REVIEW

Risk Stratification and Biomarkers

Circulating MicroRNAs as Novel Biomarkers in Risk Assessment

and Prognosis of Coronary Artery Disease

Chiara Vavassori ,1,2 Eleonora Cipriani 1

and Gualtiero Ivanoe Colombo 1

1. Unit of Immunology and Functional Genomics, Centro Cardiologico Monzino, IRCCS, Milan, Italy;
2. Cardiovascular Section, Department of Clinical Sciences and Community Health, University of Milan, Milan, Italy

Abstract
Coronary artery disease is among the leading causes of death worldwide. Nevertheless, available cardiovascular risk prediction algorithms still
miss a significant portion of individuals at-risk. Thus, the search for novel non-invasive biomarkers to refine cardiovascular risk assessment is
both an urgent need and an attractive topic, which may lead to a more accurate risk stratification and/or prognostic score definition for coronary
artery disease. A new class of such non-invasive biomarkers is represented by extracellular microRNAs (miRNAs) circulating in the blood. MiRNAs
are non-coding RNA of 22–25 nucleotides in length that play a significant role in both cardiovascular physiology and pathophysiology. Given their
high stability and conservation, resistance to degradative enzymes, and detectability in body fluids, circulating miRNAs are promising emerging
biomarkers, and specific expression patterns have already been associated with a wide range of cardiovascular conditions. In this review, an
overview of the role of blood miRNAs in risk assessment and prognosis of coronary artery disease is given.

Keywords
MicroRNA, biomarkers, prognosis, prevention, cardiovascular risk, coronary artery disease

Disclosure: GIC is supported by Fondazione Regionale per la Ricerca Biomedica (FRRB), Research Grant no. CP2_14/2018 ‘INTESTRAT-CAD’ and Italian Ministry of Health,
Project ID RCR-2019 –23669118_001. All other authors have no conflicts of interest to declare.
Received: 30 August 2021 Accepted: 5 January 2022 Citation: European Cardiology Review 2022;17:e06. DOI: https;//[Link]/10.15420/ecr.2021.47
Correspondence: Gualtiero I Colombo, Unit of Immunology and Functional Genomics, Centro Cardiologico Monzino IRCCS, Via Carlo Parea, 4 – 20138 Milan, Italy.
E: [Link]@[Link]

Open Access: This work is open access under the CC-BY-NC 4.0 License which allows users to copy, redistribute and make derivative works for non-commercial
purposes, provided the original work is cited correctly.

Cardiovascular diseases (CVDs) are the leading cause of death globally, In the past two decades, great attention has been paid to the role of
representing a significant concern for public health. Coronary artery miRNAs as diagnostic and prognostic biomarkers, as well as therapeutic
disease (CAD) accounts for nearly half of all CVD deaths worldwide.1,2 targets in heart disease. In the present review, we summarise existing
Despite improvements in primary prevention and treatment, and the knowledge on miRNAs as biomarkers for risk stratification and prognosis
development of several risk stratification algorithms to predict 10-year in CAD.
cardiovascular mortality or lifetime risk in different populations, the
prevalence of CAD continues to rise.1,3,4 Thus, the identification of novel MicroRNA Function and Biogenesis
biomarkers for early and accurate recognition of at-risk individuals is of MiRNAs are short (22–25 nucleotides), non-coding, single-stranded RNA
paramount importance. molecules that regulate the expression of 60% of protein-coding genes at
the post-transcriptional level by complementary binding to the 3´
Circulating microRNAs (miRNAs) represent optimal non-invasive potential untranslated region of the target mRNA, leading to inhibition of translation
candidates for both diagnostic and prognostic purposes.5 Due to their or mRNA degradation.11,12 They could also interact with the 5´ untranslated
high stability, phylogenetic conservation and resistance to degradative region, causing gene expression silencing (Figure 2).13
enzymes, and due to their easy detectability in body fluids thanks to
advances in molecular detection techniques, miRNAs may serve as stable MiRNA canonical biogenesis consists of several steps: transcription,
and reliable reporters of disease onset and progression. MiRNAs are non- processing, splicing, export to the cytoplasm, maturation and target
coding RNAs that play a role as ‘fine-tuners’ of gene expression.6 They are binding.14 Briefly, a pri-miRNA (a large structure composed of sequences
key regulators of the cardiovascular system, including heart muscle for several miRNAs) is synthesised by the RNA polymerase II enzyme in
contraction and growth, conductance of electrical signals, vessel wall the nucleus. Pri-miRNAs are cleaved by the RNA-specific RNase-III-type
homeostasis, response to vascular injury and tissue repair.7 Dysregulation endonuclease, Drosha, with its cofactor DGCR8, to produce pre-miRNAs
of their expression is involved in cardiovascular pathophysiology, and (70 nucleotides), which are exported in the cytoplasm via exportin-5 and
circulating miRNAs may contribute to the interaction between inflammatory RanGTP-binding protein. Pre-miRNAs are further processed by Dicer (the
and vascular cells, systemic inflammation, and oxidative stress, and thus cytoplasmic RNase-III endonuclease) into a double-stranded miRNA
to CAD pathogenesis (Figure 1).8–10 duplex and loaded on the RNA-induced silencing complex, where the

© RADCLIFFE CARDIOLOGY 2022

[Link]
 MicroRNAs as Novel Risk Biomarkers for CAD

Figure 1: Schematic Illustration of MicroRNA Involvement in Coronary Artery Disease Pathogenesis

CAD and miRNA

LUMEN
Erythrocyte Atherosclerotic
plaque
Monocytes

EC activation and
inflammation
EC apoptosis Angiogenesis
miR-10a; miR-30-5p; miR-143/145;
miR-21; miR-155; miR-146a; miR-181b; miR-100; Let-7a; miR-126-5p; miR-92a;
miR-181a, miR-351 Let-7b; Let-7g; miR-34a; miR-92a; miR-200; miR-210; miR-221/222
miR-712/205; miR-103; miR-155; miR-21

ENDOTHELIUM

HDL LDL Macrophage activation

Cholesterol efflux and
reverse transport miR-93; miR-124; miR-223; miR-125a;
miR-223; miR-9-5p; miR-27a/b; miR-33; miR-46; miR-10a; miR-147; miR-124a;
miR-96; miR-144; miR-148; miR-185; miR-758 Ox-LDL miR-150; let-7c; miR-33; miR-142-5p

Cholesterol accumulation Foam cell VSMC proliferation VSMC survival

and foam cell formation and migration in fibrous cap
miR-10a; miR-223; miR-30c; Fibrosis miR-22-3p; miR-125b; miR-24; miR-181b; miR-210;
miR-125-5p; miR-122; miR-146a miR-29a; miR-663; miR-638; miR-21
miR-21; miR-29;
miR-133; miR-424; miR-195;
INTIMA miR-30
let-7b; miR-146a
BASAL LAMINA

MEDIA

MicroRNAs known to be involved in atherosclerotic plaque formation are listed at the site of their action and/or along with the process they influence: endothelial cell apoptosis, activation and
inflammation; neo-angiogenesis; cholesterol efflux, reverse transport and accumulation; vascular smooth muscle cell proliferation, migration and survival in fibrous cap; fibrosis; macrophage activation;
and foam cell formation. MicroRNAs highlighted in bold blue have been also reported as potential circulating biomarkers for coronary artery disease. CAD = coronary artery disease; EC = endothelial
cell; miRNA = microRNA; Ox-LDL = oxidised LDL; VSMC = vascular smooth muscle cell.

