Analyzing the gene expression profile of pediatric acute myeloid leukemia with real-time PCR arrays

ABSTRACT: BACKGROUND: The Real-time PCR Array System is the ideal tool for analyzing the expression of a focused panel of genes. In this study, we will analyze the gene expression profile of pediatric acute myeloid leukemia with real-time PCR arrays. METHODS: Real-time PCR array was designed and tested firstly. Then gene expression profile of 11 pediatric AML and 10 normal controls was analyzed with real-time PCR arrays. We analyzed the expression data with MEV (Multi Experiment View) cluster software. Datasets representing genes with altered expression profile derived from cluster analyses were imported into the Ingenuity Pathway Analysis Tool. RESULTS: We designed and tested 88 real-time PCR primer pairs for a quantitative gene expression analysis of key genes involved in pediatric AML. The gene expression profile of pediatric AML is significantly different from normal control; there are 19 genes up-regulated and 25 genes down-regulated in pediatric AML. To investigate possible biological interactions of differently regulated genes, datasets representing genes with altered expression profile were imported into the Ingenuity Pathway Analysis Tool. The results revealed 12 significant networks. Of these networks, Cellular Development, Cellular Growth and Proliferation, Tumor Morphology was the highest rated network with 36 focus molecules and the significance score of 41. The IPA analysis also groups the differentially expressed genes into biological mechanisms that are related to hematological disease, cell death, cell growth and hematological system development. In the top canonical pathways, p53 and Huntington's disease signaling came out to be the top two most significant pathways with a p value of 1.5E-8 and2.95E-7, respectively. CONCLUSIONS: The present study demonstrates the gene expression profile of pediatric AML is significantly different from normal control; there are 19 genes up-regulated and 25 genes down-regulated in pediatric AML. We found some genes dyes-regulated in pediatric AML for the first time as FASLG, HDAC4, HDAC7 and some HOX family genes. IPA analysis showed the top important pathways for pediatric AML are p53 and Huntington's disease signaling. This work may provide new clues of molecular mechanism in pediatric AML.     

Gene expression profile identifies distinct molecular subtypes and potential therapeutic genes in Merkel cell carcinoma

Merkel cell carcinoma (MCC) is a rare primary cutaneous neoplasm of neuroendocrine carcinoma of the skin. About 80% of the MCC occurs due to Merkel cell polyomavirus (MCPyV) and 20% of the tumors usually occur due to severe UV exposure which is a more aggressive type of MCC. It tends to have an increased incidence rate among elderly and immunosuppressed individuals. On therapeutic level, sub-classification of MCC through molecular subtyping has emerged as a promising technique for MCC prognosis. In current study, two consistent distinct molecular subtypes of MCCs were identified using gene expression profiling data. Subtypes I MCCs were associated with spliceosome, DNA replication and cellular pathways. On the other hand, genes overexpressed in subtype II were found active in TNF signalling pathway and MAPK signalling pathway. We proposed different therapeutic targets based on subtype specificity, such as PTCH1, CDKN2A, AURKA in case of subtype I and MCL1, FGFR2 for subtype II. Such findings may provide fruitful knowledge to understand the intrinsic subtypes of MCCs and the pathways involved in distinct subtype oncogenesis, and will further advance the knowledge in developing a specific therapeutic strategy for these MCC subtypes.     

Identification of genes associated with tumorigenesis of retinoblastoma by microarray analysis

There is no report on the gene expression profile of retinoblastoma (Rb). We analyzed the gene expression profile of Rb by the microarray technique. One thousand four genes were upregulated and 481 genes were downregulated. Microarray data were confirmed by semiquantitative RT-PCR for 5 genes in Rb samples: CDC25A, C17orf75, ERBB3, LATS2, and CHFR. Clusters of differentially expressed genes were identified on chromosomes 1, 16, and 17. Based on the expression profile, we hypothesized that the PI3K/AKT/mTOR (insulin signaling) pathway might be dysregulated in Rb. Our semiquantitative RT-PCR analysis of the PIK3CA, AKT1, FRAP1, and RPS6KB1 genes in Rb samples supported this hypothesis. We suggest that known inhibitors of this pathway could be evaluated for the treatment of Rb.     

Discovering molecular pathways from protein interaction and gene expression data

In this paper, we describe an approach for identifying 'pathways' from gene expression and protein interaction data. Our approach is based on the assumption that many pathways exhibit two properties: their genes exhibit a similar gene expression profile, and the protein products of the genes often interact. Our approach is based on a unified probabilistic model, which is learned from the data using the EM algorithm. We present results on two Saccharomyces cerevisiae gene expression data sets, combined with a binary protein interaction data set. Our results show that our approach is much more successful than other approaches at discovering both coherent functional groups and entire protein complexes.     

