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.

Abstract

Background: Disease biomarkers are often identified long after initiating pathologies, hampering mechanistic understanding and development of preventative strategies. We hypothesised that aberrant cellular responses to normally-encountered stresses may be relevant to later disease states.

Aim: To model two under-explored acute cellular stresses for blood-exposed cells, and cross-reference to known biomarkers of disease.

Methods: Normal primary human endothelial cells (ECs) were treated for 1-6 h with cycloheximide 100 μg/mL to inhibit protein translation and nonsense mediated decay (modelling the integrated stress response), or 10 μmol/L ferric citrate (modelling diurnal variation in serum iron that can be augmented by treatments prescribed 8 million times/year in England). Directional whole transcriptome RNA-seq identified differentially expressed genes and micro(mi)RNAs. Customized novel scripts examined expression of 517,225 exons to predict 1 h cycloheximide-stabilized exons. Validations were by cel-miR-39-spiked qRT-PCR and RNA-seq in other endothelial cell types, peripheral blood mononuclear cells (PBMCs), and plasma.

Results: miRNAs fell transiently at 1 h after 10 μmol/L ferric citrate (p < 0.01), specifically in let-7 family member pre-miRNAs ('let-7', p < 0.05), where there was an accompanying differential 6 h increased expression of 570 let-7-target mRNAs identified through TargetScan (p < 0.0001). qRT-PCR and RNA-seq validations in other normal endothelial cells, plasma and PBMCs confirmed up to 80% falls in pre-let-7b/let-7b-5p after 1 h iron, and exon 3B of the SLC11A2 (NRAMP2/DMT1)-encoded iron/copper transporter as a novel exon most consistently stabilized following 1 h treatment with cycloheximide. Overlaps with disease biomarkers for cancer, growth retardation, and multiple organ-specific diseases were identified.

Conclusions: Biomarkers for normal, acute cellular responses overlap with disease-state biomarkers, warranting further study.