寡核苷酸阵列比较基因组杂交技术在肿瘤研究中作用

人类肿瘤的发生发展具有遗传相关性[1],几乎所有的肿瘤存在基因组DNA拷贝数异常,这些异常与原癌基因的扩增和抑癌基因的缺失密切相关.因此,利用先进的分子遗传学新技术,研究肿瘤组织中基因组DNA异常,已成为人类了解肿瘤发生机制的重心.1992年,Kallioniemi等首次提出比较基因组杂交(CGH)技术,能对染色体上DNA序列进行检测并定位.     

基因扩增与人类恶性肿瘤

基因扩增(gene amplification)是细胞内染色体上特定基因拷贝数的大量增加,常见于生物发育、肿瘤发生及抗药性的形成过程。肿瘤发生的分子遗传学标志即为基因组不稳定,其中基因扩增作为基因组不稳定的主要形式在很多人类恶性肿瘤的发生发展中起着重要作用。肿瘤细胞中基因扩增是癌基因激活的主要机制,可调控肿瘤细胞逃避生长限制产生耐药。针对基因扩增的深入研究对肿瘤遗传学的发展及肿瘤的诊断、治疗有着重要的作用。本文就人类恶性肿瘤中基因扩增的组成成分、表现形式、扩增机制及研究手段进展做一简要综述。     

非同源末端连接机制与基因扩增关联的研究进展

基因扩增是肿瘤中基因组异常形式之一,与肿瘤发生发展及耐药性有密切关系.非同源末端连接(non-homologous end joining,NHEJ)是哺乳动物DNA双链断裂(doublestrand breaks,DSBs)修复的主要机制之一,在维持基因组的稳定性中发挥着重要作用.近年来,非同源末端连接与基因扩增关系的研究受到越来越多的关注.本文分别介绍了基因扩增,两种非同源末端连接修复方式机制及其与基因扩增的研究进展.     

肌色素上皮源性因子基因与黑色素瘤的发生相关

目的：寻找与B16黑色素瘤发生相关的DNA序列或片段。方法：用105条引物对B16黑色素瘤及其来源小鼠C57BL／6J基因组DNA进行随机扩增多态DNA分析,比较肿瘤组织及其相应正常组织的DNA指纹,对差异很明显的片段收、克隆和测序,序列与GenBank数据库进行同源性分析。结果：105条引物中有24条引物扩增出的条带在肿瘤组织与其相应正常组织间存在差异。在6个差异很明显的回收DNA片段中引物AB8－5扩增后所得差异片段B8－5,肿瘤组织中此片段缺失,该片段序列长610bp,与GenBank序列数据库中鼠肌色素上皮源性因子基因具有99％（419／421）的同源性,与鼠肌色素上皮源性因子（PEDF）mRNA的同源性为100％（213／213）。可以认为该序列即为此基因,结论：黑色素瘤中存在PEDF基因缺失,提示PEDF基因与黑色素瘤发生相关。     

微阵列比较基因组杂交技术在肿瘤研究中作用

人类肿瘤的研究重心经历了从单基因水平到染色体、多基因水平的转变。许多人类肿瘤与发育异常疾病一样以基因组DNA拷贝数变化为特点。在癌症中,不利于肿瘤生长的基因（抑癌基因）可能存在于DNA拷贝数减少的区域;而有利于肿瘤发生、发展的基因（癌基因）可能存在于DNA拷贝数增加的区域。1992年,Kallioniemi等。首次描述了比较基因组杂交（CGH）技术。其基本原理是：分别用不同的荧光标记体系标记来自待检组织和正常组织的全基因组DNA,[第一段]     

