Integrated miRNA-mRNA Analysis Revealing the Potential Roles of miRNAs in Chordomas

Introduction

Emerging evidence suggests that microRNAs (miRNAs) are crucially involved in tumorigenesis and that paired expression profiles of miRNAs and mRNAs can be used to identify functional miRNA-target relationships with high precision. However, no studies have applied integrated analysis to miRNA and mRNA profiles in chordomas. The purpose of this study was to provide insights into the pathogenesis of chordomas by using this integrated analysis method.

Methods

Differentially expressed miRNAs and mRNAs of chordomas (n = 3) and notochord tissues (n = 3) were analyzed by using microarrays with hierarchical clustering analysis. Subsequently, the target genes of the differentially expressed miRNAs were predicted and overlapped with the differentially expressed mRNAs. Then, GO and pathway analyses were performed for the intersecting genes.

Results

The microarray analysis indicated that 33 miRNAs and 2,791 mRNAs were significantly dysregulated between the two groups. Among the 2,791 mRNAs, 911 overlapped with putative miRNA target genes. A pathway analysis showed that the MAPK pathway was consistently enriched in the chordoma tissue and that miR-149-3p, miR-663a, miR-1908, miR-2861 and miR-3185 likely play important roles in the regulation of MAPK pathways. Furthermore, the Notch signaling pathway and the loss of the calcification or ossification capacity of the notochord may also be involved in chordoma pathogenesis.

Conclusion

This study provides an integrated dataset of the miRNA and mRNA profiles in chordomas, and the results demonstrate that not only the MAPK pathway and its related miRNAs but also the Notch pathway may be involved in chordoma development. The occurrence of chordoma may be associated with dysfunctional calcification or ossification of the notochord.     

Decreased Pattern Recognition Receptor Signaling,Interferon-Signature,and Bactericidal/Permeability-Increasing Protein Gene Expression in Cord Blood of Term Low Birth Weight Human Newborns

Background

Morbidity and mortality rates of low birth weight (LBW) newborns at term are higher than rates in normal birth weight (NBW) newborns. LBW newborns are at greater risk to acquire recurrent bacterial and viral infections during their first few weeks of life possibly as an outcome of compromised innate immune functions. As adaptive immunity is in a naive state, increased risk of infection of LBW as compared to NBW newborns may reflect impairments in innate immunity.

Methodology

To characterize the increased susceptibility to infections in LBW newborns we used microarray technology to identify differences in gene expression in LBW newborns (n = 8) compared to NBW newborns (n = 4) using cord blood. The results obtained from the microarray study were validated on a larger number of samples using real time RT-PCR (LBW = 22, NBW = 18) and western blotting (LBW = 12, NBW = 12). The Interferome database was used to identify interferon (IFN) signature genes and ingenuity pathway analysis identified canonical pathways and biological functions associated with the differentially expressed genes in LBW newborns. ELISAs for IFNs and bactericidal/permeability-increasing protein were performed in both LBW and NBW newborns and in adults (LBW = 18, NBW = 18, Adults  = 8).

Principal Findings

Upon microarray analysis, we identified 1,391 differentially expressed genes, of which, 1,065 genes were down-regulated and 326 genes were up-regulated in the LBW compared to NBW newborns. Of note, 70 IFN-signature genes were found to be significantly down-regulated in LBW compared to NBW newborns. Ingenuity pathway analysis revealed pattern recognition receptors signaling including Toll-Like Receptors (TLRs) -1, -5, and -8 genes and IFN signaling as the most significantly impacted pathways. Respiratory infectious diseases were the most significantly affected bio-functions in LBW newborns.

Conclusion and Significance

Diminished PRRs, IFN-signature, and BPI gene expression raises the possibility that impairments in these pathways contribute to the susceptibility of LBW term infants to infection.     

Transcriptomic Meta-Analysis of Multiple Sclerosis and Its Experimental Models

Background

Multiple microarray analyses of multiple sclerosis (MS) and its experimental models have been published in the last years.

Objective

Meta-analyses integrate the information from multiple studies and are suggested to be a powerful approach in detecting highly relevant and commonly affected pathways.

Data sources

ArrayExpress, Gene Expression Omnibus and PubMed databases were screened for microarray gene expression profiling studies of MS and its experimental animal models.

Study eligibility criteria

Studies comparing central nervous system (CNS) samples of diseased versus healthy individuals with n >1 per group and publically available raw data were selected.

