Microarray, validation and gene-enrichment approach for assessing differentially expressed circulating miRNAs in obese and lean heart failure patients

Abstract

BackgroundObesity is a risk factor associated with cardiovascular diseases that may lead to heart failure (HF). However, in HF, overweight and obese patients have longer survival than underweight patients, a phenomenon known as the obesity paradox. MiRNAs play a fundamental role in gene regulation and transcription factors involved in obesity and HF. The main objective of this study was to identify and validate differentially expressed circulating miRNAs in HF-obese and HF-lean patients. Materials and MethodsThis case-control study was carried out in two parts; the discovery and validation phase. In the discovery phase, plasma samples from 20 HF patients and from 10 healthy controls for microarray analysis were selected. The miRNAs were extracted and then analyzed on the miRNA 4.0 Affymetrix GeneChip array following the manufacturers instructions. Raw data were normalized using Robust Microarray Average (RMA), batch effects were adjusted with Surrogate Variable Analysis (SVA), and differential expression analysis was performed with the Limma R package. Differentially expressed miRNAs were ranked based on effect size, p-value, and biological plausibility, and selected for validation. In the validation phase, plasma miRNAs from 80 patients and controls were extracted and miRNAs -451a, - 22-3p, and -548ac were validated by quantitative reverse transcription-PCR. Then, they were subjected to target analysis using miRTarBase release 8.0 and TarBase v8.0. We performed functional enrichment analysis of miRNA targets using pathway annotation from the KEGG Pathway Database using the ClueGO software. ResultsMiRNAs -451a, -22-3p, and -548ac were up-regulated in HF-lean and HF-obese groups compared to control, showing an association of these miRNAs with HF. Enrichment analysis showed an association of validated miRNAs with AKT1, MAPK1, GRB2, and IGF1R genes. These genes are associated with different biological processes, such as apoptosis, carbohydrate metabolism, neurogenesis, sugar transport, translation regulation, transport, cell cycle, actin cytoskeleton reorganization, cardiac neural crest cell development involved in heart development, and aging. ConclusionmiR-451a, miR-22-3p, and miR-548ac are up-regulated in HF, independent of obesity, and are associated with metabolic, morphological, and functional outcomes. These results might help in the selection of targets for studies aiming to underscore the mechanisms of HF.