激光捕获显微切割联合基因芯片在肿瘤差异表达基因研究中的应用

 目的 为激光捕获显微切割（LCM）联合基因芯片在肿瘤差异表达基因研究中应用摸索可行的技术方法。方法 采用 LCM 技术分别自原发性膀胱移行癌患者手术切除的癌组织和癌旁正常组织冻存标本获取细胞，提取 RNA，用 Agilent 2100 Bioanalyzer 芯片分析系统进行 RNA 完整度（RIN）检测。取总 RNA 100 ng 进行线性扩增和荧光标记，获得 aRNA 探针。取等量的癌组织探针与癌旁正常组织探针，与 Agilent 人全基因组寡核苷酸基因表达谱芯片杂交。通过自身比较实验分析芯片的假阳性基因数，计算假阳性率（FPR）。通过数据分析寻找癌组织与癌旁正常组织差异表达基因。 结果 LCM 所获微量 RNA 的 RIN 都在 8.0 以上，表明LCM 后 RNA 完整度较高。100 ng RNA 经过线性扩增与荧光标记，获 aRNA 产量约 16 µg，片段大小 0.5 ~ 2.5 kb。芯片自身比较实验结果良好，FPR < 1%，验证了实验系统的可靠性。相对于癌旁正常组织，癌组织发生表达上调的基因有 286 条，下调的基因 112 条。 结论 以 LCM 技术获得的细胞提取 RNA 用于制备基因芯片探针，获得了可信的芯片杂交结果，为肿瘤基因差异表达研究提供了可行的技术方法。     

表观遗传分子机制及其在肿瘤发生中的作用

表观遗传指不改变DNA序列的基因表达改变,是多细胞真核生物的重要生物学现象.在个体发生过程中,干细胞一旦分化为某种分化细胞,该分化细胞的特征就会维持.肿瘤发生中基因组相同的细胞一旦成为肿瘤细胞,就会维持肿瘤的特征,一旦成为非肿瘤细胞多数也会维持非肿瘤特性.表观遗传关系到细胞分化、分化状态维持、肿瘤发生、衰老等过程.DNA甲基化、组蛋白乙酰化、小RNA与表观遗传的分子机制有关.DNA的CpG甲基化影响基因表达,甲基化状态可以传递给子链DNA,小RNA通过改变DNA甲基化参与表观遗传;组蛋白乙酰化改变染色质构象影响基因表达.基因组的广泛低甲基化和抑癌基因高甲基化在肿瘤细胞中普遍存在,改变表观遗传的药物已试用于肿瘤治疗.     

Taq聚合酶逆转录并直接扩增细胞RNA

几年来的实践表明,PCR技术是基因分析中的一种十分有用的手段。近来结合mRNA逆转录为cDNA方法,应用PCR技术分析基因转录体(RT-PCR)。作者在用PCR技术分析基因组DNA过程中,注意到时常出现相应于cDNA片段大小的扩增产物,因而作者推测,Taq聚合酶直接合成并扩增了cDNA片段。为了证明Taq聚合酶能逆转录RNA模板,从末稍血分离4μg总RNA,用Taq聚合酶进行40轮的PCR扩增,所用引物为与人β血影蛋白cDNA 0.4kb两侧互补的寡核苷酸引物。在另一反应管中,同时用1μg基因组DNA用同一引物进行PCR扩增。PCR反应产物电泳分析表明,前者可是0.4kb扩增片     

表遗传学与肿瘤

通常认为遗传学上的基因突变是肿瘤发病机制中的关键事件,尤其是抑癌基因的体细胞突变与肿瘤的发生有着密切的关系。但是,近年来随着对肿瘤认识的深入,人们发现DNA序列以外的调控机制异常在肿瘤的发生、发展过程中更为普遍,也更为重要。这种调控机制被称为表观遗传学（Epigenetics）,研究没有DNA序列变化的,可遗传的表达改变。例如基因启动子区CpG岛甲基化模式的异常与许多肿瘤的发生有着密切的关系。除了DNA甲基化调控形式外,表观遗传学还包括基因组印迹、染色质组蛋白修饰、隔离蛋白以及非编码RNA（包括microRNA）等DNA序列本身以外的各种调控方式。本文将就表观遗传学调控机制与肿瘤发生的关系作一简要综述。     

