Statistical identification of gene association by CID in application of constructing ER regulatory network

<p>Abstract</p> <p>Background</p> <p>A variety of high-throughput techniques are now available for constructing comprehensive gene regulatory networks in systems biology. In this study, we report a new statistical approach for facilitating <it>in silico </it>inference of regulatory network structure. The new measure of association, coefficient of intrinsic dependence (CID), is model-free and can be applied to both continuous and categorical distributions. When given two variables X and Y, CID answers whether Y is dependent on X by examining the conditional distribution of Y given X. In this paper, we apply CID to analyze the regulatory relationships between transcription factors (TFs) (X) and their downstream genes (Y) based on clinical data. More specifically, we use estrogen receptor α (ERα) as the variable X, and the analyses are based on 48 clinical breast cancer gene expression arrays (48A).</p> <p>Results</p> <p>The analytical utility of CID was evaluated in comparison with four commonly used statistical methods, Galton-Pearson's correlation coefficient (GPCC), Student's <it>t</it>-test (STT), coefficient of determination (CoD), and mutual information (MI). When being compared to GPCC, CoD, and MI, CID reveals its preferential ability to discover the regulatory association where distribution of the mRNA expression levels on X and Y does not fit linear models. On the other hand, when CID is used to measure the association of a continuous variable (Y) against a discrete variable (X), it shows similar performance as compared to STT, and appears to outperform CoD and MI. In addition, this study established a two-layer transcriptional regulatory network to exemplify the usage of CID, in combination with GPCC, in deciphering gene networks based on gene expression profiles from patient arrays.</p> <p>Conclusion</p> <p>CID is shown to provide useful information for identifying associations between genes and transcription factors of interest in patient arrays. When coupled with the relationships detected by GPCC, the association predicted by CID are applicable to the construction of transcriptional regulatory networks. This study shows how information from different data sources and learning algorithms can be integrated to investigate whether relevant regulatory mechanisms identified in cell models can also be partially re-identified in clinical samples of breast cancers.</p> <p>Availability</p> <p>the implementation of CID in R codes can be freely downloaded from <url>http://homepage.ntu.edu.tw/~lyliu/BC/</url>.</p

Gene Expression Profiles of Sporadic Canine Hemangiosarcoma Are Uniquely Associated with Breed

The role an individual's genetic background plays on phenotype and biological behavior of sporadic tumors remains incompletely understood. We showed previously that lymphomas from Golden Retrievers harbor defined, recurrent chromosomal aberrations that occur less frequently in lymphomas from other dog breeds, suggesting spontaneous canine tumors provide suitable models to define how heritable traits influence cancer genotypes. Here, we report a complementary approach using gene expression profiling in a naturally occurring endothelial sarcoma of dogs (hemangiosarcoma). Naturally occurring hemangiosarcomas of Golden Retrievers clustered separately from those of non-Golden Retrievers, with contributions from transcription factors, survival factors, and from pro-inflammatory and angiogenic genes, and which were exclusively present in hemangiosarcoma and not in other tumors or normal cells (i.e., they were not due simply to variation in these genes among breeds). Vascular Endothelial Growth Factor Receptor 1 (VEGFR1) was among genes preferentially enriched within known pathways derived from gene set enrichment analysis when characterizing tumors from Golden Retrievers versus other breeds. Heightened VEGFR1 expression in these tumors also was apparent at the protein level and targeted inhibition of VEGFR1 increased proliferation of hemangiosarcoma cells derived from tumors of Golden Retrievers, but not from other breeds. Our results suggest heritable factors mold gene expression phenotypes, and consequently biological behavior in sporadic, naturally occurring tumors

Gene expression profiling identifies inflammation and angiogenesis as distinguishing features of canine hemangiosarcoma

