Journal: QJM: An International Journal of Medicine
Article Title: Acute endothelial stresses identify microRNA let-7b-5p and non-coding SLC11A2 (NRAMP2/DMT1) exon as biomarkers that overlap with those detected in malignant and non-malignant diseases
doi: 10.1093/qjmed/hcae235
Figure Lengend Snippet: Pre-miRNA and mature miR responses to ferric citrate treatment of normal primary ECs. ( A ) RNA-seq data for all pre-miRNAs detected in 1 and 6 h rRNA-depleted libraries from EC treated with media or 10 μmol/l ferric citrate, presented as fold change of respective media-treated EC represented at 0 h. One hour data are from normal HDMEC compared to paired 1 h media-treated HDMEC. Six hour data are from normal HPMEC compared to paired 6 h media-treated HPMEC. By Dunn’s test post Kruskal–Wallis, there was a fall (* P < 0.05) after 1 h treatment, and no significant change after 6 h. ( B ) RNA-seq data for let-7 pre-miRNAs as in (A) (let-7a-1, let-7a-2, let-7a-3, let-7b, let-7d, let-7f-1, let-7g, let-7i), with P values (* P < 0.05) calculated by Dunn’s test post Friedman. ( C ) RNA-seq data for mRNA changes in HPMEC after 6 h 10μmol/l ferric citrate, as fold change of respective values in paired media-treated HPMEC, categorized by whether mRNAs were identified by TargetScan 5.2[28] as a let-7 target based on mature miRNAs from miRbase: 570 of 10 851 mRNAs had 1-6 (mean 1.11) let-7 family 8mer, 7mer-m8 or 7mer-1A binding sites . ( D ) Comparisons of let-7b by qRT-PCR and RNA-seq. (i) Mature miRNA let-7b-5p, assayed at stated durations of 10μmol/l ferric citrate as quantified in HPMEC by qRT-PCR using Applied Biosystems miRNA assay 002619. Ct values were converted to concentrations following spiking of cell extracts with known concentrations of cel-miR-39 as described in the . (ii) RNA-seq comparisons (as also represented in A and B). ( E ) Dose–response curves for mature let-7b-5p in four cultures of primary HDMEC or primary HUVEC, after 1 h treatment with media (‘0’), 4 or 10 μmol/l ferric citrate treatments, quantified by qRT-PCR using miRNA assay 002619 (Applied Biosystems). Threshold cycles (Ct) values were converted to concentrations based on known concentrations of spiked cel-miR-39 (described further in ), before fold-changes from the media-treated EC were calculated for graphical presentation.
Article Snippet: For treatment duration, no morphological changes were observed in primary human pulmonary microvascular ECs (HPMEC) or primary human dermal microvascular ECs (HDMEC, PromoCell GmbH, Heidelberg) cultured for 24 h with 10 μmol/l ferric citrate: 1 h and 6 h responses were evaluated.
Techniques: RNA Sequencing, Binding Assay, Quantitative RT-PCR
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![Pre-miRNA and mature miR responses to ferric citrate treatment of normal primary ECs. ( A ) RNA-seq data for all pre-miRNAs detected in 1 and 6 h rRNA-depleted libraries from EC treated with media or 10 μmol/l ferric citrate, presented as fold change of respective media-treated EC represented at 0 h. One hour data are from normal HDMEC compared to paired 1 h media-treated HDMEC. Six hour data are from normal <t>HPMEC</t> compared to paired 6 h media-treated HPMEC. By Dunn’s test post Kruskal–Wallis, there was a fall (* P < 0.05) after 1 h treatment, and no significant change after 6 h. ( B ) RNA-seq data for let-7 pre-miRNAs as in (A) (let-7a-1, let-7a-2, let-7a-3, let-7b, let-7d, let-7f-1, let-7g, let-7i), with P values (* P < 0.05) calculated by Dunn’s test post Friedman. ( C ) RNA-seq data for mRNA changes in HPMEC after 6 h 10μmol/l ferric citrate, as fold change of respective values in paired media-treated HPMEC, categorized by whether mRNAs were identified by TargetScan 5.2[28] as a let-7 target based on mature miRNAs from miRbase: 570 of 10 851 mRNAs had 1-6 (mean 1.11) let-7 family 8mer, 7mer-m8 or 7mer-1A binding sites . ( D ) Comparisons of let-7b by qRT-PCR and RNA-seq. (i) Mature miRNA let-7b-5p, assayed at stated durations of 10μmol/l ferric citrate as quantified in HPMEC by qRT-PCR using Applied Biosystems miRNA assay 002619. Ct values were converted to concentrations following spiking of cell extracts with known concentrations of cel-miR-39 as described in the . (ii) RNA-seq comparisons (as also represented in A and B). ( E ) Dose–response curves for mature let-7b-5p in four cultures of primary HDMEC or primary HUVEC, after 1 h treatment with media (‘0’), 4 or 10 μmol/l ferric citrate treatments, quantified by qRT-PCR using miRNA assay 002619 (Applied Biosystems). Threshold cycles (Ct) values were converted to concentrations based on known concentrations of spiked cel-miR-39 (described further in ), before fold-changes from the media-treated EC were calculated for graphical presentation.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_8437/pmc12668437/pmc12668437__hcae235f2.jpg)