guide and passenger strands are selected by Argonaute protein.15 The signatures in cells, tissues or biological fluids, demonstrating their
RNA-induced silencing complex leads the guide strand to bind to its target association with human diseases and/or their prognosis.22 Although the
mRNA through a complementary interaction between the miRNA and vast majority did not prove to be clinically relevant, specific miRNA
target mRNA seeding sequences.14 expression signatures, especially in serum or plasma, have shown
promise as minimally invasive tools for diagnostic/prognostic purposes.
Extracellular miRNAs are secreted into the blood circulation through The potential use of circulating miRNAs for diagnostic purposes has been
several carriers to protect them from digestion: packed in exosomes, extensively explored in CAD.23,24
microparticles or lipid vesicles; bound to Argonaute-2 or nucleophosmin-1
proteins; or linked with high- or low-density lipoproteins.16–18 Circulating MicroRNAs in Coronary
Artery Disease Risk Assessment
MicroRNAs as Circulating Biomarkers Several risk algorithms have been developed for CVD primary prevention,
Extracellular miRNAs have been detected in different body fluids, such as mostly based on traditional cardiovascular risk factors (TRFs; e.g. sex,
pleural, peritoneal, cerebrospinal, seminal and amniotic fluids, urine, age, smoking habit, total and HDL cholesterol, systolic blood pressure,
breast milk, colostrum, saliva, tears and blood.19 Differently from treatment, diabetes).3 However, although they represent very useful tools
intracellular mRNAs, extracellular miRNAs circulating in the blood show to help clinicians stratify patients based on risk and guide treatment
great stability under harsh conditions (such as extreme environmental options, the performance of these models is suboptimal, showing the
basic pH, high temperature, multiple repeated freeze–thaw cycles or need for new refined and accurate risk stratification models, including
prolonged storage) and resistance to endogenous RNase activity.20 novel biomarkers.25 Yet, studies on the role of circulating miRNAs in
Moreover, miRNAs are evolutionarily conserved among species, allowing cardiovascular risk stratification are rather sparse.
translation from preclinical models to clinical practice.21 Finally, they are
easily detectable by sensitive techniques, such as reverse transcription Primary Prevention Settings
quantitative real-time polymerase chain reaction (RT-qPCR). The main results of studies assessing circulating miRNAs potential as
predictors of future cardiovascular events in the general population (i.e. in
These properties raised enormous interest in their use as biomarkers for primary prevention) are shown in Table 1, along with sample type,
various diseases. Indeed, since 2008, when Lawrie et al. first demonstrated detection and normalisation methods, miRNA regulation, primary
the potential of serum miRNAs as biomarkers for diffuse large B-cell outcome, and factors and covariates for adjustment in multivariable
lymphoma, several studies have identified specific miRNA expression analyses.

EUROPEAN CARDIOLOGY REVIEW

[Link]
 MicroRNAs as Novel Risk Biomarkers for CAD

Figure 2: Schematic Illustration of MicroRNA Biogenesis, Mechanisms of Action and Extracellular Release

LDL
Lipoproteins EXTRACELLULAR
Lipid vesicle SPACE
HDL
NPM1 Nucleophosmin
Exosomes

Argonaute-2 Ago2

RISC
NUCLEUS

Pol-II Translational repression

Mature miRNA or deadenylation
miRNA gene
Dicer RISC
5´ Cap AAAAAAA
Partial
Passenger complementarity
Pri-miRNA miRNA duplex strand
mRNA target
Drosha, degradation
Dgcr8
Exportin 5 RISC
5´ Cap AAAAAAA
Pre-miRNA Pre-miRNA Perfect
CYTOPLASM complementarity
mRNA degradation

MiRNA genes are transcribed by RNA polymerase II in primary miRNA transcripts. These hairpin loop structures are recognised by the DiGeorge Syndrome Critical Region 8 protein, which associates with
the Drosha enzyme, a double-stranded RNA-specific ribonuclease III. The cleavage product, the precursor miRNA, is exported by Exportin-5 to the cytoplasm, where it is processed by the Dicer enzyme,
first in a duplex conformation and then in a mature miRNA. This could be incorporated into the RNA-induced silencing complex through which, depending on the total or partial complementarity with the
target, it leads to mRNA degradation or mRNA translational repression, respectively. Both pre-miRNAs and mature miRNAs can be secreted into the extracellular space through several carriers, such as
lipid vesicles, exosomes, LDLs and HDLs, or bind to RNA-binding proteins nucleophosmin 1 and Ago2. Ago2 = Argonaute 2; Dgcr8 = DiGeorge syndrome critical region 8; mRNA = messenger RNA;
miRNA = microRNA; NPM1 = nucleophosmin 1; Pol II = polymerase II; pre-miRNA = precursor microRNA; pri-miRNA = primary microRNA transcript; RISC = RNA-induced silencing complex.

An early study that prospectively sought an association between basal platelets, and an interventional study by subjecting 11 healthy volunteers to
miRNA levels and incident MI was that of Zampetaki et al., reporting in limb ischaemia-reperfusion (which induces platelet activation), assessing
2012 a significant relationship between plasma levels of three miRNAs plasma levels of 30 candidate miRNAs at various time-points. Through
and MI (miR-126-3p positively associated, while miR-197 and miR-223 computational analysis, they identified a temporal cluster containing all
inversely associated).26 Using RT-qPCR, they evaluated the baseline three MI-predictive miRNAs, characterised by early and sustained
plasma expression of seven miRNA candidates (miR-24, miR-126, miR-140, elevation, and consisting of miRNAs predominantly expressed in platelets.
miR-150, miR-197, miR-223 and miR-486) in a population-based cohort of
820 participants (aged 40–79 years) in the Bruneck Study.27 A total of 47 In 2016, Bye et al. described a signature of five miRNAs (miR-106a-5p,
participants experienced fatal or non-fatal MI within the 10-year follow-up miR-424-5p, let-7g-5p, miR-144-3p and miR-660-5p) that could predict
period. Seven miRNAs emerged as promising from an initial screening future fatal acute MI (AMI) in healthy individuals.29 They assessed by RT-
using Taqman miRNA arrays, covering 754 small non-coding RNA, on eight qPCR the baseline serum levels of a panel of 179 miRNAs in a prospective
pooled samples of participants with or without atherosclerotic vascular nested case–control study cohort of 112 healthy individuals (aged 40–70
disease matched for various TRFs. They used two distinct approaches to years) participating in the HUNT study with a 10-year observation period.30
select the subset of miRNAs with the highest predictive ability for future This derivation cohort consisted of 56 cases suffering from fatal AMI
MI (i.e. stepwise Cox regression analyses followed by comparison of the within 10 years and 56 controls remaining healthy matched for risk factors.
emerging models’ Akaike information criterion; L1 penalisation, based on MiRNAs found to be differentially expressed in the screening were then
least absolute shrinkage and selection operator algorithm), which identify analysed in an independent validation cohort of 100 healthy participants
the same miRNA signature. from the same study, with 50 suffering from fatal AMI and 50 risk factor-
matched controls reporting no cardiovascular events: 10 miRNAs were
Then, risk estimates for the three miRNAs selected were computed by confirmed to be differentially expressed in cases versus controls.
standard Cox regression analysis adjusting for a set of TRFs. They showed
that adding the three miRNAs to a Framingham Risk Score (FRS) model for The exclusion criteria for both cohorts included previous cardiovascular
hard endpoints of coronary heart disease slightly increased both the events and several comorbidities. Interestingly, a sex-specific association
C-index, the net reclassification index, and the integrated discrimination was observed, as miR-424-5p and miR-26a-5p were exclusively related to
improvement.28 risk in men and women, respectively.

Finally, to determine the cellular origin of these three miRNAs, they Finally, a model consisting of the five miRNAs with the highest predictive
performed a miRNA screening in preparations of thrombin-activated ability for future AMI in both sexes was identified by conditional logistic

EUROPEAN CARDIOLOGY REVIEW

[Link]
 MicroRNAs as Novel Risk Biomarkers for CAD

regression followed by a comparison of the Akaike information criterion of The addition of the 5-miRNA panel significantly improved risk
the models, providing 77.6% overall correct classification. Adding the five stratification models based on the FRS or the SCORE in predicting
miRNAs to the FRS significantly increased the receiver operating mortality, increasing both net reclassification index and integrated
characteristic area under the curve (AUC). discrimination improvement.