脱氧核酶的研究进展

脱氧核酶是近年来利用体外分子进化技术合成的一种具有催化功能的单链 DNA片段 ,它能催化 RNA特定部位的切割反应 ,从 m RNA水平对基因灭活 ,从而调控蛋白的表达 ,可能成为对抗 RNA病毒感染、肿瘤等疾病的新型工具     

Examination of oncogene amplification by genomic DNA microarray in hepatocellular carcinomas: comparison with comparative genomic hybridization analysis

To identify amplified oncogenes involved in hepatocellular carcinomas (HCC), we applied a genomic DNA microarray spotted with 57 oncogenes to 20 HCCs. Aberrations in DNA copy number also were analyzed by comparative genomic hybridization (CGH) using an aliquot of DNA samples. In 5 of 20 HCCs, only 6 oncogenes (CCND1, FGF3/FGF4, SAS/CDK4, TERC, MET, and MYC) were amplified, whereas in the remaining 15 tumors no oncogenes were amplified. A comparison of DNA microarray and conventional CGH analyses showed that, although 5 of 6 amplified oncogenes shown by microarray were located in chromosomal regions shown by CGH to have increased DNA copy numbers, not all genes located in such chromosomal regions were affected. One of the amplified oncogenes (SAS/CDK4) was found in a chromosomal region that was undetected by CGH. We, therefore, conclude that amplification of the oncogenes examined in this series is not directly implicated in hepatocellular carcinogenesis.     

Gene expression analysis of chromosomal regions with gain or loss of genetic material detected by comparative genomic hybridization

Comparative genomic hybridization (CGH) has been widely used to detect copy number alterations in cancer and to identify regions containing candidate tumor-responsible genes; however, gene expression changes have been described only in highly amplified regions (amplicons). To study the overall impact of slight copy number changes on gene expression, we analyzed 16 T-cell lymphomas by using CGH and a custom-designed cDNA microarray containing 7,657 genes and expressed sequence tags related to tumorigenesis. We evaluated mean gene expression and variability within CGH-altered regions and explored the relationship between the effects of the gene and its position within these regions. Minimally overlapping CGH candidate areas (6q25, 13q21-q22, and 19q13.1) revealed a weak relationship between altered genomic content and gene expression. However, some candidate genes showed modified expression within these regions in the majority of tumors; these candidate genes were evaluated and confirmed in another independent series of 23 T-cell lymphomas by use of the same cDNA microarray and by FISH on a tissue microarray. When all the CGH regions detected for each tumor were considered, we found a significant increase or decrease in the mean expression of the genes contained in gained or lost regions, respectively. In addition, we found that the expression of a gene was dependent not only on its position within an altered region but also on its own mechanism of regulation: genes in the same altered region responded very differently to the gain or loss of genetic material. Supplementary material for this article can be found on the Genes, Chromosomes, and Cancer website at http://www.interscience.wiley.com/jpages/1045-2257/suppmat/index.html.     

Amplification of MGC2177, PLAG1, PSMC6P, and LYN in a malignant mixed tumor of salivary gland detected by cDNA microarray with tyramide signal amplification

Gene amplifications have been observed in many different tumor cells, and many of these changes are related to tumor pathogenesis. Comparative genomic hybridization (CGH) using metaphase chromosomes can detect changes in chromosome copy number with a resolution of 10-20 Mb. Current advances in CGH analysis in a microarray format allow us to refine such changes down to the gene level. We applied microarray technology to detect novel gene amplification in a malignant mixed tumor of salivary gland. Besides detecting previously known gene amplifications (MDM2 and MYC), we identified four other highly amplified genes located at 8q11.2 approximately q13: MGC2177, PLAG1, PSMC6P, and LYN. The amplification was further validated with real-time quantitative polymerase chain reaction.     