弥漫大B 淋巴瘤12 号染色体基因表达的研究

目的:研究弥漫大B淋巴瘤(Diffuse Large B-Cell Lymphoma,DLBCL)12号染色体基因表达情况。方法:收取临床DLBCL病人淋巴结标本液氮速冻,快速冷冻切片,采用激光显微切割技术分离单纯淋巴瘤细胞,提取淋巴瘤细胞中的mRNA与表达谱芯片杂交,通过信号扫描、处理后获得表达基因杂交信号强度。每基因设11-20对探针。杂交信号与错配探针对比,扣除背景值后,使用Wilcoxon符号秩和检验选取与错配杂交信号有显著差异的基因作为分析结果(P=0.05)。随机选取两个检测到的基因,使用PCR方法检验基因芯片结果的可靠性。结果:成功地从快速冷冻保存的DLBCL标本中提取了RNA。使用表达谱芯片进行研究,发现了共164条12号染色体编码的基因在淋巴瘤细胞中表达。并根据胞内定位,基因功能和基因所属的代谢通路三种分类方法对所得基因进行分类分析。基因表达密度分析显示12号染色体上的基因表达情况与编码基因分布情况比较一致。结论:使用表达谱芯片研究了12号染色体上的基因表达情况,为研究DLBCL提供了依据。     

Identification of oscillatory genes in somitogenesis from functional genomic analysis of a human mesenchymal stem cell model

During somitogenesis, oscillatory expression of genes in the notch and wnt signaling pathways plays a key role in regulating segmentation. These oscillations in expression levels are elements of a species-specific developmental mechanism. To date, the periodicity and components of the human clock remain unstudied. Here we show that a human mesenchymal stem/stromal cell (MSC) model can be induced to display oscillatory gene expression. We observed that the known cycling gene HES1 oscillated with a 5 h period consistent with available data on the rate of somitogenesis in humans. We also observed cycling of Hes1 expression in mouse C2C12 myoblasts with a period of 2 h, consistent with previous in vitro and embryonic studies. Furthermore, we used microarray and quantitative PCR (Q-PCR) analysis to identify additional genes that display oscillatory expression both in vitro and in mouse embryos. We confirmed oscillatory expression of the notch pathway gene Maml3 and the wnt pathway gene Nkd2 by whole mount in situ hybridization analysis and Q-PCR. Expression patterns of these genes were disrupted in Wnt3a(tm1Amc) mutants but not in Dll3(pu) mutants. Our results demonstrate that human and mouse in vitro models can recapitulate oscillatory expression observed in embryo and that a number of genes in multiple developmental pathways display dynamic expression in vitro.     

Early gene expression profile in mouse brain after exposure to ionizing radiation

Acute changes in the gene expression profile in mouse brain after exposure to ionizing radiation were studied using microarray analysis. RNA was isolated at 0.25, 1, 5 and 24 h after exposure to 20 Gy and at 5 h after exposure of the whole brain of adult mice to 2 or 10 Gy. RNA was hybridized onto 15K cDNA microarrays, and data were analyzed using GeneSpring and Significant Analysis of Microarray. Radiation modulated the expression of 128, 334, 325 and 155 genes and ESTs at 0.25, 1, 5 and 24 h after 20 Gy and 60 and 168 at 5 h after 2 and 10 Gy, respectively. The expression profiles showed dose- and time-dependent changes in both expression levels and numbers of differentially modulated genes and ESTs. Seventy-eight genes were modulated at two or more times. Differentially modulated genes were associated with 12 different classes of molecular function and 24 different biological pathways and showed time- and dose-dependent changes. The change in expression of four genes (Jak3, Dffb, Nsep1 and Terf1) after irradiation was validated using quantitative real-time PCR. Up-regulation of Jak3 was observed in another mouse strain. In mouse brain, there was an increase of Jak3 immunoreactivity after irradiation. In conclusion, changes in the gene profile in the brain after irradiation are complex and are dependent on time and dose, and genes with diverse functions and pathways are modulated.     

Genetic profile of Egyptian hepatocellular-carcinoma associated with hepatitis C virus Genotype 4 by 15 K cDNA microarray: Preliminary study

Background

Hepatocellular carcinoma (HCC) is a preventable disease rather than a curable one, since there is no well-documented effective treatment modality until now, making the molecular study of this disease mandatory.

