is overexpressed in a glioma-grade-specific pattern, and correlates with Aurora kinase overexpression in glioblastomas. Our multifaceted genomic evaluation of glioblastoma establishes ERRFI1 as a potential candidate tumor suppressor gene and TACC3 as a potential oncogene, and provides insight on targets for oncogenic pathway-based therapy.     

Identification of a human glioma-associated growth factor gene, granulin, using differential immuno-absorption

Identification of the genes that are differentially expressed in brain tumor cells but not in normal brain cells is important for understanding the molecular basis of these neurological cancers and for defining possible targets for therapeutic intervention. In an effort to discover potentially antigenic proteins that may be involved in the malignant transformation and progression of human glioblastomas, a novel antibody-based approach was developed to identify and isolate gene products that are expressed in brain tumors versus normal brain tissue. Using this method, whereby tumor-specific antibodies were isolated and used to screen a glioblastoma cDNA expression library, 28 gene products were identified. Nine of these clones had homology to known gene products, and 19 were novel. The expression of these genes in multiple different human gliomas was then evaluated by cDNA microarray hybridization. One of the isolated clones had consistently higher levels of expression (3-30-fold) in brain tumors compared with normal brain. Northern blot analysis and in situ hybridization confirmed this differential overexpression. cDNA sequence analysis revealed that this gene was identical to a relatively new class of growth regulators known as granulins, which have tertiary structures resembling the epidermal growth factor-like proteins. The 2.1-kb granulin mRNA was expressed predominantly in glial tumors, with lower levels in spleen, kidney, and testes, whereas expression was not detected in non-tumor brain tissues. Functional assays using [3H]thymidine incorporation indicated that granulin may be a glial mitogen, as addition of synthetic granulin peptide to primary rat astrocytes and three different early-passage human glioblastoma cultures increased cell proliferation in vitro, whereas increasing concentrations of granulin antibody inhibited cell growth in a dose-dependent manner. The differential expression pattern, tissue distribution, and implication of this glioma-associated molecule in growth regulation suggest a potentially important role for granulin in the pathogenesis and/or malignant progression of primary brain neoplasms.     

Topoisomerase-II alpha is upregulated in malignant peripheral nerve sheath tumors and associated with clinical outcome.

PURPOSE: To identify target genes of clinical significance for patients with malignant peripheral-nerve sheath tumor (MPNST), an aggressive cancer for which no consensus therapy exists. MATERIALS AND METHODS: Biopsies and clinical data from 51 patients with MPNST were included in this study. Based on our previous research implicating chromosome arm 17q amplification in MPNST, we performed gene expression analyses of 14 MPNSTs using chromosome 17-specific cDNA microarrays. Copy numbers of selected gene probes and centromere probes were then determined by interphase fluorescence in situ hybridization in 16 MPNSTs. Finally, we generated a tissue microarray containing 79 samples from 44 MPNSTs, on which in situ protein expressions of candidate genes were examined and related to clinical end points. RESULTS: Among several deregulated genes found by cDNA microarray analyses, topoisomerase II alpha (TOP2A) was the most overexpressed gene in MPNSTs compared with benign neurofibromas. Excess copies of the TOP2A were also seen at the DNA level in 10 of 16 cases, and high expression of the TOP2A protein was seen in 83% of the tumors on the tissue microarray. The TOP2A-expressing tumors were associated with poor cancer-specific survival and presence of metastases. CONCLUSION: We have identified TOP2A as a target gene in MPNST, using a focused gene expression profiling followed by a DNA copy number evaluation and clinical validation of the encoded protein using a tissue microarray. This study is the first to suggest that TOP2A expression may be a predictive factor for adverse outcome in MPNST.