Differential expression of microRNA in serum fractions and association of Argonaute 1 microRNAs with heart failure

Abstract

The serum or plasma microRNA (miRNA) molecules have been suggested as diagnostic and prognostic biomarkers, in various pathological conditions. However, these molecules are also found in different serum fractions, such as exosomes and Argonaute (Ago) protein complexes. Ago1 is the predominant Ago protein expressed in heart tissue. The objective of the study was to examine the hypothesis that Ago1-associated miRNAs may be more relevant to cardiac disease and heart failure compared with the serum. In total, 84 miRNA molecules were screened for their expression in the whole serum, exosomes and Ago1, and Ago2 complexes. Ago1-bound miR-222-3p, miR-497-5p and miR-21-5p were significantly higher, and let-7a-5p was significantly lower in HF patients compared with healthy controls, whereas no such difference was observed for those markers in the serum samples among the groups. A combination of these 4 miRNAs into an Ago1-HF score provided a ROC curve with an AUC of 1, demonstrating clear discrimination between heart failure patients and healthy individuals. Ago1 fraction might be a better and more specific platform for identifying HF-related miRNAs compared with the whole serum.

Keywords: argonaute; heart failure; microRNA.

FIGURE 1

Global miRNA expression in whole serum, exosomes or in Ago1 and/or Ago2 protein complexes. Values shown are median miRNA expression in inverted C_t units (50‐C_t) for each donor. For each sample type, global expression was adjusted based on the initial amount of serum used, as described in the Methods Section

FIGURE 2

miRNA hierarchical clustering analysis. Forty out of the 84 measured miRNAs with the highest coefficient of variation were chosen for the analysis (selected miRNAs names are indicated on the Y‐axis in the left). The colored miRNAs names are RBC‐ (red) and WBC‐ (grey) associated miRNAs. The expression level of these 40 miRNAs is represented as a colored heat‐map (Y‐axis bar in the right). The correlation of miRNAs expression was used as a parameter for clustering distance. Three major clusters were formed: Ago1 + Ago2 RIP miRNAs, whole serum and exosomal miRNA and Ago1 associated miRNAs (indicated on X‐axis top). The numbers on the X‐axis in the lower bar indicate donors (n = 3)

FIGURE 3

miRNAs differentially associated with Ago1 vs Ago2 proteins in serum and plasma samples. Ago1‐ and Ago2‐assosiated miRNAs detected in serum and plasma samples were selected based on the following criteria: median fold change > 2 and P < .05 between Ago1 and Ago2 serum samples, and C_t < 37.5 (for both serum and plasma samples of all 3 donors). The colored miRNAs are RBC (red) and WBC (grey) associated miRNAs. Samples of three healthy donors were used for the analysis

FIGURE 4

Relative expression of miR‐222‐3p (A,B), let‐7a‐5p (C,D), miR‐497‐5p (E,F) and miR‐21‐5p (G,H) in Ago1 RIP (A,C,E,G) and whole serum (B,D,F,H) of healthy individuals (n = 10) and HF patients (n = 8)

FIGURE 5

(A,B) Values of Ago1‐HF score, calculated as a sum of miR‐222‐3p, let‐7a‐5p, miR‐497‐5p and miR‐21‐5p relative expression values, in Ago1 RIP (A) and whole serum (B). (C,D) ROC curve for HF score of healthy individuals (n = 10) vs HF patients (n = 8) in Ago1 RIP (C) and whole serum (D)