Identification of novel circulating microRNAs in advanced heart failure by next-generation sequencing

Abstract

Aims: Risk stratification in patients with advanced chronic heart failure (HF) is an unmet need. Circulating microRNA (miRNA) levels have been proposed as diagnostic and prognostic biomarkers in several diseases including HF. The aims of the present study were to characterize HF-specific miRNA expression profiles and to identify miRNAs with prognostic value in HF patients.

Methods and results: We performed a global miRNome analysis using next-generation sequencing in the plasma of 30 advanced chronic HF patients and of matched healthy controls. A small subset of miRNAs was validated by real-time PCR (P < 0.0008). Pearson's correlation analysis was computed between miRNA expression levels and common HF markers. Multivariate prediction models were exploited to evaluate miRNA profiles' prognostic role. Thirty-two miRNAs were found to be dysregulated between the two groups. Six miRNAs (miR-210-3p, miR-22-5p, miR-22-3p, miR-21-3p, miR-339-3p, and miR-125a-5p) significantly correlated with HF biomarkers, among which N-terminal prohormone of brain natriuretic peptide. Inside the cohort of advanced HF population, we identified three miRNAs (miR-125a-5p, miR-10b-5p, and miR-9-5p) altered in HF patients experiencing the primary endpoint of cardiac death, heart transplantation, or mechanical circulatory support implantation when compared with those without clinical events. The three miRNAs added substantial prognostic power to Barcelona Bio-HF score, a multiparametric and validated risk stratification tool for HF (from area under the curve = 0.72 to area under the curve = 0.82).

Conclusions: This discovery study has characterized, for the first time, the advanced chronic HF-specific miRNA expression pattern. We identified a few miRNAs able to improve the prognostic stratification of HF patients based on common clinical and laboratory values. Further studies are needed to validate our results in larger populations.

Keywords: Biomarkers; Circulating microRNAs; Heart failure; Risk stratification.

Figure 1

Validation by real‐time PCR of a subset of four microRNAs differentially expressed between heart failure (HF) cases and controls in the next‐generation sequencing analyses (P‐value <0.0008 and log₂ fold change >0.6). Blue: controls. Red: HF patients. **P‐value ≤0.01.

Figure 2

Differentially expressed microRNAs significantly correlated with heart failure (HF) biomarkers [copeptin, N‐terminal prohormone of brain natriuretic peptide (NT‐proBNP), soluble ST2 (sST2), and high‐sensitivity cardiac troponin T (Hs‐cTnT)] and Barcelona (BCN) Bio‐HF score. Bright red (R > 0.40) and bright green (R < −0.40) colours are associated with stronger positive and negative correlations, respectively. Dark red and dark green colours are weak correlation values. MicroRNA expression and clinical variable values are reported as base 2 logarithms. The correlation coefficients are shown (R).

Figure 3

Circulating microRNAs (miRNAs) related to clinical outcomes. (A) miRNAs differentially expressed among heart failure (HF) patients experiencing primary endpoint (HF‐PE, red) and those without clinical events (HF‐noPE, green). *Adjusted P‐value = 0.04. ^♦Outlier sample values. (B) Correlation levels of the three differentially expressed miRNAs in HF‐PE (red) vs. HF‐noPE (green) with clinical variable significant predictive markers (Table  2 ). miRNA expression and clinical variable values are reported as base 2 logarithms. The correlation coefficients are shown (R). (C) Receiver operating characteristic curves built for the three differentially expressed miRNAs among HF‐PE and HF‐noPE groups (green), Barcelona (BCN) Bio‐HF score (blue), and three miRNAs + BCN Bio‐HF score (red). The value of area under the curve (AUC) was reported. TPG, trans‐pulmonary gradient.