Elizabeth Guruceaga

Correlation between Gene Expression and GO Semantic Similarity

This research analyzes some aspects of the relationship between gene expression, gene function, and gene annotation. Many recent studies are implicitly based on the assumption that gene products that are biologically and functionally related would maintain this similarity both in their expression profiles as well as in their Gene Ontology (GO) annotation. We analyze how accurate this assumption proves to be using real publicly available data. We also aim to validate a measure of semantic similarity for GO annotation. We use the Pearson correlation coefficient and its absolute value as a measure of similarity between expression profiles of gene products. We explore a number of semantic similarity measures (Resnik, Jiang, and Lin) and compute the similarity between gene products annotated using the GO. Finally, we compute correlation coefficients to compare gene expression similarity against GO semantic similarity. Our results suggest that the Resnik similarity measure outperforms the others and seems better suited for use in Gene Ontology. We also deduce that there seems to be correlation between semantic similarity in the GO annotation and gene expression for the three GO ontologies. We show that this correlation is negligible up to a certain semantic similarity value; then, for higher similarity values, the relationship trend becomes almost linear. These results can be used to augment the knowledge provided by clustering algorithms and in the development of bioinformatic tools for finding and characterizing gene products.

A small noncoding RNA signature found in exosomes of GBM patient serum as a diagnostic tool

BACKGROUND Glioblastoma multiforme (GBM) is the most frequent malignant brain tumor in adults, and its prognosis remains dismal despite intensive research and therapeutic advances. Diagnostic biomarkers would be clinically meaningful to allow for early detection of the tumor and for those cases in which surgery is contraindicated or biopsy results are inconclusive. Recent findings show that GBM cells release microvesicles that contain a select subset of cellular proteins and RNA. The aim of this hypothesis-generating study was to assess the diagnostic potential of miRNAs found in microvesicles isolated from the serum of GBM patients. METHODS To control disease heterogeneity, we used patients with newly diagnosed GBM. In the discovery stage, PCR-based TaqMan Low Density Arrays followed by individual quantitative reverse transcriptase polymerase chain reaction were used to test the differences in the miRNA expression levels of serum microvesicles among 25 GBM patients and healthy controls paired by age and sex. The detected noncoding RNAs were then validated in another 50 GBM patients. RESULTS We found that the expression levels of 1 small noncoding RNA (RNU6-1) and 2 microRNAs (miR-320 and miR-574-3p) were significantly associated with a GBM diagnosis. In addition, RNU6-1 was consistently an independent predictor of a GBM diagnosis. CONCLUSIONS Altogether our results uncovered a small noncoding RNA signature in microvesicles isolated from GBM patient serum that could be used as a fast and reliable differential diagnostic biomarker.

Adenovirus VA RNA-derived miRNAs target cellular genes involved in cell growth, gene expression and DNA repair

Adenovirus virus-associated (VA) RNAs are processed to functional viral miRNAs or mivaRNAs. mivaRNAs are important for virus production, suggesting that they may target cellular or viral genes that affect the virus cell cycle. To look for cellular targets of mivaRNAs, we first identified genes downregulated in the presence of VA RNAs by microarray analysis. These genes were then screened for mivaRNA target sites using several bioinformatic tools. The combination of microarray analysis and bioinformatics allowed us to select the splicing and translation regulator TIA-1 as a putative mivaRNA target. We show that TIA-1 is downregulated at mRNA and protein levels in infected cells expressing functional mivaRNAs and in transfected cells that express mivaRNAI-138, one of the most abundant adenoviral miRNAs. Also, reporter assays show that TIA-1 is downregulated directly by mivaRNAI-138. To determine whether mivaRNAs could target other cellular genes we analyzed 50 additional putative targets. Thirty of them were downregulated in infected or transfected cells expressing mivaRNAs. Some of these genes are important for cell growth, transcription, RNA metabolism and DNA repair. We believe that a mivaRNA-mediated fine tune of the expression of some of these genes could be important in adenovirus cell cycle.

Identification of Tissue microRNAs Predictive of Sunitinib Activity in Patients with Metastatic Renal Cell Carcinoma