Gene amplifications at chromosome 7 of the human gastric cancer genome

Genetic aberrations at chromosome 7 are known to be related with diverse human diseases, including cancer and autism. In a number of cancer research areas involving gastric cancer, several comparative genomic hybridization studies employing metaphase chromosome or BAC clone microarrays have repeatedly identified human chromosome 7 as containing 'regions of changes' related with cancer progression. cDNA microarray-based comparative genomic hybridization can be used to directly identify individual target genes undergoing copy number variations. Copy number change analysis for 17,000 genes on a microarray format was performed with tumor and normal gastric tissues from 30 patients. A group of 90 genes undergoing copy number increases (gene amplification) at the p11 approximately p22 or q21 approximately q36 region of chromosome 7 is reported. The list of genes includes wingless-type MMTV integration site family member 2 (WNT2), a proto-oncogene and acyloxyacyl hydrolase (AOAH) that was amplified in >80% of the tested cases. The amplified genes are those functioning in the biological processes such as signal transduction pathways, cell proliferation, metabolism, transport, inflammatory response and protein folding or proteolysis. Also found in the list are genes that are targets for drug development, such as maltase-glucoamylase (MGAM), cyclin-dependent kinase 5 (CDK5), neuropeptide Y (NPY) and dopa decarboxylase (DDC). The current dataset can be used as one of the resources in understanding genetic aberrations of chromosome 7 in human gastric cancer.     

Gene expression profiling in hepatocellular carcinoma: upregulation of genes in amplified chromosome regions.

Cytogenetics of hepatocellular carcinoma and adenoma have revealed gains of chromosome 1q as a significant differentiating factor. However, no studies are available comparing these amplification events with gene expression. Therefore, gene expression profiling was performed on tumours cytogenetically well characterized by array-based comparative genomic hybridisation. For this approach analysis was carried out on 24 hepatocellular carcinoma and 8 hepatocellular adenoma cytogenetically characterised by array-based comparative genomic hybridisation. Expression profiles of mRNA were determined using a genome-wide microarray containing 43,000 spots. Hierarchical clustering analysis branched all hepatocellular adenoma from hepatocellular carcinoma. Significance analysis of microarray demonstrated 722 dysregulated genes in hepatocellular carcinoma. Gene set enrichment analysis detected groups of upregulated genes located in chromosome bands 1q22-42 seen also as the most frequently gained regions by comparative genomic hybridisation. Comparison of significance analysis of microarray and gene set enrichment analysis narrowed down the number of dysregulated genes to 18, with 7 genes localised on 1q22 (SCAMP3, IQGAP3, PYGO2, GPATC4, ASH1L, APOA1BP, and CCT3). In hepatocellular adenoma 26 genes in bands 11p15, 11q12, and 12p13 were upregulated. However, the respective chromosome bands were not gained in hepatocellular adenoma. Expression analysis and comparative genomic hybridisation identified an upregulation of genes in amplified regions of 1q. These results may serve to further narrow down the number of candidate driver genes in hepatocarcinogenesis.     

RAD21 and KIAA0196 at 8q24 are amplified and overexpressed in prostate cancer

To detect genes that are overexpressed in prostate cancer, a subtracted cDNA library was first constructed from the PC-3 cell line and subsequently screened by using cDNA microarray hybridization. Sixty-eight genes were found to be overexpressed (ratio>3) in PC-3. Half of these genes were in chromosomal regions, which, using comparative genomic hybridization, we previously showed to be gained in PC-3. Subsequently, the expression and copy number of selected genes were studied by quantitative RT-PCR and fluorescence in situ hybridization in prostate cancer cell lines, xenografts, and clinical tumor specimens of benign prostate hyperplasia and untreated as well as hormone-refractory prostate carcinomas. Two genes from chromosomal region 8q24-RAD21 and KIAA0196-showed increased expression in clinical prostate carcinomas and were also amplified in 30-40% of xenografts and hormone-refractory tumors. In addition, the expression of KIAA0196 was significantly (P=0.0051) higher in tumors with the gene amplification than in those without it. The data suggest that KIAA0196 and possibly RAD21 are putative target genes for the common amplification of 8q23-24 in prostate cancer.     

