Bioinformatic Analysis of Altered microRNA Production in Normal Adrenal Tissue and Aldosterone-Producing Adenoma Nur Izah Ab Razak1,2, Louise A. Diver1, Samantha Alvarez-Madrazo1, Stacy Robertson1, Martin McBride1, Paul M. Stewart3, John M. Connell4, Scott M. MacKenzie1 & Eleanor Davies1 1Institute of Cardiovascular and Medical Sciences, University of Glasgow, U.K.; 2Faculty of Medicine & Health Sciences, University Putra Malaysia, Serdang, Selangor, Malaysia; 3Faculty of Medicine & Health, University of Leeds, U.K.; 4College of Medicine, Dentistry & Nursing, University of Dundee, U.K. Corresponding author: n.ab-razak.1@research.gla.ac.uk Figure 1: Biosynthesis of aldosterone in the adrenal cortex. To use a combination of microarray and bioinformatic analyses to identify miRNAs likely to have a role in the aetiology and pathophysiology of APA. Objective Introduction Hypertension is a common risk factor for cardiovascular disease. Up to 15% of hypertensive patients are now known to have primary aldosteronism (PA), where the adrenal glands secrete inappropriately high levels of aldosterone. Almost half of these cases are due to unilateral aldosterone-producing adenoma (APA). Aldosterone is synthesized in the zona glomerulosa of the adrenal cortex, with the final stages of production catalysed by aldosterone synthase, an enzyme encoded by the CYP11B2 gene (Figure 1). Previous studies (Robertson et al.,Hypertension 2013:62;572) show CYP11B2 expression is subject to regulation by microRNAs (miRNAs), which are small, single-stranded RNAs 20–25 nucleotides in size that target particular messenger RNAs (mRNAs), negatively modulating gene expression at the post-transcriptional level. We identified miRNAs dysregulated in APA compared to normal adrenal (NA) and examined their possible effects. Methods microRNA isolated from human APA and normal adrenal (NA) tissue samples (each n=4) was analysed by microarray. The resulting data were subjected to bioinformatic analysis, including use of the DIANA micro-T database and Ingenuity Pathway Analysis (IPA) software package. Results Microarray analysis of APA vs NA samples shows marked consistency in miRNA levels between the two tissue types (Figure 2). 33 miRNAs detected above the arbitrary threshold level (>500AU) were differentially expressed between the two tissue types (p<0.05): 19 were higher in NA and 14 in APA . DIANA MicroT CDS database analysis predicts 5 of the expressed miRNAs to target CYP11B2 mRNA (Figure 3)., so may have a direct regulatory influence on aldosterone production More sophisticated bioinformatic analysis using the IPA package showed Endocrine System Disorders to be one of the top 3 disease states associated with these miRNAs, alongside Cancer and Cardiovascular. Differentially expressed miRNAs are predicted to target mRNAs with functions related to APA, i.e. steroidogenesis, cholesterol biosynthesis, apoptosis, cell proliferation (Figure 4). Plausible mRNA targets for these 33 miRNAs were predicted using IPA. Highlights of the analysis are shown in Figures 5 and 6. Figure 5A shows selected miRNAs expressed at higher levels in NA samples; their predicted targets, including VEGF and members of the WNT family, are shown in Figure 6A. Of particular interest is 3-hydroxy-3-methylglutaryl-CoA-reductase (HMGCR) mRNA, which is predicted to bind four miRNAs each significantly downregulated in APA. HMGCR represents a rate-limiting step in cholesterol production and may be an important determinant of steroid biosynthesis. Figure 5B shows miRNAs expressed at higher levels in APA samples; predicted targets, again including VEGF, are shown in Figure 6B. Figure 2: Heat map of differentially expressed miRNAs in NA and APA. Comparison of human NA and APA samples (each n=4) analysed by microarray. miRNAs shown were differentially expressed (p<0.05). Green colour corresponds to relative downregulation, red to upregulation. Figure 3: Tissue miRNAs predicted by DIANA MicroT to target CYP11B2. Figure 4: Summary of differentially expressed miRNA analysis using Ingenuity Pathway Analysis (IPA). Figure 5: selected miRNAs differentially expressed between APA and NA samples. A: miRNAs expressed at significantly greater levels in NA samples. B: miRNAs expressed at significantly greater levels in NA samples. All p<0.05 between groups. A B A B Figure 6: : Selected mechanisms of miRNA action predicted by IPA software. A: Mechanisms involving miRNAs present at higher levels in NA tissue (see Figure 5A, above). B: Mechanisms involving miRNAs present at higher levels in APA (see Figure 5B, above). Conclusions miRNAs dysregulated in APAs are bioinformatically predicted to have functional effects on steroidogenesis, cell proliferation and apoptosis. These findings will direct future in vitro analyses designed to confirm these predicted mRNA targets and understand mechanisms underlying the aetiology of APA. miRNAs and their targets may be valuable biomarkers in the diagnosis and treatment of APA.