Findings

We studied gene expression profile of 17 Egyptian HCC patients associated with HCV genotype-4 infection by c-DNA microarray. Out of the 15,660 studied genes, 446 were differentially expressed; 180 of them were up regulated and 134 were down regulated. Seventeen genes out of the 180 up-regulated genes are involved in 28 different pathways. Protein phosphatase 3 (PPP3R1) is involved in 10 different pathways followed by fibroblast growth factor receptor 1 (FGFR1), Cas-Br-M ecotropic retroviral transforming sequence b (CBLB), spleen tyrosine kinase (SYK) involved in three pathways; bone morphogenetic protein 8a (BMP8A), laminin alpha 3 (LAMA3), cell division cycle 23 (CDC23) involved in 2 pathways and NOTCH4 which regulate Notch signaling pathway. On the other hand, 25 out of the 134 down-regulated genes are involved in 20 different pathways. Integrin alpha V alpha polypeptide antigen CD51 (ITGVA) is involved in 4 pathways followed by lymphotoxin alpha (TNF superfamily, member 1) (LTA) involved in 3 pathways and alpha-2-macroglobulin (A2M), phosphorylase kinase alpha 2-liver (PHKA2) and MAGI1 membrane associated guanylate kinase 1 (MAGI1) involved in 2 pathways. In addition, 22 genes showed significantly differential expression between HCC cases with cirrhosis and without cirrhosis. Confirmation analysis was performed on subsets of these genes by RT-PCR, including some up-regulated genes such as CDK4, Bax, NOTCH4 and some down-regulated genes such as ISGF3G, TNF, and VISA.

Conclusion

This is the first preliminary study on gene expression profile in Egyptian HCC patients associated with HCV-Genotype-4 using the cDNA microarray. The identified genes could provide a new gate for prognostic and diagnostic markers for HCC associated with HCV. They could also be used to identify candidate genes for molecular target therapy.     

调控通路内基因表达的相关性分析

本研究从基因表达调控通路的角度分析了基因功能与基因表达之间的关系,利用7套酿酒酵母基因芯片表达谱数据和通路数据库（KEGG和CYGD）所提供的信息,应用我们研制的Genehub软件分析研究了同一基因表达调控通路内的基因在mRNA表达水平上的相关性,共涉及16条通路,495个基因。通过Pearson相关系数和Spearman相关系数两种相似性测度的分析,我们发现有94%（15条）的基因表达调控通路内的基因在大于等于4套的表达谱数据中是共表达的,以上结果从基因表达调控通路的角度,证实了基因功能与基因表达之间存在着一定的相关性。     

Impaired expression of NER gene network in sporadic solid tumors

Nucleotide repair genes are not generally altered in sporadic solid tumors. However, point mutations are found scattered throughout the genome of cancer cells indicating that the repair pathways are dysfunctional. To address this point, in this work we focus on the expression pathways rather than in the DNA structure of repair genes related to either genome stability or essential metabolic functions. We present here a novel statistical analysis comparing ten gene expression pathways in human normal and cancer cells using serial analysis of gene expression (SAGE) data. We find that in cancer cells nucleotide-excision repair (NER) and apoptosis are the most impaired pathways and have a highly altered diversity of gene expression profile when compared to normal cells. We propose that genome point mutations in sporadic tumors can be explained by a structurally conserved NER with a functional disorder generated from its entanglement with the apoptosis gene network.     

Gene expression is highly correlated on the chromosome level in urinary bladder cancer

Objective: Chromosome correlation maps display correlations between gene expression patterns on the same chromosome. Our goal was to map the genes on chromosome regions and to identify correlations through their location on chromosome regions.

Materials and Methods: Following microarray analysis we used Ingenuity Pathway Analysis (IPA) to construct gene networks of the co-deregulated genes in bladder cancer. Chromosome mapping, mathematical modeling and data simulations were performed using the WebGestalt and Matlab^® softwares.

Results: The top deregulated molecules among 129 bladder cancer samples were implicated in the PI3K/AKT signaling, cell cycle, Myc-mediated apoptosis signaling and ERK5 signaling pathways. Their most prominent molecular and cellular functions were related to cell cycle, cell death, gene expression, molecular transport and cellular growth and proliferation. Chromosome correlation maps allowed us to detect significantly co-expressed genes along the chromosomes. We identified strong correlations among tumors of Tα-grade 1, as well as for those of Tα-grade 2, in chromosomes 1, 2, 3, 7, 12 and 19. Chromosomal domains of gene co-expression were revealed for the normal tissues, as well. The expression data were further simulated, exhibiting an excellent fit (0.7 < R² < 0.9). The simulations revealed that along the different samples, genes on same chromosomes are expressed in a similar manner.