Three years later (Velle-Forbord et al.), leveraging the population-based Using a different approach, in 2020, Gigante et al. tried to identify
HUNT cohort, the same group showed that a combination of another five circulating miRNA signatures that predict major adverse cardiovascular
miRNAs (miR-21-5p, miR-26a-5p, mir-29c-3p, miR-144-3p and miR-151a- events (MACE; defined as sudden cardiac death, first-time MI or angina
5p) added to the FRS was the best predictive risk model for both fatal and requiring hospitalisation) in middle-aged men and women.41 To this end,
non-fatal MI.31 They performed a case–control study, testing by RT-qPCR they took advantage of a prospective Swedish cohort of 60-year-old men
in baseline serum samples of 195 participants (aged 60–79 years) the 10 and women from the Stockholm Study, selecting the first 100 MACE-
candidate miRNAs (let-7g-5p, miR-21-5p, miR-26a-5p, miR-29c-3p, miR- presenting individuals and 100 MACE-free individuals in an 11-year follow-
106a-5p, miR-144-3p, miR-151a-5p, miR-191-5p, miR-424-5p and miR-451a) up (matched for sex and time of inclusion in the cohort).42
previously explored for their potential to predict future fatal MI.29
They initially screened a panel of 754 miRNAs, using TaqMan OpenArrays,
Specifically, during the 10-year follow-up, 36 and 60 participants in baseline plasma samples. Using a random effects logistic regression,
experienced either a fatal or a non-fatal MI, respectively, whereas 99 age- adjusted for common TRFs, they identified nine miRNAs potentially
and sex-matched controls remained healthy. They used the same logistic associated with an increased rate of future MACEs, being miR-145-3p
regression approach for the best subset to identify the miRNA signature associated with the largest estimated risk increase, and miR-720
that, along with the FRS for hard CAD, most effectively predicted future associated with reduced MACE risk.
MI. The model was evaluated by 10-fold cross-validation for the AUC, and
the miRNA panel was shown to add predictive value to the FRS, leading to Then, they identified 16 interacting miRNA pairs associated with an
a significant increase in the AUC. Of note, the addition of other important increased probability of an event, by random effects logistic regression
CAD risk factors (waist:hip ratio, triglycerides, glucose, creatinine) to the models that included only two miRNAs and their interaction (without
FRS did not significantly improve risk prediction. covariates). Notably, miR-320b was present in all interacting miRNA pairs
associated with the risk of MACE, and the expression level of miR-320b
In 2017, Keller et al. tested the ability of another panel of five candidate influenced the strength of the association of the paired miRNA (the higher
miRNAs in predicting cardiovascular outcome.32 Selected miRNAs miR-320b expression level, the greater the risk associated with each
included miR-34a for its role in cardiac ageing, miR-223 as a regulator of miRNA).
inflammation, and the cardiac-enriched miR-378, miR-133 and miR-499
associated with either cardiac hypertrophy or myocardial damage.33–37 Finally, they performed target prediction of miR-320b and the 16 identified
They used as the derivation cohort a sample of low-to-intermediate-risk interacting miRNAs, and grouped miRNAs in four clusters: three of these
primary care patients (178 German healthy individuals, median age 55.5 clusters (cluster 1: miR-320b plus miR-145-3p, miR-128a, miR-548d-3p;
years, interquartile range: 46–69 years) randomly chosen from the cluster 2: miR-320b plus let-7g-5p, let-7d-5p, let-7e-5p, miR-196b-5p,
prospective longitudinal DETECT study, and as the independent validation miR-191-5p, miR-324-3p; and cluster 4: miR-320b plus miR-301b,miR-340-
cohort, a general population sample (129 healthy participants, mean age 3p, miR-376a) were linked with cardiovascular development and function,
74.7 ± 8.2 years) from the SHIP.38,39 and CVDs, as documented by enrichment pathway analysis of the targets.
They then sought validation using an external prospective cohort (58
Endpoints were overall mortality and/or cardiovascular events patients with incident MI and 60 sex-matched controls within 1 year from
(cardiovascular death, non-fatal MI or need for coronary revascularisation). initial serum sampling, mean age 60 ± 5 years) from the HUNT study.30
Twenty-one participants from the DETECT study reached the combined Although not significant, they observed a similar pattern of association
endpoint within a 5-year follow-up period, 12 of whom died. Baseline with the risk of MI, and a trend in a progressive increase in MI risk
plasma levels of the five selected miRNAs were assessed by RT-qPCR. estimates for miRNAs from clusters 2 and 4.
Cox proportional hazards models, adjusted for age, sex and FRS, showed
that 5-year all-cause mortality was associated with reduced miR-133 The same year, Wang et al. reported a negative correlation of miR-423-3p
levels and that there was a trend for the association with lower miR-223 with the risk of CAD events, showing added predictive ability to TRFs.43 To
levels. Importantly, a score from the multivariate miRNA panel, calculated identify the most specific miRNAs associated with CAD, they implemented
by a logistic regression model, showed a robust association with overall a multistep design. They initially performed a screening by miRNA
mortality, withstanding adjustment for the same covariates. No significant sequencing on pooled samples of serum or peripheral blood monocytes
association with the combined outcome was observed for either individual from 10 CAD patients and 10 healthy controls, as well as hypoxia-treated
miRNAs or the entire panel. However, the 5-miRNA panel improved the vascular endothelial cells (ECs). Out of the 1,022 miRNAs detected in
ability of the FRS to identify patients at risk to die within 5 years, as patient serum and 976 in controls, they selected 48 candidates for
indexed by a significant increase in AUC. detection in serum, which were also dysregulated in CAD monocytes and
hypoxic ECs. By PCR product sequencing, the authors found that five
Then, the authors explored the use of the miRNA panel in primary miRNAs (miR-10a-5p, miR-126-3p, miR-210-3p, miR-423-3p and miR-92a-
prevention: 64 individuals without a CVD history, who died within a 12- 3p) were specifically detectable in serum.
year follow-up, were randomly chosen from the SHIP study, and age-
and sex-matched 1:1 with 65 controls. The 5-miRNA panel confirmed in Finally, the serum levels of these five miRNAs were measured by RT-qPCR
this validation cohort its ability to predict all-cause mortality, in two cohorts of sex-matched CAD patients and controls (n=39 versus
independently of established risk scores, such as the FRS or SCORE.28,40 n=39 and n=30 versus n=21, respectively): miR-10a-5p and miR-423-3p