<p>Abstract</p> <p>Background</p> <p>The etiology of hemangiosarcoma remains incompletely understood. Its common occurrence in dogs suggests predisposing factors favor its development in this species. These factors could represent a constellation of heritable characteristics that promote transformation events and/or facilitate the establishment of a microenvironment that is conducive for survival of malignant blood vessel-forming cells. The hypothesis for this study was that characteristic molecular features distinguish hemangiosarcoma from non-malignant endothelial cells, and that such features are informative for the etiology of this disease.</p> <p>Methods</p> <p>We first investigated mutations of VHL and Ras family genes that might drive hemangiosarcoma by sequencing tumor DNA and mRNA (cDNA). Protein expression was examined using immunostaining. Next, we evaluated genome-wide gene expression profiling using the Affymetrix Canine 2.0 platform as a global approach to test the hypothesis. Data were evaluated using routine bioinformatics and validation was done using quantitative real time RT-PCR.</p> <p>Results</p> <p>Each of 10 tumor and four non-tumor samples analyzed had wild type sequences for these genes. At the genome wide level, hemangiosarcoma cells clustered separately from non-malignant endothelial cells based on a robust signature that included genes involved in inflammation, angiogenesis, adhesion, invasion, metabolism, cell cycle, signaling, and patterning. This signature did not simply reflect a cancer-associated angiogenic phenotype, as it also distinguished hemangiosarcoma from non-endothelial, moderately to highly angiogenic bone marrow-derived tumors (lymphoma, leukemia, osteosarcoma).</p> <p>Conclusions</p> <p>The data show that inflammation and angiogenesis are important processes in the pathogenesis of vascular tumors, but a definitive ontogeny of the cells that give rise to these tumors remains to be established. The data do not yet distinguish whether functional or ontogenetic plasticity creates this phenotype, although they suggest that cells which give rise to hemangiosarcoma modulate their microenvironment to promote tumor growth and survival. We propose that the frequent occurrence of canine hemangiosarcoma in defined dog breeds, as well as its similarity to homologous tumors in humans, offers unique models to solve the dilemma of stem cell plasticity and whether angiogenic endothelial cells and hematopoietic cells originate from a single cell or from distinct progenitor cells.</p

Low-Coverage Whole Genome Sequencing Using Laser Capture Microscopy with Combined Digital Droplet PCR: An Effective Tool to Study Copy Number and Kras Mutations in Early Lung Adenocarcinoma Development

Defining detailed genomic characterization of early tumor progression is critical to identifying key regulators and pathways in carcinogenesis as potentially druggable targets. In human lung cancer, work to characterize early cancer development has mainly focused on squamous cancer, as the earliest lesions are more proximal in the airways and often accessible by repeated bronchoscopy. Adenocarcinomas are typically located distally in the lung, limiting accessibility for biopsy of pre-malignant and early stages. Mouse lung cancer models recapitulate many human genomic features and provide a model for tumorigenesis with pre-malignant atypical adenomatous hyperplasia and in situ adenocarcinomas often developing contemporaneously within the same animal. Here, we combined tissue characterization and collection by laser capture microscopy (LCM) with digital droplet PCR (ddPCR) and low-coverage whole genome sequencing (LC-WGS). ddPCR can be used to identify specific missense mutations in Kras (Kirsten rat sarcoma viral oncogene homolog, here focused on Kras Q61) and estimate the percentage of mutation predominance. LC-WGS is a cost-effective method to infer localized copy number alterations (CNAs) across the genome using low-input DNA. Combining these methods, the histological stage of lung cancer can be correlated with appearance of Kras mutations and CNAs. The utility of this approach is adaptable to other mouse models of human cancer

Low-Coverage Whole Genome Sequencing Using Laser Capture Microscopy with Combined Digital Droplet PCR: An Effective Tool to Study Copy Number and Kras Mutations in Early Lung Adenocarcinoma Development

