Expression of Regulatory Platelet MicroRNAs in Patients with Sickle Cell Disease

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. © 2013 Jain et al

Prenatal care programs in a clinic based center in El Paso, Texas: Evaluating outcomes among Hispanic women

Early prenatal care has been found to improve pregnancy and birth outcomes by reducing the risk of low birth weight and preterm births. Statistics point to lower utilization rates of prenatal care in El Paso, Texas and increasing rates of low birth weight. This study was a secondary data analysis, which explored the perceived benefits and barriers known to influence the utilization of prenatal care, and the birth outcomes among participating subjects attending three different prenatal care programs offered at the Centro San Vincent Clinic during the period of December 2006 to February 2008. A survey was used to collect data on demographics and assess perceived benefits and barriers regarding early prenatal care and self-efficacy of the participants. Post-partum birth records were used to gather data about the participant\u27s newborns to include birth weight, gestational age, and the type of delivery (i.e. vaginal versus Cesarean birth). Chi square analysis was conducted to test for significant differences and associations. The sample of women in the study had high risk factors for inadequate utilization of prenatal care. In spite of high demographic risk factors, these women had higher perceived benefits, lower perceived barriers and high self-efficacy associated with prenatal care utilization. The perceived benefits and barriers to prenatal care were associated with place of birth and language preferred, with women from Mexico and who preferred Spanish having higher perceived benefits and lower barriers. There was no significant difference in birth outcomes among the women in different prenatal care programs. The findings of this study suggest that of all participants in the study, those born in Mexico demonstrated higher rates of perceived benefits of early prenatal care. The analysis also suggested that healthy birth outcomes were evenly distributed among women participating in one of the three prenatal care programs

Central nervous system metastases from primary epithelial ovarian cancer

This article does not have an abstract

Plasma thrombospondin-1 is increased during acute sickle cell vaso-occlusive events and associated with acute chest syndrome, hydroxyurea therapy, and lower hemolytic rates

Platelets are activated in sickle cell disease (SCD), and particularly during vaso-occlusive episodes (VOE). Thrombospondin-1 (TSP1), a major secretory product of activated platelets, is increased in the circulation in VOE and binds to sickle red blood cells (RBC) promoting vascular adhesion. Thus, we hypothesized that TSP1 may represent a plasma biomarker of disease severity in SCD. We tested the plasma collected from patients in steady state (n = 27) and VOE (n = 14), as well as healthy controls (n = 17) at the University of Pittsburgh Medical Center (UPMC), and from patients in steady state enrolled in the walk-PHaSST clinical trial (n = 483). We found that TSP1 levels were increased in VOE in the UPMC cohort. Among steady-state patients at UPMC, TSP1 values correlated positively with lifetime history of acute chest syndrome (r = 0.72, P < 0.0001) and hemoglobin concentration (r = 0.49, P = 0.01), and negatively with markers of hemolysis, such as LDH (r = −0.50, P = 0.009). Analysis of the walk-PHaSST cohort also showed a positive association between TSP1 levels and hydroxyurea use (r = 0.14, P = 0.003), and confirmed the negative associations with the severity of hemolysis. Our results suggest that TSP1 levels are associated with more VOE, hydroxyurea use and lower rates of hemolysis. High TSP1 concentrations may indicate higher risk of the viscosity/vaso-occlusion phenotype of SCD

Characteristics of Study Participants.^*

*<p>Age and Laboratory values indicate median (range).</p

MiRNAs in platelets of SCD patients are differentially expressed.

<p>(<b>A</b>) Characterization of purified platelet preparation by flow cytometry. (<b>B</b>) Bioanalyzer assessment of RNA samples from purified platelet preps showing good quality with RIN of 7.8. (<b>C</b>) Venn diagram comparing differentially expressed miRNAs from two independent study cohorts. Comparison of 259 differentially expressed miRNAs from NHLBI samples and 67 differentially expressed miRNAs from UPMC samples. In total, there are 40 miRNAs overlapping between the two cohorts. (<b>D</b>) The heatmap depicts the 40 statistically significant (<i>p</i>-value<0.05, FC>2) differentially expressed miRNAs between SCD and controls that were common between the two cohorts. Columns represent individual samples and each represents a miRNA. Upregulated miRNAs expression levels are shown in progressively brighter shades of yellow, depending on the fold difference. Downregulated miRNAs are shown in progressively brighter shades of purple. No difference is represented as grey. The names of the miRNAs are displayed to the right of the heatmap.</p

miR-1225-3p regulates gene expression after transfection in MEG-01 cells.

<p>(<b>A</b>) Expression of the flourescent marker pmaxGFP plasmid as determined by flow cytometry demonstrates 76% GFP expressing cells out of the viable cell population. (<b>B</b>) qRT-PCR assay confirms overexpression of miR-1225-3p in MEG-01 cells (n = 5) vs scrambled (scr) (n = 5). (<b>C</b>) MT1X, PTPN6, IFI6, FCER1G and RAP2A are significantly downregulated in MEG-01 transfected cells as validated by qRT-PCR. (<b>D</b>) Schematic representation of overlap in differentially expressed genes between miR-1225-3p transfected MEG-01 cells and platelets from SCD patients. The numbers in the circles denote the differentially expressed genes with the leftmost figure representing genes using a FDR of 5% and the rightmost figure after applying a stringent statistical filter of FC>2. Further overlap with ComiR predicted target list of the 40 differentially expressed miRNAs resulted in a list of 7 genes potentially regulated by miR-1225-3p. (<b>E</b>) Out of the 7 genes, PLAGL2 and PBXIP1 genes are significantly downregulated and PHF20L1 is significantly upregulated in miR-1225-3p transfected cells vs scrambled. In the qRT-PCR figures (B, C and D), for each gene, the blue bar represents the cells transfected with scrambled RNA (n = 5) and the red bar represents the miR-1225-3p transfected cells (n = 5). Y-axis shows fold change of miR-1225-3p in transfected cells with the expression level in scrambled set to 1. Error bars are based on standard deviation. *denotes p-value<0.05.</p

Three distinct microRNA families among the differentially expressed microRNAs.

<p>Three distinct microRNA families among the differentially expressed microRNAs.</p

Functional annotation clustering of predicted target genes to the differentially expressed microRNAs belonging to three distinct families located at chromosome 14q32.31.

<p>Functional annotation clustering of predicted target genes to the differentially expressed microRNAs belonging to three distinct families located at chromosome 14q32.31.</p