EUROPEAN CARDIOLOGY REVIEW

[Link]
 MicroRNAs as Novel Risk Biomarkers for CAD

emerged as the best-associated candidates, consistently showing a Circulating MicroRNAs and Coronary
significantly lower expression in CAD patients than in controls. Therefore, Artery Disease Prognosis
the authors used Cox regression analysis to assess the ability of these two Risk stratification for future events among patients with CAD is critically
miRNAs to predict CAD events in a large-scale general population of 2,812 important for prognostication. Risk assessment for recurrent
individuals (China-CVD cohort, mean age 51.2 ± 7.9 years), with a median cardiovascular events currently relies on myocardial injury markers, such
follow-up of 6 years. CAD events (n=64) were non-fatal, and included AMI as troponins or N-terminal prohormone of brain natriuretic peptide, but
and subsequent MI. After adjustment for age, sex and TRFs, they found none of them are specific for any particular outcome. This has prompted
that only miR-423-3p was associated with CAD event risk. The addition of the search for new reliable biomarkers.
miR-423-3p to TRF-based models improved prediction performance,
including AUC and net reclassification index. Secondary Prevention Settings
The main findings and characteristics of studies assessing the potential of
Challenges of MicroRNAs as Predictive Biomarkers circulating miRNAs in this context are shown in Table 2, presenting data
The potential of circulating miRNAs as non-invasive biomarkers for risk on acute coronary syndromes (ACS) first and then on chronic coronary
stratification is an attractive field. However, miRNAs detected so far have syndromes (stable CAD).
shown poor reproducibility between studies (Table 1). Specifically, miR-
233 was shown to be predictive of MI or all-cause mortality in two different In an early study (2011), Widera et al. reported an association of miR-133a
studies.26,32 Surprisingly, miR-144-3p was part of two different panels and miR-208b levels with the risk of all-cause death in an ACS cohort from
reported as predictive of MI by the same group in two subsequent studies Hannover.47 They focused on cardiomyocyte-enriched miRNAs (miR-1,
of samples from the HUNT cohort, but in the first its levels were increased miR-133a, miR-133b, miR208a, miR-208b and miR-499), known to be
in cases, in the second its levels were decreased.29,31 Similarly, let-7g-5p cardiac selective and rapidly released after AMI.37,48,49 To assess their
had decreased levels in cases with future fatal MI in the study by Bye et prognostic value, the authors determined plasma concentrations of the
al., and instead increased in patients who developed MACE in the study six miRNAs on admission by RT-qPCR in a cohort of 444 consecutive
by Gigante et al.29,41 patients (aged 55–73 years) with a final diagnosis of ACS (n=117 unstable
angina, n=131 non-ST-segment elevation MI [NSTEMI] and n=196 ST-
The conflicting results reported above could be due to several factors. segment elevation MI [STEMI]). All-cause mortality at 6 months (n=34
Sampling is a critical issue; specifically, the choice of anticoagulant and patients) was the primary endpoint. They found that miR-133a and miR-
blood fraction could affect the quality of results. Indeed, variability in 208b baseline levels were significantly related to the outcome at the log-
miRNA detection levels has been observed between serum and plasma.44 rank test, but stepwise Cox regression analyses indicated that they
An utmost critical step affecting result reproducibility is the normalisation withstood adjustment for age and sex, but not for high-sensitivity troponin
method used. Normalisation strategies include the use of endogenous T (hsTnT). Moreover, they did not increase the ability of hsTnT in
reference miRNAs, spiking-in of exogenous miRNAs or global mean discriminating survivors from non-survivors.
normalisation to account for technical variability.
Gidlof et al. used a similar approach to assess the prognostic potential of
Although miR-16-5p and small nucleolar RNA U6 have commonly been three cardio-enriched miRNAs (miR-1, miR-208b and miR-499-5p) in
used in several studies, there is a substantial lack of consensus on patients with suspected ACS, and found that increased baseline plasma
endogenous miRNAs.45 Their amount in plasma or serum samples may be levels of miR-208b and miR-499-5p were associated with the risk of death
affected by several factors, including haemolysis, physical activity and or heart failure (HF) and reduction in systolic function after MI.50 They
fasting, and these miRNAs have been shown to be invariant only in analysed by RT-qPCR 407 Swedish patients presenting with chest pain
specific situations.46 Conversely, a major drawback of the spike-in method suspicious for ACS (n=88 non-MI, n=146 NSTEMI, n=173 STEMI; median
is that it is unreliable when quantification of the extracted RNA is not age 65 years), after interventional therapies and within 24-72 hours from
possible, which is often the case for circulating miRNAs. In fact, equal presentation. A total of 74 patients experienced the combined primary
volumes of serum/plasma may contain variable amounts of miRNAs.46 endpoint (death within 30 days of hospitalisation, heart failure, ejection
fraction <40%, or cardiogenic shock). Plasma levels of miR-208b and miR-
Finally, mean/median normalisation methods are another possibility for 499-5p were associated with the outcome, with significant odds ratios
quantifying miRNA expression, but produce data that are difficult to adjusted for age, sex and time from admission to sampling. However,
compare with other normalisation methods. It is clear that different again, they did not withstand adjustment for TnT, and their prognostic
normalisation methods, as those used in the above-mentioned studies, accuracy was similar to TnT.
profoundly affect the final results.
In keeping with this line of research, Olivieri et al. described an association
Another issue of fundamental importance is that the clinical outcome was between admission circulating levels of the cardio-miRNA miR-499-5p and
often different among primary prevention studies, ranging from MACE to 12-month cardiovascular mortality in elderly NSTEMI patients.51 They
fatal or non-fatal MI to death from all causes. Clearly, different miRNAs can elected to test the prognostic potential of two miRNAs (miR-499-5p and
be expected to predict different events. Furthermore, these studies were miR-21) based on previous observations about their diagnostic performance
conducted on populations of diverse ethnicity, had varying approaches in in the same setting.52 They analysed the baseline expression of these
study design and detection methods, and adopted different statistical miRNAs by RT-qPCR in plasma samples of 142 Italian NSTEMI patients
models. In addition, there are strong correlations between circulating admitted to the coronary care unit with an interval of 4–9 hours from
miRNA levels; thus, different studies may report different, but highly symptoms onset to admission. Cardiovascular mortality at 12 and 24
correlated, miRNAs with approximately the same chance of being included months was prospectively defined as the primary endpoint. A total of 54
in prediction models. Finally, physiological or normal miRNA concentration patients (mean age 85.9 ± 5.4 years) died within 12 months, and 88
ranges in blood have yet to be established. survived (mean age 81.7 ± 6.0 years). By multivariate analysis using Cox

EUROPEAN CARDIOLOGY REVIEW

[Link]
 MicroRNAs as Novel Risk Biomarkers for CAD

Table 1: MicroRNAs as Potential Biomarkers in Coronary Artery Disease Primary Prevention

miRNA ID Sample Detection Normalisation Regulation Population Risk Adjustment/ Reference

matching
miR-126-3p ↑ Age, sex, smoking,
SBP, LDL-C, diabetes,
miR-197 Specific U6 ↓
↑ Fatal and history of CVD, other
Plasma TaqMan or General Zampetaki et al.10
non-fatal MI miRNAs, BMI, WHR,
probes average Ct
miR-223 ↓ HDL-C, CRP,
fibrinogen
miR-34a ↑
miR-133 ↓
Specific
Primary care and ↑ All-cause Age, sex, FRS or
miR-223 Plasma TaqMan None ↓ Keller et al.32
general mortality SCORE
probes
miR-378 ↓
miR-499 ↑
miR-106a-5p ↓ Cases and controls
were matched for
miR-424-5p ↑
LNA age, sex, smoking,
let-7g-5p Global mean or ↓
Serum primers, General ↑ Fatal MI BMI, TG, total Bye et al.29
miR-425-5p
miR-144-3p SYBR Green ↑ cholesterol, HDL-C,
glucose, creatinine,
miR-660-5p ↑ SBP
miR-21-5p ↑
miR-26a-5p ↑
LNA Cases and controls
↑ Fatal and
miR-29c-3p Serum primers, miR-425-5p ↓ General were matched for age Velle-Forbord et al.31
non-fatal MI
SYBR Green and sex
miR-144-3p ↓
miR-151a-5p ↑
miR-320b plus
miR-145-3p
miR-128a ↑
miR-548d-3p
let-7g-5p Matched for age;
let-7d-5p adjusted for sex,
LNA
diabetes,
let-7e-5p Serum primers, miR-16-5p General ↑ MACE Gigante et al.41
↑ hypertension,
SYBR Green
miR-196b-5p hyperlipidaemia,
miR-191-5p smoking and obesity

miR-324-3p
miR-301b
miR-340-3p ↑
miR-376a
Specific
Age, sex, CV risk
miR-423-3p Serum primers, Global mean ↓ General ↑ Non-fatal MI Wang et al.43
factors
SYBR green

