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

miRNAs in platelets have a unique expression profile in SCD patients based on Doppler-estimated TRV and HU treatment.

<p>(A) The heatmap represents 23 statistically significant (<i>p</i>-value<0.1, FC>2) differentially expressed miRNAs between subjects with (N = 18) and without (N = 8) elevated TRV (>2.5 m/sec). (B) The heatmap represents 10 statistically significant (<i>p</i>-value<0.1, FC>2) differentially expressed miRNAs between the subjects on (N = 14) and off HU (N = 12). Every row represents a gene and every column is a patient. Upregulated miRNAs are shown in yellow color, downregulated miRNAs are shown in purple and no difference is grey. The names of the miRNAs are displayed to the right of heatmap.</p

Most significant predicted canonical pathways regulated by the target genes of the 3 miRNAs: 376a, 409-3p and 1225-3p (p-value<0.01).

<p>Orange squares depict ratio which is the number of target genes associated with each pathway. The vertical line across the bars represents the cut-off of the p-value.</p

Twenty most differentially expressed (A) up- and (B) down-regulated microRNAs (P<0.05, fold change>2) between sickle cell disease (N = 26) and controls (N = 17) ranked according to p-value (low to high).

<p>Twenty most differentially expressed (A) up- and (B) down-regulated microRNAs (P<0.05, fold change>2) between sickle cell disease (N = 26) and controls (N = 17) ranked according to p-value (low to high).</p

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

List of novel putative target genes of miR-1225-3p from the differentially expressed genes (FDR<5%, FC>2).

<p>List of novel putative target genes of miR-1225-3p from the differentially expressed genes (FDR<5%, FC>2).</p

Validation of RNA-sequencing data by quantitative real-time PCR.

<p>Representative up-regulated and down-regulated target genes from the RNA-Seq data set were validated with real time quantitative PCR using the same samples used for RNA-Seq assays (*<i>p</i><0.05 two-tailed t-test, vs. respective control, n = 4–5). Values represent mean (+/-) SEM of relative gene expression changes vs. 18S housekeeping gene, calculated by the ΔΔCt method. Human alveolar macrophages were stimulated in the presence or absence of LPS for 6 h.</p