Dataset Information

Expression of regulatory platelet microRNAs in patients with sickle cell disease.

ABSTRACT:

Background

Increased platelet activation in sickle cell disease (SCD) contributes to a state of hypercoagulability and confers a risk of thromboembolic complications. The role for post-transcriptional regulation of the platelet transcriptome by microRNAs (miRNAs) in SCD has not been previously explored. This is the first study to determine whether platelets from SCD exhibit an altered miRNA expression profile.

Methods and findings

We analyzed the expression of miRNAs isolated from platelets from a primary cohort (SCD?=?19, controls?=?10) and a validation cohort (SCD?=?7, controls?=?7) by hybridizing to the Agilent miRNA microarrays. A dramatic difference in miRNA expression profiles between patients and controls was noted in both cohorts separately. A total of 40 differentially expressed platelet miRNAs were identified as common in both cohorts (p-value 0.05, fold change>2) with 24 miRNAs downregulated. Interestingly, 14 of the 24 downregulated miRNAs were members of three families - miR-329, miR-376 and miR-154 - which localized to the epigenetically regulated, maternally imprinted chromosome 14q32 region. We validated the downregulated miRNAs, miR-376a and miR-409-3p, and an upregulated miR-1225-3p using qRT-PCR. Over-expression of the miR-1225-3p in the Meg01 cells was followed by mRNA expression profiling to identify mRNA targets. This resulted in significant transcriptional repression of 1605 transcripts. A combinatorial approach using Meg01 mRNA expression profiles following miR-1225-3p overexpression, a computational prediction analysis of miRNA target sequences and a previously published set of differentially expressed platelet transcripts from SCD patients, identified three novel platelet mRNA targets: PBXIP1, PLAGL2 and PHF20L1.

Conclusions

We have identified significant differences in functionally active platelet miRNAs in patients with SCD as compared to controls. These data provide an important inventory of differentially expressed miRNAs in SCD patients and an experimental framework for future studies of miRNAs as regulators of biological pathways in platelets.

SUBMITTER: Jain S

PROVIDER: S-EPMC3625199 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Publications

Expression of regulatory platelet microRNAs in patients with sickle cell disease.

Jain Shilpa S Kapetanaki Maria G MG Raghavachari Nalini N Woodhouse Kimberly K Yu Guoying G Barge Suchitra S Coronnello Claudia C Benos Panayiotis V PV Kato Gregory J GJ Kaminski Naftali N Gladwin Mark T MT

PloS one 20130412 4

<h4>Background</h4>Increased platelet activation in sickle cell disease (SCD) contributes to a state of hypercoagulability and confers a risk of thromboembolic complications. The role for post-transcriptional regulation of the platelet transcriptome by microRNAs (miRNAs) in SCD has not been previously explored. This is the first study to determine whether platelets from SCD exhibit an altered miRNA expression profile.<h4>Methods and findings</h4>We analyzed the expression of miRNAs isolated from ...[more]

PMID: 23593351

Similar Datasets

Project description:BackgroundIn sickle cell disease, ischemia-reperfusion injury and intravascular hemolysis produce endothelial dysfunction and vasculopathy characterized by reduced nitric oxide and arginine bioavailability. Recent functional studies of platelets in patients with sickle cell disease reveal a basally activated state, which suggests that pathological platelet activation may contribute to sickle cell disease vasculopathy.Methods and resultsStudies were therefore undertaken to examine transcriptional signaling pathways in platelets that may be dysregulated in sickle cell disease. We demonstrate and validate in the present study the feasibility of comparative platelet transcriptome studies on clinical samples from single donors by the application of RNA amplification followed by microarray-based analysis of 54,000 probe sets. Data mining an existing microarray database, we identified 220 highly abundant genes in platelets and a subset of 72 relatively platelet-specific genes, defined by >10-fold increased expression compared with the median of other cell types in the database with amplified transcripts. The highly abundant platelet transcripts found in the present study included 82% or 70% of platelet-abundant genes identified in 2 previous gene expression studies on nonamplified mRNA from pooled or apheresis samples, respectively. On comparing the platelet gene expression profiles in 18 patients with sickle cell disease in steady state to those of 12 black control subjects, at a 3-fold cutoff and 5% false-discovery rate, we identified approximately 100 differentially expressed genes, including multiple genes involved in arginine metabolism and redox homeostasis. Further characterization of these pathways with real-time polymerase chain reaction and biochemical assays revealed increased arginase II expression and activity and decreased platelet polyamine levels.ConclusionsThe present studies suggest a potential pathogenic role for platelet arginase and altered arginine and polyamine metabolism in sickle cell disease and provide a novel framework for the study of disease-specific platelet biology.

Project description:In sickle cell disease, ischemia-reperfusion injury and intravascular hemolysis produce endothelial dysfunction and vasculopathy characterized by reduced nitric oxide (NO) and arginine bioavailability. Recent functional studies of platelets in patients with sickle cell disease reveal a basally activated state, suggesting that pathological platelet activation may contribute to sickle cell disease vasculopathy. Studies were therefore undertaken to examine transcriptional signaling pathways in platelets that may be dysregulated in sickle cell disease. We demonstrate and validate here the feasibility of comparative platelet transcriptome studies on clinical samples from single donors, by the application of RNA amplification followed by microarray-based analysis of 54,000 probe sets. Data mining an existing microarray database, we identified 220 highly abundant genes in platelets and a subset of 72 relatively platelet-specific genes, defined by more than 10-fold increased expression compared to the median of other cell types in the database with amplified transcripts. The highly abundant platelet transcripts found in the current study included 82% or 70% of platelet abundant genes identified in two previous gene expression studies on non-amplified mRNA from pooled or apheresis samples, respectively. On comparing the platelet gene expression profiles in 18 patients with sickle cell disease in steady state to 12 African American controls, at a 3-fold cut-off and 5% false discovery rate, we identified ~100 differentially expressed genes, including multiple genes involved in arginine metabolism and redox homeostasis. Further characterization of these pathways using real time PCR and biochemical assays revealed increased arginase II expression and activity and decreased platelet polyamine levels. These studies suggest a potential pathogenic role for platelet arginase and altered arginine and polyamine metabolism in sickle cell disease and provide a novel framework for the study of disease-specific platelet biology. Keywords: Platelets, genes, enzymes, metabolism, signal transduction, sickle cell disease

Dataset Information

Expression of regulatory platelet microRNAs in patients with sickle cell disease.

Background

Methods and findings

Conclusions

Publications

Expression of regulatory platelet microRNAs in patients with sickle cell disease.

Similar Datasets

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