CAD = coronary artery disease; CRP = C-reactive protein; Ct = cycle threshold; CV = cardiovascular; HDL-C = HLD cholesterol; LDL-C = LDL cholesterol; LNA = locked nucleic
acids; MACE = major adverse cardiovascular events; miRNA = microRNA; SBP = systolic blood pressure; TG = triglycerides; WHR = waist:hip ratio.

proportional hazards model adjusting for all significantly different variables setting, to stratify the risk of future cardiovascular events in patients with
at admission, they reported a significant increase in the 12-month risk of documented CAD.26,53 Indeed, miR-126, miR-197 and miR-223 were known
death among NSTEMI patients whose miR-499-5p admission level was to be involved in endovascular inflammation and platelet activation, and
higher than the median. In particular, patients presenting with congestive as biomarkers in the diagnosis of CAD-related conditions.5,23,24 They
heart failure had a twofold greater risk of dying within the first 12 months measured the serum levels of these miRNAs by RT-qPCR in a cohort of 873
when baseline miR-499-5p exceeded the median. However, the German patients with manifest CAD (median age 64 years [interquartile
performance of this model was far from optimal, and miR-499-5p was not range 57–69 years], selected from the AtheroGene study), of whom 340
associated with cardiovascular mortality at 24 months. had ACS and 533 had stable angina (SA).54 After a median follow-up of 4
years, 18 cardiovascular deaths were recorded, eight among ACS and 10
Starting with a different hypothesis, Schulte et al. investigated the ability among SA patients. The authors used ACS-SA stratified Cox regression
of the three miRNAs described by Zampetaki et al. in a primary prevention models, adjusted for age, sex and further TRFs, followed by 10-fold cross-

EUROPEAN CARDIOLOGY REVIEW

[Link]
 MicroRNAs as Novel Risk Biomarkers for CAD

Table 2: MicroRNAs as Potential Biomarkers in Coronary Artery Disease Secondary Prevention

miRNA ID Sample Detection Normalisation Regulation Population Risk Adjustment/ Reference

matching
miR-133a Specific ↑
Spike-in ↑ All-cause
Plasma TaqMan ACS Age, sex, (hsTnT) Widera et al.47
miR-208b cel-miR-54 ↑ mortality
probe sets
miR-208b ↑ ↑ Mortality
LNA primers, Age, sex, time from
Plasma miR-17 Suspected ACS and HF, Gidlof et al.50
miR-499-5p SYBR Green ↑ admission to sampling
↓ LVEF
Specific
Age, hsCRP, WBC,
miR-499-5p Plasma TaqMan miR-17 ↑ NSTEMI ↑ CV mortality Olivieri et al.51
homocysteine, BMI
probes
miR-197 ↑ Age, sex, BMI, diabetes,
Specific
Spike-in CAD (ACS + SA) hypertension, history of
Serum TaqMan ↑ CV Mortality Shulte et al.53
miR-223 cel-miR-39 ↑ or ACS MI, hyperlipidaemia,
probes
ever smoker
miR-19b ↑
Age and sex and
miR-132 ↑ hypertension, smoking
CAD (ACS + SA)
miR-140-3p ↑ or status, hyperlipidaemia,
Specific
Spike-in ACS diabetes, history of MI,
miR-150 Serum TaqMan ↑ ↑ CV mortality Karakas et al.55
cel-miR-39 or cTnI, or NT-proBNP, or
probes
miR-186 ↑ LVEF and number of
diseased vessels, or
miR-19a ↑
ACS type of ACS
miR-210 ↑
miR-126-3p ↑ Age, sex, BMI, diabetes,
Specific hypertension,
Spike-in
MVs TaqMan Stable CAD ↓ MACE hyperlipoproteinaemia, Jansen et al.57
miR-199a cel-miR-39 ↑
probes CKD, use of ACE-
inhibitors and statins
Bulge-loop Age, sex, diabetes,
Spike-in CAD undergoing
miR-142 Plasma primers, ↑ ↑ MACE hypertension, HF, Tang et al.59
cel-miR-39 PCI
SYBR Green medications
miR-1 ↓ Age, sex, primary
diagnosis, history of
miR-19a ↓
Stable chronic coronary
miR-126 Specific ↓
Spike-in CVD (post-ACS, ↑ All-cause and revascularisation, time
Plasma TaqMan Mayer et al.60
miR-133a cel-miR-39 ↓ CABG, PCI or CV mortality to interview, current
probes
stroke) smoking, BMI, SBP, DBP,
miR-223 ↓ LDL-C, glucose, HbA1c,
cTnI, BNP, medications
ACE = angiotensin-converting enzyme; ACS = acute coronary syndrome; BNP = brain natriuretic peptide; CABG = coronary artery bypass graft; CAD = coronary artery disease;
cTnI = cardiac troponin I; CKD = chronic kidney disease; CV = cardiovascular; CVD = cardiovascular diseases; DBP = diastolic blood pressure; hsCRP = high-sensitivity C-reactive
protein; hsTnT = high-sensitivity troponin T; HF = heart failure; LDL-C = LDL cholesterol; LNA = locked nucleic acids; LVEF = left ventricular ejection fraction; MACE = major adverse
cardiovascular events; NSTEMI = non-ST-elevation MI; NT-proBNP = N-terminal prohormone of brain natriuretic peptide; PCI = percutaneous coronary intervention;
SA = stable angina; SBP = systolic blood pressure; WBC = white blood cells.

validation of the C-indices, to explore the association with each miRNA, derived from the above-mentioned study. After a median 4-year follow-
and found that elevated levels of miR-197 and miR-223 were able to up, 78 cardiovascular deaths or non-fatal MI were recorded (43 among
predict future cardiovascular death in the overall group. Considering only ACS and 35 among SA patients).
the ACS subgroup, the prognostic ability of the two miRNAs was even
higher, but in SA patients it was not significant. Of note, there was no Specifically, the authors assessed the association of circulating miRNAs
incremental benefit in prognostic power using any combination of the with cardiovascular mortality both in the ACS and the overall cohort by
three miRNAs compared with single miR-197 or miR-223. Cox regression correcting for multiple testing (controlling the false
discovery rate). All measured miRNAs, except miR-142-5p, were likely to
The same group (Karakas et al.), followed up on a similar working predict cardiovascular death in the ACS group, even after adjustment for
hypothesis and evaluated the prognostic potential of eight miRNAs they age, sex plus TRFs, other emerging risk factors or ACS diagnosis (unstable
had previously identified as diagnostic of unstable angina.55,56 Using RT- angina, STEMI, NSTEMI). The best predictors were miR-132, miR-140-3p
qPCR, they assessed baseline serum concentrations of miR-19a, miR-19b, and miR-210, which were able to markedly increase the c-statistics in the
miR-132, miR-140-3p, miR-142-5p, miR-150, miR-186 and miR-210 in a ACS cohort. In the overall CAD cohort, five miRNAs (miR-19b, miR-132, miR-
cohort of 1,112 patients with an angiographically documented CAD (430 140-3p, miR-150 and miR-186) were associated with an increased risk of
with ACS, mean age 63 years, and 682 with SA, mean age 64 years) cardiovascular mortality, again, independent of risk factors and covariates.

EUROPEAN CARDIOLOGY REVIEW

[Link]
 MicroRNAs as Novel Risk Biomarkers for CAD

Table 3: MicroRNAs as Potential Predictors of Postoperative AF

miRNA ID Sample Detection Normalisation Regulation Population Risk Adjustment/matching Reference

Matched for age, sex, family
history, BMI, previous CV
miRNA-specific
events, hypertension,
miR-483-5p Plasma TaqMan primer miR-186 ↑ CABG ↑POAF Harling et al.64
hypercholesterolaemia,
sets
diabetes, smoking, alcohol
consumption
miR-23a Specific primers, Median of miR-423, Matched for sex, hypertension,
Serum ↓ CABG ↑POAF Feldman et al.65
miR-26a SYBR green miR-16, cel-miR-39 hyperlipaemia, diabetes, smoking
Specific primers, Age, BMI, COPD, hypertension,
miR-29a Serum Spike-in cel-miR-39 ↓ CABG ↑POAF Rizvi et al.67
SYBR green sleep apnoea
CABG = coronary artery bypass graft; CV = cardiovascular; COPD = chronic obstructive pulmonary disease; POAF = postoperative AF.

