High miR-126-3p levels associated with cardiovascular events in a general population

Abstract

Background: Endothelial dysfunction is a forerunner of atherosclerosis, leading to cardiovascular disease, and albuminuria is a marker of endothelial dysfunction. Circulating levels of microRNAs are emerging as potential biomarkers for cardiovascular disease. Here we estimate the predictive value of a plasma microRNAs signature associated with albuminuria in the incidence of cardiovascular events.

Methods: Plasma microRNAs quantified in hypertensive patients by next generation sequencing were validated in a cohort of patients and controls by real-time quantitative PCR. The microRNAs found to be associated with albuminuria were analysed for their prognostic value in predicting cardiovascular events incidence on a retrospective, population-based study (Hortega Study), using Cox proportional hazard models.

Results: A plasma microRNA profile was identified in the discovery cohort (n = 48) associated with albuminuria and three microRNAs (miR-126-3p, miR-1260b and miR-374a-5p) were confirmed in the validation cohort (n = 98). The microRNA signature discriminates urinary albumin excretion at baseline (n = 1025), and predicts the incidence of cardiovascular events and coronary heart disease and stroke in a general population retrospective study within a 14-year follow-up (n = 926). High miR-126-3p levels were associated with a shorter time free of both cardiovascular events (HR=1.48, (1.36-1.62), p < 0.0001), as well as coronary artery disease and stroke combined (HR=2.49, (2.19-2.83), p < 0.0001).

Conclusions: An increased plasma microRNAs profile was identified in hypertensive patients with albuminuria. Increased miR-126-3p suggest it may serve as a prognostic marker for cardiovascular events in a long-term general population. Further studies will assess the potential role of miR-126-3p as a guide for the status of endothelial dysfunction.

Keywords: Albuminuria; Biomarker; Cardiovascular event; Cardiovascular risk; Endothelial dysfunction; MicroRNA.

Graphical abstract

Fig. 1

Study design for miRNA identification associated with albuminuria and cardiovascular events. The study design involved a discovery phase including hypertensives (HTN) (n = 48) with urinary albumin excretion (UAE) or normoalbuminuric (Non-UAE) by small RNA sequencing (Small RNA-Seq). In a second validation stage with a large hypertensive cohort (n = 69), including UAE or Non-UAE, and healthy controls (CNT, n = 29), 5 microRNA (miRNAs) were validated by real-time quantitative polymerase chain reaction (RT-qPCR). Finally, in a testing population cohort (Hortega's cohort), at baseline (n = 1025), the associations with 3 miRNAs (miR-374a-5p, miR-1260b and miR-126–3p) and albuminuria were tested. Then, at the 14-year follow-up (n = 926), the predictive power of miRNA levels with cardiovascular disease (CVD) events and combined coronary artery disease (CAD) + stroke incidence was investigated.

Fig. 2

Discriminatory power of plasma miRNAs for urinary albumin excretion. (a) Plasma levels of miR-374a-5p, miR-1260b and miR-126–3p in testing cohort 1025 participants, 83 with urinary albumin excretion (UAE). The microRNA (miRNA) levels have been quantified by real-time quantitative polymerase chain reaction (RT-qPCR), expressed as log10-transformed of absolute copy number and was normalized to miR-125a and miR-186 as internal controls. The miR-374a-5p, miR-1260b and miR-126–3p were significantly increased in UAE subjects. Data was compared using the Mann-Whitney U test. The ends of the boxes indicate the quartile 1(upper) and 3 (down), the middle line represents the median, and the whiskers are the minimum and maximum values of the data series (b) The areas under the curve (AUC) and receiver operating characteristics (ROC) curves were calculated for plasma miRNAs in the testing cohort. The diagnostic signature was calculated by a logistic regression model. The AUC of 3-miRNAs and 2-miRNAs signatures were significantly higher when compared to individual miRNAs (p < 0.05). (c) ROC curves are shown for individual and miRNA signatures.

Fig. 3

Kaplan–Meier of cardiovascular event-free survival. Testing cohort was grouped according to plasma miR-126–3p, with the value -0.92 as the cut-off obtained from receiver operating characteristic (ROC) analysis during the follow-up period of 14-years for cardiovascular disease (CVD) incidence (a) and for combined coronary artery disease (CAD) and stroke (b). Higher levels of miR-126–3p were associated with a significantly lower incidence rate of CVD and combined CAD and stroke. Weighted long-rank test: p-values at bottom survival curves are shown.