Defining detailed genomic characterization of early tumor progression is critical to identifying key regulators and pathways in carcinogenesis as potentially druggable targets. In human lung cancer, work to characterize early cancer development has mainly focused on squamous cancer, as the earliest lesions are more proximal in the airways and often accessible by repeated bronchoscopy. Adenocarcinomas are typically located distally in the lung, limiting accessibility for biopsy of pre-malignant and early stages. Mouse lung cancer models recapitulate many human genomic features and provide a model for tumorigenesis with pre-malignant atypical adenomatous hyperplasia and in situ adenocarcinomas often developing contemporaneously within the same animal. Here, we combined tissue characterization and collection by laser capture microscopy (LCM) with digital droplet PCR (ddPCR) and low-coverage whole genome sequencing (LC-WGS). ddPCR can be used to identify specific missense mutations in Kras (Kirsten rat sarcoma viral oncogene homolog, here focused on Kras Q61) and estimate the percentage of mutation predominance. LC-WGS is a cost-effective method to infer localized copy number alterations (CNAs) across the genome using low-input DNA. Combining these methods, the histological stage of lung cancer can be correlated with appearance of Kras mutations and CNAs. The utility of this approach is adaptable to other mouse models of human cancer

Platelet Gene Expression as a Biomarker Risk Stratification Tool in Acute Myocardial Infarction: A Pilot Investigation

Platelets play a major role in the pathophysiology of acute myocardial infarction (AMI). Recent evidence reveals megakaryocyte-derived platelet pre-mRNA is spliced to mRNA and then translated into functional proteins in response to external stimulation. An exon microarray analyzes pre-mRNA alternative splicing and is thus applicable for studying gene expression in the anucleate platelet. We hypothesized a subset of megakaryocyte/platelet genes exists that are significantly over or underexpressed in AMI compared with stable coronary artery disease (CAD), yielding a gene expression profile for further study. Microarray analysis employing platelet mRNA was used to generate gene expression data in the above two patient groups. Unsupervised hierarchical clustering has revealed an expression profile that includes 95 over- or under-expressed genes depicted in a heat map where separation of both sets takes place. This preliminary study reveals a platelet-based gene expression signature that differentiates between AMI and stable CAD, and further study may yield a prognostic tool for a future AMI event in atherosclerosis risk factor-based subsets of CAD patients

Murine trophoblast-derived and pregnancy-associated exosome-enriched extracellular vesicle microRNAs: Implications for placenta driven effects on maternal physiology.

The role of extracellular vesicles (EVs), specifically exosomes, in intercellular communication likely plays a key role in placental orchestration of pregnancy and maternal immune sensing of the fetus. While murine models are powerful tools to study pregnancy and maternal-fetal immune interactions, in contrast to human placental exosomes, the content of murine placental and pregnancy exosomes remains largely understudied. Using a recently developed in vitro culture technique, murine trophoblast stem cells derived from B6 mice were differentiated into syncytial-like cells. EVs from the conditioned media, as well as from pregnant and non-pregnant sera, were enriched for exosomes. The RNA composition of these murine trophoblast-derived and pregnancy-associated exosome-enriched-EVs (ExoE-EVs) was determined using RNA-sequencing analysis and expression levels confirmed by qRT-PCR. Differentially abundant miRNAs were detected in syncytial differentiated ExoE-EVs, particularly from the X chromosome cluster (mmu-miR-322-3p, mmu-miR-322-5p, mmu-miR-503-5p, mmu-miR-542-3p, and mmu-miR-450a-5p). These were confirmed to be increased in pregnant mouse sera ExoE-EVs by qRT-PCR analysis. Interestingly, fifteen miRNAs were only present within the pregnancy-derived ExoE-EVs compared to non-pregnant controls. Mmu-miR-292-3p and mmu-miR-183-5p were noted to be some of the most abundant miRNAs in syncytial ExoE-EVs and were also present at higher levels in pregnant versus non-pregnant sera ExoE-EVs. The bioinformatics tool, MultiMir, was employed to query publicly available databases of predicted miRNA-target interactions. This analysis reveals that the X-chromosome miRNAs are predicted to target ubiquitin-mediated proteolysis and intracellular signaling pathways. Knowing the cargo of placental and pregnancy-specific ExoE-EVs as well as the predicted biological targets informs studies using murine models to examine not only maternal-fetal immune interactions but also the physiologic consequences of placental-maternal communication