In 2014, Jansen et al. used a different approach, and observed that high cause and cardiovascular death in stable chronic cardiovascular patients.
levels of miR-126 and miR-199a contained in circulating microvesicles 47,50,53,55,60
This study originated as a secondary analysis from the Czech
(MVs) were associated with a lower rate of MACE in patients with stable EUROASPIRE Survey data.61 A prospective cohort of 826 patients with
CAD.57 In two German cohorts with a total of 181 patients (mean age 66.7 chronic vascular disease (mean age 65.2 ± 9.3 years) was enrolled
± 10.2 years) affected by angiographically documented stable CAD, they 6–36 months after a vascular event (ACS and/or coronary revascularisation
quantified by RT-qPCR the baseline arterial blood plasma and MV levels of for 487 CAD patients, first-ever ischaemic stroke for the other 339
10 miRNAs involved in the regulation of vascular function and/or expressed patients). During a median follow-up of 5.6 years, 167 patients died,
by vascular smooth muscle cells or ECs (miR-126, miR-222, miR-let7d, including 126 from events considered to be of cardiovascular origin.
miR-21, miR-20a, miR-27a, miR-92a, miR-17, miR-130 and miR-199a).58
Occurrence of a first MACE, including non-fatal MI, need for Based on previous literature, the authors initially quantified by RT-qPCR
revascularisation and death from cardiac causes, was evaluated within a the plasmatic levels of 10 miRNA (miR-1, miR-19a, miR-21, miR-34a, miR-
median follow-up period of 6 years, and 55 events were recorded. No 126, miR-133a, miR-197, miR-214, miR-223 and miR-499) in a pilot cohort of
association between miRNA plasma levels and MACE was observed. On 100 patients, 50 dead and 50 age- and sex-matched survivors. Baseline
the contrary, above-median expression of miR-126 and miR-199a in levels of the five miRNAs showing statistical differences between the two
circulating MVs was associated with a reduced risk of MACE, at univariate groups (see above) were then assessed in the full cohort. Using Cox
analysis. Binary logistic regression, adjusting models for TRFs and proportional hazards regression, they found an inverse association
medication used, showed that the two miRNAs were not associated with between each of the five miRNAs, and a significant increase in both all-
baseline characteristics. Importantly, fluorescence-activated cell sorting cause and cardiovascular mortality, despite adjustment for TRFs, primary
analysis revealed that ECs and platelets were the main sources of MVs diagnosis, treatments, increased cardiac troponin I and BNP. Noteworthy,
containing miR-126 and miR-199a, respectively. if all five miRNAs were included in a single regression model, only low
miR-19a was a significant predictor of both all-cause and cardiovascular
At variance with the study by Karakas et al., in 2019, Tang et al. reported mortality, independently of all the other risk factors and covariates. A
that plasma miR-142 was a potential marker for MACE prediction in CAD subgroup analysis showed that low miR-19a predicted all-cause mortality
patients undergoing percutaneous coronary intervention on dual risk only in chronic CAD, but not in post-stroke, patients.
antiplatelet therapy with clopidogrel and aspirin.55,59 To select miRNA
candidates, the authors first performed a screening by miRNA sequencing Circulating MicroRNAs and Postoperative AF
of four pooled plasma samples from 115 Chinese CAD patients stratified Postoperative AF (POAF) is a common complication in CAD patients
according to their sensitivity or resistance to aspirin and/or clopidogrel undergoing coronary artery bypass grafting (CABG) surgery, and is
treatment, as documented by platelet aggregation tests. Differential associated with increased 10-year mortality risk.62 Preoperative estimation
expression analysis between the four subgroups, followed by RT-qPCR of the risk for POAF remains a great challenge.63 Circulating miRNAs have
validation in plasma pools and 115 individual samples, revealed that six been proposed as potential biomarkers to predict the risk of POAF after
miRNAs (miR-126, miR-130a, miR-142, miR-27a, miR-21 and miR-106a) CABG surgery (Table 3).
were positively associated with clopidogrel antiplatelet efficacy. Based on
these results, the six miRNAs were tested by RT-qPCR after percutaneous In 2017, Harling et al. found that increased preoperative serum levels of
coronary intervention in a prospective cohort of 1,199 Chinese CAD miR-483-5p could help to predict the risk of developing POAF in patients
patients (aged 40–80 years). The primary outcome was MACE, including undergoing CABG revascularisation.64 They enrolled a prospective cohort
cardiovascular death, MI or stent thrombosis, within a 3-year follow-up of 34 UK patients undergoing non-emergent, on-pump CABG, including 13
period. Multivariable Cox regression analysis, after correction for multiple who subsequently developed POAF (mean age 64.6 ± 11.3 years) and 21
comparisons by false discovery rate, revealed that a high plasma level of without POAF (mean age 59.6 ± 12.1 years), matched for age, sex and
miR-142 was an independent risk factor for MACE, irrespective of age, sex, TRFs, and without previous history of AF. Using a microarray approach,
comorbidities and medications. they first investigated the entire miRNAome in the atrial tissue, taken
intraoperatively before bypass instigation, of a subgroup of 11 patients
Also, quite at odds with several of the abovementioned studies, Mayer et with POAF and 11 matched patients without POAF. Then, they quantified by
al. reported that low plasma levels of miR-1, miR-19a, miR-126, miR-133a RT-qPCR in the whole cohort the preoperative serum levels of the most
and miR-223 were associated with a significant increase in 5-year all- upregulated and the most downregulated of the 16 differentially