Material and Methods

Included conditions for re-analysis of differentially expressed genes (DEGs) were MS, myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (EAE) in rats, proteolipid protein-induced EAE in mice, Theiler’s murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD), and a transgenic tumor necrosis factor-overexpressing mouse model (TNFtg). Since solely a single MS raw data set fulfilled the inclusion criteria, a merged list containing the DEGs from two MS-studies was additionally included. Cross-study analysis was performed employing list comparisons of DEGs and alternatively Gene Set Enrichment Analysis (GSEA).

Results

The intersection of DEGs in MS, EAE, TMEV-IDD, and TNFtg contained 12 genes related to macrophage functions. The intersection of EAE, TMEV-IDD and TNFtg comprised 40 DEGs, functionally related to positive regulation of immune response. Over and above, GSEA identified substantially more differentially regulated pathways including coagulation and JAK/STAT-signaling.

Conclusion

A meta-analysis based on a simple comparison of DEGs is over-conservative. In contrast, the more experimental GSEA approach identified both, a priori anticipated as well as promising new candidate pathways.     

Clustered gene expression changes flank targeted gene loci in knockout mice

Background

Gene expression profiling using microarrays is a powerful technology widely used to study regulatory networks. Profiling of mRNA levels in mutant organisms has the potential to identify genes regulated by the mutated protein.

Methodology/Principle Findings

Using tissues from multiple lines of knockout mice we have examined genome-wide changes in gene expression. We report that a significant proportion of changed genes were found near the targeted gene.

Conclusions/Significance

The apparent clustering of these genes was explained by the presence of flanking DNA from the parental ES cell. We provide recommendations for the analysis and reporting of microarray data from knockout mice     

GWAS Meets Microarray: Are the Results of Genome-Wide Association Studies and Gene-Expression Profiling Consistent? Prostate Cancer as an Example

Background

Genome-wide association studies (GWASs) and global profiling of gene expression (microarrays) are two major technological breakthroughs that allow hypothesis-free identification of candidate genes associated with tumorigenesis. It is not obvious whether there is a consistency between the candidate genes identified by GWAS (GWAS genes) and those identified by profiling gene expression (microarray genes).

Methodology/Principal Findings

We used the Cancer Genetic Markers Susceptibility database to retrieve single nucleotide polymorphisms from candidate genes for prostate cancer. In addition, we conducted a large meta-analysis of gene expression data in normal prostate and prostate tumor tissue. We identified 13,905 genes that were interrogated by both GWASs and microarrays. On the basis of P values from GWASs, we selected 1,649 most significantly associated genes for functional annotation by the Database for Annotation, Visualization and Integrated Discovery. We also conducted functional annotation analysis using same number of the top genes identified in the meta-analysis of the gene expression data. We found that genes involved in cell adhesion were overrepresented among both the GWAS and microarray genes.

Conclusions/Significance

We conclude that the results of these analyses suggest that combining GWAS and microarray data would be a more effective approach than analyzing individual datasets and can help to refine the identification of candidate genes and functions associated with tumor development.     

Gene Expression in Peripheral Immune Cells following Cardioembolic Stroke Is Sexually Dimorphic

Aims

Epidemiological studies suggest that sex has a role in the pathogenesis of cardioembolic stroke. Since stroke is a vascular disease, identifying sexually dimorphic gene expression changes in blood leukocytes can inform on sex-specific risk factors, response and outcome biology. We aimed to examine the sexually dimorphic immune response following cardioembolic stroke by studying the differential gene expression in peripheral white blood cells.

Methods and Results

Blood samples from patients with cardioembolic stroke were obtained at ≤3 hours (prior to treatment), 5 hours and 24 hours (after treatment) after stroke onset (n = 23; 69 samples) and compared with vascular risk factor controls without symptomatic vascular diseases (n = 23, 23 samples) (ANCOVA, false discovery rate p≤0.05, |fold change| ≥1.2). mRNA levels were measured on whole-genome Affymetrix microarrays. There were more up-regulated than down-regulated genes in both sexes, and females had more differentially expressed genes than males following cardioembolic stroke. Female gene expression was associated with cell death and survival, cell-cell signaling and inflammation. Male gene expression was associated with cellular assembly, organization and compromise. Immune response pathways were over represented at ≤3, 5 and 24 h after stroke in female subjects but only at 24 h in males. Neutrophil-specific genes were differentially expressed at 3, 5 and 24 h in females but only at 5 h and 24 h in males.

Conclusions

There are sexually dimorphic immune cell expression profiles following cardioembolic stroke. Future studies are needed to confirm the findings using qRT-PCR in an independent cohort, to determine how they relate to risk and outcome, and to compare to other causes of ischemic stroke.     