Purpose To identify tissue microRNAs predictive of sunitinib activity in patients with metastatic renal-cell-carcinoma (MRCC) and to evaluate in vitro their mechanism of action in sunitinib resistance. Methods We screened 673 microRNAs using TaqMan Low-density-Arrays (TLDAs) in tumors from MRCC patients with extreme phenotypes of marked efficacy and resistance to sunitinib, selected from an identification cohort (n = 41). The most relevant differentially expressed microRNAs were selected using bioinformatics-based target prediction analysis and quantified by qRT-PCR in tumors from patients presenting similar phenotypes selected from an independent cohort (n = 101). In vitro experiments were conducted to study the role of miR-942 in sunitinib resistance. Results TLDAs identified 64 microRNAs differentially expressed in the identification cohort. Seven candidates were quantified by qRT-PCR in the independent series. MiR-942 was the most accurate predictor of sunitinib efficacy (p = 0.0074). High expression of miR-942, miR-628-5p, miR-133a, and miR-484 was significantly associated with decreased time to progression and overall survival. These microRNAs were also overexpressed in the sunitinib resistant cell line Caki-2 in comparison with the sensitive cell line. MiR-942 overexpression in Caki-2 up-regulates MMP-9 and VEGF secretion which, in turn, promote HBMEC endothelial migration and sunitinib resistance. Conclusions We identified differentially expressed microRNAs in MRCC patients presenting marked sensitivity or resistance to sunitinib. MiR-942 was the best predictor of efficacy. We describe a novel paracrine mechanism through which high miR-942 levels in MRCC cells up-regulates MMP-9 and VEGF secretion to enhance endothelial migration and sunitinib resistance. Our results support further validation of these miRNA in clinical confirmatory studies.

Involvement of miRNAs in the Differentiation of Human Glioblastoma Multiforme Stem-Like Cells

Glioblastoma multiforme (GBM)-initiating cells (GICs) represent a tumor subpopulation with neural stem cell-like properties that is responsible for the development, progression and therapeutic resistance of human GBM. We have recently shown that blockade of NFκB pathway promotes terminal differentiation and senescence of GICs both in vitro and in vivo, indicating that induction of differentiation may be a potential therapeutic strategy for GBM. MicroRNAs have been implicated in the pathogenesis of GBM, but a high-throughput analysis of their role in GIC differentiation has not been reported. We have established human GIC cell lines that can be efficiently differentiated into cells expressing astrocytic and neuronal lineage markers. Using this in vitro system, a microarray-based high-throughput analysis to determine global expression changes of microRNAs during differentiation of GICs was performed. A number of changes in the levels of microRNAs were detected in differentiating GICs, including over-expression of hsa-miR-21, hsa-miR-29a, hsa-miR-29b, hsa-miR-221 and hsa-miR-222, and down-regulation of hsa-miR-93 and hsa-miR-106a. Functional studies showed that miR-21 over-expression in GICs induced comparable cell differentiation features and targeted SPRY1 mRNA, which encodes for a negative regulator of neural stem-cell differentiation. In addition, miR-221 and miR-222 inhibition in differentiated cells restored the expression of stem cell markers while reducing differentiation markers. Finally, miR-29a and miR-29b targeted MCL1 mRNA in GICs and increased apoptosis. Our study uncovers the microRNA dynamic expression changes occurring during differentiation of GICs, and identifies miR-21 and miR-221/222 as key regulators of this process.

SPACE: an algorithm to predict and quantify alternatively spliced isoforms using microarrays

Exon and exon+junction microarrays are promising tools for studying alternative splicing. Current analytical tools applied to these arrays lack two relevant features: the ability to predict unknown spliced forms and the ability to quantify the concentration of known and unknown isoforms. SPACE is an algorithm that has been developed to (1) estimate the number of different transcripts expressed under several conditions, (2) predict the precursor mRNA splicing structure and (3) quantify the transcript concentrations including unknown forms. The results presented here show its robustness and accuracy for real and simulated data.

Type I Interferon Regulates the Expression of Long Non-Coding RNAs

Interferons (IFNs) are key players in the antiviral response. IFN sensing by the cell activates transcription of IFN-stimulated genes (ISGs) able to induce an antiviral state by affecting viral replication and release. IFN also induces the expression of ISGs that function as negative regulators to limit the strength and duration of IFN response. The ISGs identified so far belong to coding genes. However, only a small proportion of the transcriptome corresponds to coding transcripts and it has been estimated that there could be as many coding as long non-coding RNAs (lncRNAs). To address whether IFN can also regulate the expression of lncRNAs, we analyzed the transcriptome of HuH7 cells treated or not with IFNα2 by expression arrays. Analysis of the arrays showed increased levels of several well-characterized coding genes that respond to IFN both at early or late times. Furthermore, we identified several IFN-stimulated or -downregulated lncRNAs (ISRs and IDRs). Further validation showed that ISR2, 8, and 12 expression mimics that of their neighboring genes GBP1, IRF1, and IL6, respectively, all related to the IFN response. These genes are induced in response to different doses of IFNα2 in different cell lines at early (ISR2 or 8) or later (ISR12) time points. IFNβ also induced the expression of these lncRNAs. ISR2 and 8 were also induced by an influenza virus unable to block the IFN response but not by other wild-type lytic viruses tested. Surprisingly, both ISR2 and 8 were significantly upregulated in cultured cells and livers from patients infected with HCV. Increased levels of ISR2 were also detected in patients chronically infected with HIV. This is relevant as genome-wide guilt-by-association studies predict that ISR2, 8, and 12 may function in viral processes, in the IFN pathway and the antiviral response. Therefore, we propose that these lncRNAs could be induced by IFN to function as positive or negative regulators of the antiviral response.