EUROPEAN CARDIOLOGY REVIEW

[Link]
 MicroRNAs as Novel Risk Biomarkers for CAD

expressed miRNAs; that is, miR-483-5p and miR-208a, respectively. populations examined, different methodological approaches and
Interestingly, they observed significantly higher levels of miR-483-5p in analytical strategies.
preoperative serum of POAF patients. The authors reported a predictive
ability of this miRNA, as indicated by a significant AUC. Limitations of MicroRNAs in
Modern Cardiology Practice
Feldman et al. in 2017 investigated the potential of serum miR-1, miR-23a Despite their potential, several problems need to be overcome before
and miR-26a in identifying patients who develop PAOF after CABG.65 circulating miRNAs can be used in clinical practice. An important issue to
These miRNAs were selected based on previously reported associations be addressed is the implementation of standard operating procedures for
with AF.66 Using RT-qPCR, they measured preoperative and postoperative sample preparation, miRNA isolation, quantification and normalisation
(48 h) serum levels of the three miRNA in a cohort of 48 Brazilian patients across multiple samples, as the sensitivity of diverse methodologies could
undergoing CABG surgery, including 24 POAF (mean age 64.7 ± 7.5 years) be substantially different and could lead to inconsistent results.46,69
and 24 non-POAF (mean age 56.3 ± 9.2 years) matched for sex and TRFs. Measuring circulating miRNAs is still challenging due to their low
They found no difference in preoperative circulating miRNA levels concentration, and a univocal consensus on miRNA quantification
between the two groups. Conversely, they observed that miR-23a and methods is still lacking. The main detection methods available are RT-
miR-26a were significantly reduced in POAF patients after surgery, and qPCR, next-generation sequencing and microarrays. RT-qPCR is the most
showed a moderate predictive ability. widely used method, but the various approaches can yield significantly
different results: universal RT or RT with specific primers; preamplification;
In 2020, Rizvi et al. proposed a preoperative multiparameter biomarker to specific amplification primers with a DNA-binding dye (SYBR Green), or
identify the risk of new-onset POAF, which included serum levels of miR- target-specific primers combined with dual-labelled probes that hybridise
29a.67 They hypothesised that combining clinical risk factors for AF with to complementary target sequences (TaqMan). Next-generation
markers of the atrial pathophysiological substrate (fibrosis) and its sequencing is highly processive, has no probe-related bias, and enables
regulatory miRNAs might have incremental value in identifying patients at the discovery of novel miRNAs, but protocols are still cumbersome
risk of POAF. They measured preoperative serum concentrations of compared with the other two techniques. Finally, microarrays are relatively
peptides reflecting collagen synthesis and degradation, extracellular inexpensive probe-based assays that require a preamplification step and
matrix protein synthesis and deposition, and of miR-29s (miR-29a, miR- are less sensitive than the other two techniques.70
29b and miR-29c) known to modulate fibrosis, in a US cohort of 90
patients scheduled for an elective CABG procedure with no prior AF (34 In addition to poorly defined laboratory standards, another crucial issue is
with POAF, mean age 72.4 ± 10.8 years, and 56 without POAF, mean age the lack of sufficient evidence.
67.3 ± 10.7 years).68 They observed that procollagen III N-terminal and
procollagen I C-terminal peptides were elevated in POAF compared with The data presented here underline the need for well-designed clinical
non-POAF patients, while miR-29 levels were reduced. These markers studies in larger prospective cohorts of patients, using standardised
showed a correlation with atrial fibrosis extent. The model with the protocols. Larger sample sizes are crucial to distinguish between healthy
highest accuracy in identifying POAF patients was obtained by combining or diseased status, and dissect the contribution of confounders, such as
age with procollagen III N-terminal peptide and miR-29a, as assessed age, sex, ethnicity, lifestyle, pre-treatment and disease history. Despite
using the receiver operating characteristic curve by fitting logistic encouraging results, miRNAs are not currently used for the risk
regression with POAF as the outcome. stratification or prognosis of CAD. Indeed, although circulating miRNAs
have been shown to add predictive capacity to TRFs-based risk scores in
Challenges of MicroRNAs as Prognostic Biomarkers several primary prevention studies, the poor reproducibility of the results
These studies highlighted the potential of circulating miRNAs as promising and the different clinical endpoints considered limit their prospective use
biomarkers with prognostic value for MACE, mortality or POAF in patients in clinical practice.26,29,31,32,43
with CAD. Specifically, higher plasmatic levels of miR-208b and miR-499-
5p were found to be associated with an increased risk of mortality in Furthermore, single miRNAs are rarely disease-specific, whereas
different studies on ACS cohorts (Table 2).47,50,51 However, there is little combining miRNAs in panels increases specificity and slightly/moderately
overlap in many experimental findings and there is no agreement in the increases the predictive power of conventional scores. In contrast,
studies on POAF. Surprisingly, in different studies, miR-19a, miR-133a and secondary prevention studies have also provided conflicting results. In
miR-223 showed opposite regulation in baseline samples of future cases two of them, the miRNAs identified in ACS patients did not add prognostic
in patients with ACS compared with those with chronic coronary power to hsTnT.47,50 In other studies, single miRNAs or miRNA panels have
syndrome.47,53,55,60 Intriguingly, miR-126-3p, miR-197 and miR-223 were shown prognostic ability independent of various TRFs and established
identified as predictors of all-cause or cardiovascular death in both biomarkers, but once again there was little consistency and different
general population cohorts and cohorts of documented CAD patients, associations with mortality, positive in ACS patients and negative in
thus extending the applicability of these circulating miRNAs to primary patients with chronic CAD.53,55,60 In general, there is a lack of evidence that
and secondary prevention.26,32,53,57,60 circulating miRNAs can increase the prognostic power of established
biomarkers, such as LDL cholesterol, high sensitivity troponins and
The inconsistent results mentioned above are due to several factors. As N-terminal prohormone of brain natriuretic peptide.
with studies on the predictive role of miRNAs, the most critical issues are
sampling, normalisation method and clinical endpoint. The clinical Conclusion
outcomes considered were diverse, including either all-cause or MiRNAs are highly investigated for their role in the pathogenesis of CAD.
cardiovascular death, or a composite endpoint, such as MACE. Other Nevertheless, evidence for clinical implementation is still lacking. The
factors influencing reproducibility could be the limited number of cases in results of our review show that there is currently insufficient support for
many of these studies, the different composition and/or ethnicity of the the use of any of the presented miRNAs as predictive or prognostic

EUROPEAN CARDIOLOGY REVIEW

[Link]
 MicroRNAs as Novel Risk Biomarkers for CAD

biomarkers in clinical settings. However, evidence highlights the potential miRNA selection approaches, normalisation strategies, and adjustment
of circulating miRNAs as novel biomarkers for both primary and secondary for potential confounders will ensure improved reliability and
CAD prevention. Only a few miRNAs showed risk-predicting ability in more reproducibility of results, and allow adequate comparison between
than one study, underscoring the need for further investigation in larger studies. Once consensus is reached, miRNAs could be used for the
cohorts with standardised methodologies. In particular, standardising development of novel risk stratification algorithms.