Microarray analysis reveals distinct gene expression profiles among different tumor histology, stage and disease outcomes in endometrial adenocarcinoma

Background

Endometrial cancer is the most common gynecologic malignancy in developed countries and little is known about the underlying mechanism of stage and disease outcomes. The goal of this study was to identify differentially expressed genes (DEG) between late vs. early stage endometrioid adenocarcinoma (EAC) and uterine serous carcinoma (USC), as well as between disease outcomes in each of the two histological subtypes.

Methodology/Principal Finding

Gene expression profiles of 20 cancer samples were analyzed (EAC = 10, USC = 10) using the human genome wide illumina bead microarrays. There was little overlap in the DEG sets between late vs. early stages in EAC and USC, and there was an insignificant overlap in DEG sets between good and poor prognosis in EAC and USC. Remarkably, there was no overlap between the stage-derived DEGs and the prognosis-derived DEGs for each of the two histological subtypes. Further functional annotation of differentially expressed genes showed that the composition of enriched function terms were different among different DEG sets. Gene expression differences for selected genes of various stages and outcomes were confirmed by qRT-PCR with a high validation rate.

Conclusion

This data, although preliminary, suggests that there might be involvement of distinct groups of genes in tumor progression (late vs. early stage) in each of the EAC and USC. It also suggests that these genes are different from those involved in tumor outcome (good vs. poor prognosis). These involved genes, once clinically verified, may be important for predicting tumor progression and tumor outcome.     

Gene Expression Profiling Unravels Cancer-Related Hepatic Molecular Signatures in Steatohepatitis but Not in Steatosis

Background

Pathogenesis and factors for determining progression of alcoholic and non-alcoholic steatosis to steatohepatitis with risk of further progression to liver cirrhosis and cancer are poorly understood. In the present study, we aimed to identify potential molecular signatures for discrimination of steatohepatitis from steatosis.

Methodology and Results

Global microarray gene expression analysis was applied to unravel differentially expressed genes between steatohepatitis compared to steatosis and control samples. For functional annotation as well as the identification of disease-relevant biological processes of the differentially expressed genes the gene ontology (GO) database was used. Selected candidate genes (n = 46) were validated in 87 human liver samples from two sample cohorts by quantitative real-time PCR (qRT-PCR). The GO analysis revealed that genes down-regulated in steatohepatitis were mainly involved in metabolic processes. Genes up-regulated in steatohepatitis samples were associated with cancer progression and proliferation. In surgical liver resection samples, 39 genes and in percutaneous liver biopsies, 30 genes were significantly up-regulated in steatohepatitis. Furthermore, immunohistochemical investigation of human liver tissue revealed a significant increase of AKR1B10 protein expression in steatohepatitis.

Conclusions

The development of steatohepatitis is characterized by distinct molecular changes. The most striking examples in this respect were KRT23 and AKR1B10, which we found to be highly differentially expressed in steatohepatitis compared to steatosis and normal liver. We propose that KRT23 and AKR1B10 may serve as future potential biomarkers for steatohepatitis as well as markers for progression to HCC.     

Spotlight on differentially expressed genes in urinary bladder cancer

Introduction

We previously identified common differentially expressed (DE) genes in bladder cancer (BC). In the present study we analyzed in depth, the expression of several groups of these DE genes.

Materials and Methods

Samples from 30 human BCs and their adjacent normal tissues were analyzed by whole genome cDNA microarrays, qRT-PCR and Western blotting. Our attention was focused on cell-cycle control and DNA damage repair genes, genes related to apoptosis, signal transduction, angiogenesis, as well as cellular proliferation, invasion and metastasis. Four publicly available GEO Datasets were further analyzed, and the expression data of the genes of interest (GOIs) were compared to those of the present study. The relationship among the GOI was also investigated. GO and KEGG molecular pathway analysis was performed to identify possible enrichment of genes with specific biological themes.

Results

Unsupervised cluster analysis of DNA microarray data revealed a clear distinction in BC vs. control samples and low vs. high grade tumors. Genes with at least 2-fold differential expression in BC vs. controls, as well as in non-muscle invasive vs. muscle invasive tumors and in low vs. high grade tumors, were identified and ranked. Specific attention was paid to the changes in osteopontin (OPN, SPP1) expression, due to its multiple biological functions. Similarly, genes exhibiting equal or low expression in BC vs. the controls were scored. Significant pair-wise correlations in gene expression were scored. GO analysis revealed the multi-facet character of the GOIs, since they participate in a variety of mechanisms, including cell proliferation, cell death, metabolism, cell shape, and cytoskeletal re-organization. KEGG analysis revealed that the most significant pathway was that of Bladder Cancer (p = 1.5×10⁻³¹).

Conclusions

The present work adds to the current knowledge on molecular signature identification of BC. Such works should progress in order to gain more insight into disease molecular mechanisms.     