Gene expression patterns and gene copy number changes in dermatofibrosarcoma protuberans

Dermatofibrosarcoma protuberans (DFSP) is an aggressive spindle cell neoplasm. It is associated with the chromosomal translocation, t(17:22), which fuses the COL1A1 and PDGFbeta genes. We determined the characteristic gene expression profile of DFSP and characterized DNA copy number changes in DFSP by array-based comparative genomic hybridization (array CGH). Fresh frozen and formalin-fixed, paraffin-embedded samples of DFSP were analyzed by array CGH (four cases) and DNA microarray analysis of global gene expression (nine cases). The nine DFSPs were readily distinguished from 27 other diverse soft tissue tumors based on their gene expression patterns. Genes characteristically expressed in the DFSPs included PDGF beta and its receptor, PDGFRB, APOD, MEOX1, PLA2R, and PRKCA. Array CGH of DNA extracted either from frozen tumor samples or from paraffin blocks yielded equivalent results. Large areas of chromosomes 17q and 22q, bounded by COL1A1 and PDGF beta, respectively, were amplified in DFSP. Expression of genes in the amplified regions was significantly elevated. Our data shows that: 1) DFSP has a distinctive gene expression profile; 2) array CGH can be applied successfully to frozen or formalin-fixed, paraffin-embedded tumor samples; 3) a characteristic amplification of sequences from chromosomes 17q and 22q, demarcated by the COL1A1 and PDGF beta genes, respectively, was associated with elevated expression of the amplified genes.     

Comparison of Campylobacter jejuni isolates implicated in Guillain-Barré syndrome and strains that cause enteritis by a DNA microarray

We asked whether Campylobacter jejuni isolated from patients with Guillain-Barré syndrome (GBS) differ from isolates isolated from patients with uncomplicated gastrointestinal infection using DNA microarray analysis. We found that specific GBS genes or regions were not identified, and microarray analysis confirmed significant genomic heterogeneity among the isolates.     

High-level DNA amplifications are common genetic aberrations in B-cell neoplasms.

Gene amplification is one of the molecular mechanisms resulting in the up-regulation of gene expression. In non-Hodgkin's lymphomas, such gene amplifications have been identified rarely. Using comparative genomic hybridization, a technique that has proven to be very sensitive for the detection of high-level DNA amplifications, we analyzed 108 cases of B-cell neoplasms (42 chronic B-cell leukemias, 5 mantle cell lymphomas, and 61 aggressive B-cell lymphomas). Twenty-four high-level amplifications were identified in 13% of the patients and mapped to 15 different genomic regions. Regions most frequently amplified were bands Xq26-28, 2p23-24, and 2p14-16 as well as 18q21 (three times each). Amplification of several proto-oncogenes and a cell cycle control gene (N-MYC (two cases), BCL2, CCND2, and GLI) located within the amplified regions was demonstrated by Southern blot analysis or fluorescence in situ hybridization to interphase nuclei of tumor cells. These data demonstrate that gene amplifications in B-cell neoplasms are much more frequent than previously assumed. The identification of highly amplified DNA regions and genes included in the amplicons provides important information for further analyses of genetic events involved in lymphomagenesis.     

Electrophoretic isolation of extrachromosomal DNA from tumor cells

Gene amplification allows transformed cells to overexpress specific genes and gain a survival advantage. For this reason, cloning and characterization of amplified genes can improve our understanding of the biology of transformed cells. The techniques of in-gel renaturation and chromosome microdissection can enrich for amplified DNA sequences, but both are labor intensive and have other drawbacks. We have developed an alternative strategy of enriching for amplified DNA sequences that involves two-directional agarose gel electrophoresis of extrachromosomal circular DNA. Extrachromosomal circles can be detected with repetitive DNA probes and can be used to produce DNA probes suitable for fluorescence in situ hybridization for location of genomic origin. The ability to enrich for amplified DNA without specialized equipment or transformed cell metaphases should prove useful in the search for new genes which are important in tumor cell progression.