Conclusions: Gene expression is highly correlated on the chromosome level. Chromosome correlation maps of gene expression signatures can provide further information on gene regulatory mechanisms. Gene expression data can be simulated using polynomial functions.     

Interaction of MCC2, a novel homologue of MCC tumor suppressor, with PDZ-domain Protein AIE-75

AIE-75 is a protein identified as an autoantigen in patients with autoimmune enteropathy and as a colon cancer-related antigen. It has recently been assigned to be a causative gene for Usher type 1C congenital syndromic hearing loss. The novel protein has three PSD-95/Dlg/ZO-1 (PDZ) protein-protein interaction domains and is therefore implicated to function as a molecular anchor or sorter. We have identified a novel protein that binds to AIE-75 by yeast two-hybrid screening. The protein has a high homology to the tumor suppressor MCC (mutated in colon cancer; or MCC1 hereafter) and was named MCC2. MCC2 protein binds the first PDZ domain of AIE-75 with its C-terminal amino acids -DTFL. Since the MCC1 does not bind to AIE-75 and the MCC2 displays different expression patterns in various organs compared to MCC1, they appear to play distinct roles in cells. The MCC2 gene is located on chromosome 19p13 in the vicinity of APCL gene, while MCC1 maps near to APC tumor suppressor gene. Because of negative expression of MCC2 in a panel of cancer cell-lines compared to the corresponding normal tissues, we suggest that further study is necessary to investigate a possible role of MCC2 as a tumor suppressor.     

三棱内酯B调控人冠状动脉内皮细胞基因表达谱的生物信息学分析

目的:分析三棱内酯B在人冠状动脉内皮细胞中的表达谱数据集,寻找三棱内酯B调控血管内皮功能的关键作用靶点。方法:基于GEO公共数据库,下载原始表达谱数据集(GSE44598),经过差异基因筛选,功能注释,通路富集,信号通路网络以及基因互作网络分析,找出三棱内酯B对人冠状动脉内皮细胞基因表达谱产生影响的关键基因和信号通路。结果:同对照组相比,三棱内酯B给药组共有5224个基因有显著性差异,包括2628个上调基因和2596个下调基因。基因功能注释和信号通路富集分析表明,差异基因主要参与了细胞周期过程。网络分析显示,MAPK信号通路、细胞周期通路以及PLCG2,PRKACA和ADCY4等为关键信号通路和基因。结论:三棱内酯B通过影响PLCG2,PRKACA,ADCY4等基因的表达,参与MAPK和细胞周期等信号通路,从而调节人冠状内皮细胞的功能。这些关键基因和信号通路是三棱内酯B在心血管疾病治疗应用中潜在的作用靶点。     

A computational approach to measuring coherence of gene expression in pathways

This study uses a computational approach to analyze coherence of expression of genes in pathways. Microarray data were analyzed with respect to coherent gene expression in a group of genes defined as a pathway in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Our hypothesis is that genes in the same pathway are more likely to be coordinately regulated than a randomly selected gene set. A correlation coefficient for each pair of genes in a pathway was estimated based on gene expression in normal or tumor samples, and statistically significant correlation coefficients were identified. The coherence indicator was defined as the ratio of the number of gene pairs in the pathway whose correlation coefficients are significant, divided by the total number of gene pairs in the pathway. We defined all genes that appeared in the KEGG pathways as a reference gene set. Our analysis indicated that the mean coherence indicator of pathways is significantly larger than the mean coherence indicator of random gene sets drawn from the reference gene set. Thus, the result supports our hypothesis. The significance of each individual pathway of n genes was evaluated by comparing its coherence indicator with coherence indicators of 1000 random permutation sets of n genes chosen from the reference gene set. We analyzed three data sets: two Affymetrix microarrays and one cDNA microarray. For each of the three data sets, statistically significant pathways were identified among all KEGG pathways. Seven of 96 pathways had a significant coherence indicator in normal tissue and 14 of 96 pathways had a significant coherence indicator in tumor tissue in all three data sets. The increase in the number of pathways with significant coherence indicators may reflect the fact that tumor cells have a higher rate of metabolism than normal cells. Five pathways involved in oxidative phosphorylation, ATP synthesis, protein synthesis, or RNA synthesis were coherent in both normal and tumor tissue, demonstrating that these are essential genes, a high level of expression of which is required regardless of cell type.