Comparison of Microarray Platforms for Measuring Differential MicroRNA Expression in Paired Normal/Cancer Colon Tissues

Background

Microarray technology applied to microRNA (miRNA) profiling is a promising tool in many research fields; nevertheless, independent studies characterizing the same pathology have often reported poorly overlapping results. miRNA analysis methods have only recently been systematically compared but only in few cases using clinical samples.

Methodology/Principal Findings

We investigated the inter-platform reproducibility of four miRNA microarray platforms (Agilent, Exiqon, Illumina, and Miltenyi), comparing nine paired tumor/normal colon tissues. The most concordant and selected discordant miRNAs were further studied by quantitative RT-PCR. Globally, a poor overlap among differentially expressed miRNAs identified by each platform was found. Nevertheless, for eight miRNAs high agreement in differential expression among the four platforms and comparability to qRT-PCR was observed. Furthermore, most of the miRNA sets identified by each platform are coherently enriched in data from the other platforms and the great majority of colon cancer associated miRNA sets derived from the literature were validated in our data, independently from the platform. Computational integration of miRNA and gene expression profiles suggested that anti-correlated predicted target genes of differentially expressed miRNAs are commonly enriched in cancer-related pathways and in genes involved in glycolysis and nutrient transport.

Conclusions

Technical and analytical challenges in measuring miRNAs still remain and further research is required in order to increase consistency between different microarray-based methodologies. However, a better inter-platform agreement was found by looking at miRNA sets instead of single miRNAs and through a miRNAs – gene expression integration approach.     

Distinct Gene Expression Signatures in Lynch Syndrome and Familial Colorectal Cancer Type X

Introduction

Heredity is estimated to cause at least 20% of colorectal cancer. The hereditary nonpolyposis colorectal cancer subset is divided into Lynch syndrome and familial colorectal cancer type X (FCCTX) based on presence of mismatch repair (MMR) gene defects.

Purpose

We addressed the gene expression signatures in colorectal cancer linked to Lynch syndrome and FCCTX with the aim to identify candidate genes and to map signaling pathways relevant in hereditary colorectal carcinogenesis.

Experimental design

The 18 k whole-genome c-DNA-mediated annealing, selection, extension, and ligation (WG-DASL) assay was applied to 123 colorectal cancers, including 39 Lynch syndrome tumors and 37 FCCTX tumors. Target genes were technically validated using real-time quantitative RT-PCR (qRT-PCR) and the expression signature was validated in independent datasets.

Results

Colorectal cancers linked to Lynch syndrome and FCCTX showed distinct gene expression profiles, which by significance analysis of microarrays (SAM) differed by 2188 genes. Functional pathways involved were related to G-protein coupled receptor signaling, oxidative phosphorylation, and cell cycle function and mitosis. qRT-PCR verified altered expression of the selected genes NDUFA9, AXIN2, MYC, DNA2 and H2AFZ. Application of the 2188-gene signature to independent datasets showed strong correlation to MMR status.

Conclusion

Distinct genetic profiles and deregulation of different canonical pathways apply to Lynch syndrome and FCCTX and key targets herein may be relevant to pursue for refined diagnostic and therapeutic strategies in hereditary colorectal cancer.     

Spatial and Temporal Gene Expression Differences in Core and Periinfarct Areas in Experimental Stroke: A Microarray Analysis

Background

A large number of genes are regulated to promote brain repair following stroke. The thorough analysis of this process can help identify new markers and develop therapeutic strategies. This study analyzes gene expression following experimental stroke.

Methodology/Principal Findings

A microarray study of gene expression in the core, periinfarct and contralateral cortex was performed in adult Sprague-Dawley rats (n = 60) after 24 hours (acute phase) or 3 days (delayed stage) of permanent middle cerebral artery (MCA) occlusion. Independent qRT-PCR validation (n = 12) was performed for 22 of the genes. Functional data were evaluated by Ingenuity Pathway Analysis. The number of genes differentially expressed was 2,612 (24 h) and 5,717 (3 d) in the core; and 3,505 (24 h) and 1,686 (3 d) in the periinfarct area (logFC>|1|; adjP<0.05). Expression of many neurovascular unit development genes was altered at 24 h and 3 d including HES2, OLIG2, LINGO1 and NOGO-A; chemokines like CXCL1 and CXCL12, stress-response genes like HIF-1A, and trophic factors like BDNF or BMP4. Nearly half of the detected genes (43%) had not been associated with stroke previously.

Conclusions

This comprehensive study of gene regulation in the core and periinfarct areas at different times following permanent MCA occlusion provides new data that can be helpful in translational research.