Lack of the matricellular protein SPARC (secreted protein, acidic and rich in cysteine) attenuates liver fibrogenesis in mice.

Introduction Secreted Protein, Acidic and Rich in Cysteine (SPARC) is a matricellular protein involved in many biological processes and found over-expressed in cirrhotic livers. By mean of a genetic approach we herein provide evidence from different in vivo liver disease models suggesting a profibrogenic role for SPARC. Methods Two in vivo models of liver fibrosis, based on TAA administration and bile duct ligation, were developed on SPARC wild-type (SPARC+/+) and knock-out (SPARC−/−) mice. Hepatic SPARC expression was analyzed by qPCR. Fibrosis was assessed by Sirius Red staining, and the maturation state of collagen fibers was analyzed using polarized light. Necroinflammatory activity was evaluated by applying the Knodell score and liver inflammatory infiltration was characterized by immunohistochemistry. Hepatic stellate cell activation was assessed by α-SMA immunohistochemistry. In addition, pro-fibrogenic genes and inflammatory cytokines were measured by qPCR and/or ELISA. Liver gene expression profile was analyzed in SPARC−/− and SPARC+/+ mice using Affymetrix Mouse Gene ST 1.0 array. Results SPARC expression was found induced in fibrotic livers of mouse and human. SPARC−/− mice showed a reduction in the degree of inflammation, mainly CD4+ cells, and fibrosis. Consistently, collagen deposits and mRNA expression levels were decreased in SPARC−/− mice when compared to SPARC+/+ mice; in addition, MMP-2 expression was increased in SPARC−/− mice. A reduction in the number of activated myofibroblasts was observed. Moreover, TGF-β1 expression levels were down-regulated in the liver as well as in the serum of TAA-treated knock-out animals. Ingenuity Pathway Analysis (IPA) analysis suggested several gene networks which might involve protective mechanisms of SPARC deficiency against liver fibrogenesis and a better established machinery to repair DNA and detoxify from external chemical stimuli. Conclusions Overall our data suggest that SPARC plays a significant role in liver fibrogenesis. Interventions to inhibit SPARC expression are suggested as promising approaches for liver fibrosis treatment.

BCR‐ABL1‐induced expression of HSPA8 promotes cell survival in chronic myeloid leukaemia

In order to determine new signal transduction pathways implicated in chronic myeloid leukaemia (CML), we performed a gene expression profile comparison between CD34+ cells from CML patients and healthy donors. Functional studies were performed using the Mo7e and Mo7e‐p210 cell lines. Expression of CCND1 (Cyclin D1), as well as the chaperone HSPA8, which is important for regulation of CCND1, were significantly upregulated in CD34+ CML cells. Upregulation of HSPA8 was dependent, at least in part, on STAT5 (signal transducer and activator of transcrition 5)‐dependent transcriptional activation, as demonstrated by chromatin immunoprecipitation. The presence of HSPA8 in the nuclear protein fraction as well as its binding to CCND1 suggests that it may contribute to stabilization of the CCND1/CDK4 complex, which, in turn, may participate in proliferation of CML cells. Treatment of CML cells with the specific HSPA8 inhibitor 15‐deoxyspergualin induced inhibition of CML cell viability but did not induce apoptosis. In conclusion, our studies suggest that STAT5‐mediated activation of HSPA8 induces nuclear translocation and activation of the CCND1/CDK4 complex leading to increased proliferation of CML cells, deciphering a new pathway implicated in CML and supporting a potential role of chaperone inhibitors in the treatment of CML.

Spanish human proteome project: dissection of chromosome 16

The Chromosome 16 Consortium forms part of the Human Proteome Project that aims to develop an entire map of the proteins encoded by the human genome following a chromosome-centric strategy (C-HPP) to make progress in the understanding of human biology in health and disease (B/D-HPP). A Spanish consortium of 16 laboratories was organized into five working groups: Protein/Antibody microarrays, protein expression and Peptide Standard, S/MRM, Protein Sequencing, Bioinformatics and Clinical healthcare, and Biobanking. The project is conceived on a multicenter configuration, assuming the standards and integration procedures already available in ProteoRed-ISCIII, which is encompassed within HUPO initiatives. The products of the 870 protein coding genes in chromosome 16 were analyzed in Jurkat T lymphocyte cells, MCF-7 epithelial cells, and the CCD18 fibroblast cell line as it is theoretically expected that most chromosome 16 protein coding genes are expressed in at least one of these. The transcriptome and proteome of these cell lines was studied using gene expression microarray and shotgun proteomics approaches, indicating an ample coverage of chromosome 16. With regard to the B/D section, the main research areas have been adopted and a biobanking initiative has been designed to optimize methods for sample collection, management, and storage under normalized conditions and to define QC standards. The general strategy of the Chr-16 HPP and the current state of the different initiatives are discussed.