1. Roth GA, Mensah GA, Johnson CO, et al. Global burden of 20. Mitchell PS, Parkin RK, Kroh EM, et al. Circulating microRNAs 39. Volzke H, Alte D, Schmidt CO, et al. Cohort profile: the study
cardiovascular diseases and risk factors, 1990-2019: update as stable blood-based markers for cancer detection. Proc of health in Pomerania. Int J Epidemiol 2011;40:294–307.
from the GBD 2019 study. J Am Coll Cardiol 2020;76:2982– Natl Acad Sci U S A 2008;105:10513–8. [Link] [Link] PMID: 20167617.
3021. [Link] pnas.0804549105; PMID: 18663219. 40. Conroy RM, Pyorala K, Fitzgerald AP, et al. Estimation of ten-
PMID: 33309175. 21. Ambros V. The functions of animal microRNAs. Nature year risk of fatal cardiovascular disease in Europe: the
2. Timmis A, Townsend N, Gale C, et al. European Society of 2004;431:350–5. [Link] SCORE project. Eur Heart J 2003;24:987–1003. [Link]
Cardiology: cardiovascular disease statistics 2017. Eur Heart PMID: 15372042. org/10.1016/s0195-668x(03)00114-3; PMID: 12788299.
J 2018;39:508–79. [Link] 22. Lawrie CH, Gal S, Dunlop HM, et al. Detection of elevated 41. Gigante B, Papa L, Bye A, et al. MicroRNA signatures predict
PMID: 29190377. levels of tumour-associated microRNAs in serum of patients early major coronary events in middle-aged men and
3. Rossello X, Dorresteijn JA, Janssen A, et al. Risk prediction with diffuse large B-cell lymphoma. Br J Haematol women. Cell Death Dis 2020;11:74. [Link]
tools in cardiovascular disease prevention: a report from the 2008;141:672–5. [Link] s41419-020-2291-9; PMID: 32001669.
ESC Prevention of CVD Programme led by the European org/10.1111/j.1365-2141.2008.07077.x; PMID: 18318758. 42. Gigante B, Leander K, Vikstrom M, et al. Elevated ApoB
Association of Preventive Cardiology (EAPC) in collaboration 23. Navickas R, Gal D, Laucevicius A, et al. Identifying serum levels strongly predict early cardiovascular events.
with the Acute Cardiovascular Care Association (ACCA) and circulating microRNAs as biomarkers of cardiovascular Heart 2012;98:1242–5. [Link]
the Association of Cardiovascular Nursing and Allied disease: a systematic review. Cardiovasc Res 2016;111:322– heartjnl-2012-301852; PMID: 22760866.
Professions (ACNAP). Eur J Prev Cardiol 2019;26:1534–44. 37. [Link] PMID: 27357636. 43. Wang X, Dong Y, Fang T, et al. Circulating microRNA-423-3p
[Link] PMID: 31234648. 24. Kaur A, Mackin ST, Schlosser K, et al. Systematic review of improves the prediction of coronary artery disease in a
4. Thomas H, Diamond J, Vieco A, et al. Global atlas of microRNA biomarkers in acute coronary syndrome and general population – six-year follow-up results from the
cardiovascular disease 2000-2016: the path to prevention stable coronary artery disease. Cardiovasc Res China-Cardiovascular Disease study. Circ J 2020;84:1155–
and control. Glob Heart 2018;13:143–63. [Link] 2020;116:1113–24. [Link] 62. [Link] PMID: 32404537.
org/10.1016/[Link].2018.09.511; PMID: 30301680. PMID: 31782762. 44. Wang K, Yuan Y, Cho JH, et al. Comparing the microRNA
5. Fichtlscherer S, Zeiher AM, Dimmeler S. Circulating 25. Hoefer IE, Steffens S, Ala-Korpela M, et al. Novel spectrum between serum and plasma. PLoS One
microRNAs: biomarkers or mediators of cardiovascular methodologies for biomarker discovery in atherosclerosis. 2012;7:e41561. [Link]
diseases? Arterioscler Thromb Vasc Biol 2011;31:2383–90. Eur Heart J 2015;36:2635–42. [Link] PMID: 22859996.
[Link] PMID: 22011751. eurheartj/ehv236; PMID: 26049157. 45. Donati S, Ciuffi S, Brandi ML. Human Circulating miRNAs
6. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, 26. Zampetaki A, Willeit P, Tilling L, et al. Prospective study on real-time qRT-PCR-based analysis: an overview of
and function. Cell 2004;116:281–97. [Link] circulating MicroRNAs and risk of myocardial infarction. J Am endogenous reference genes used for data normalization.
S0092-8674(04)00045-5; PMID: 14744438. Coll Cardiol 2012;60:290–9. [Link] Int J Mol Sci 2019;20. [Link]
7. Small EM, Frost RJ, Olson EN. MicroRNAs add a new jacc.2012.03.056; PMID: 22813605. PMID: 31491899.
dimension to cardiovascular disease. Circulation 27. Willeit J, Kiechl S, Oberhollenzer F, et al. Distinct risk 46. Moldovan L, Batte KE, Trgovcich J, et al. Methodological
2010;121:1022–32. [Link] profiles of early and advanced atherosclerosis: prospective challenges in utilizing miRNAs as circulating biomarkers. J
CIRCULATIONAHA.109.889048; PMID: 20194875. results from the Bruneck study. Arterioscler Thromb Vasc Biol Cell Mol Med 2014;18:371–90. [Link]
8. Creemers EE, Tijsen AJ, Pinto YM. Circulating microRNAs: 2000;20:529–37. [Link] jcmm.12236; PMID: 24533657.
novel biomarkers and extracellular communicators in PMID: 10669653. 47. Widera C, Gupta SK, Lorenzen JM, et al. Diagnostic and
cardiovascular disease? Circ Res 2012;110:483–95. https:// 28. D’Agostino RB Sr, Vasan RS, Pencina MJ, et al. General prognostic impact of six circulating microRNAs in acute
[Link]/10.1161/CIRCRESAHA.111.247452; PMID: 22302755. cardiovascular risk profile for use in primary care: the coronary syndrome. J Mol Cell Cardiol 2011;51:872–5. https://
9. Hulsmans M, De Keyzer D, Holvoet P. MicroRNAs regulating Framingham Heart Study. Circulation 2008;117:743–53. [Link]/10.1016/[Link].2011.07.011; PMID: 21806992.
oxidative stress and inflammation in relation to obesity and [Link] 48. D’Alessandra Y, Devanna P, Limana F, et al. Circulating
atherosclerosis. FASEB J 2011;25:2515–27. [Link] PMID: 18212285. microRNAs are new and sensitive biomarkers of myocardial
org/10.1096/fj.11-181149; PMID: 21507901. 29. Bye A, Rosjo H, Nauman J, et al. Circulating microRNAs infarction. Eur Heart J 2010;31:2765–73. [Link]
10. Zampetaki A, Willeit P, Drozdov I, et al. Profiling of predict future fatal myocardial infarction in healthy org/10.1093/eurheartj/ehq167; PMID: 20534597.
circulating microRNAs: from single biomarkers to re-wired individuals – The HUNT study. J Mol Cell Cardiol 49. Wang GK, Zhu JQ, Zhang JT, et al. Circulating microRNA: a
networks. Cardiovasc Res 2012;93:555–62. [Link] 2016;97:162–8. [Link] novel potential biomarker for early diagnosis of acute
org/10.1093/cvr/cvr266; PMID: 22028337. PMID: 27192016. myocardial infarction in humans. Eur Heart J 2010;31:659–
11. Huntzinger E, Izaurralde E. Gene silencing by microRNAs: 30. Krokstad S, Langhammer A, Hveem K, et al. Cohort profile: 66. [Link]
contributions of translational repression and mRNA decay. the HUNT Study, Norway. Int J Epidemiol 2013;42:968–77. PMID: 20159880.
Nat Rev Genet 2011;12:99–110. [Link] [Link] PMID: 22879362. 50. Gidlof O, Smith JG, Miyazu K, et al. Circulating cardio-
nrg2936; PMID: 21245828. 31. Velle-Forbord T, Eidlaug M, Debik J, et al. Circulating enriched microRNAs are associated with long-term
12. Friedman RC, Farh KK, Burge CB, Bartel DP. Most microRNAs as predictive biomarkers of myocardial prognosis following myocardial infarction. BMC Cardiovasc
mammalian mRNAs are conserved targets of microRNAs. infarction: evidence from the HUNT study. Atherosclerosis Disord 2013;13:12. [Link]
Genome Res 2009;19:92–105. [Link] 2019;289:1–7. [Link] PMID: 23448306.
gr.082701.108; PMID: 18955434. atherosclerosis.2019.07.024; PMID: 31437610. 51. Olivieri F, Antonicelli R, Spazzafumo L, et al. Admission
13. Zhang J, Zhou W, Liu Y, et al. Oncogenic role of microRNA- 32. Keller T, Boeckel JN, Gross S, et al. Improved risk levels of circulating miR-499-5p and risk of death in elderly
532-5p in human colorectal cancer via targeting of the stratification in prevention by use of a panel of selected patients after acute non-ST elevation myocardial infarction.
5’UTR of RUNX3. Oncol Lett 2018;15:7215–20. [Link] circulating microRNAs. Sci Rep 2017;7:4511. [Link] Int J Cardiol 2014;172:e276–8. [Link]
org/10.3892/ol.2018.8217; PMID: 29849790. org/10.1038/s41598-017-04040-w; PMID: 28674420. ijcard.2013.12.203; PMID: 24461971.
14. O’Brien J, Hayder H, Zayed Y, Peng C. Overview of 33. Boon RA, Iekushi K, Lechner S, et al. MicroRNA-34a 52. Olivieri F, Antonicelli R, Lorenzi M, et al. Diagnostic potential
microRNA biogenesis, mechanisms of actions, and regulates cardiac ageing and function. Nature 2013;495:107– of circulating miR-499-5p in elderly patients with acute non
circulation. Front Endocrinol (Lausanne) 2018;9:402. https:// 10. [Link] PMID: 23426265. ST-elevation myocardial infarction. Int J Cardiol 2013;167:531–
[Link]/10.3389/fendo.2018.00402; PMID: 30123182. 34. Johnnidis JB, Harris MH, Wheeler RT, et al. Regulation of 6. [Link]
15. Kobayashi H, Tomari Y. RISC assembly: coordination progenitor cell proliferation and granulocyte function by PMID: 22330002.
between small RNAs and Argonaute proteins. Biochim microRNA-223. Nature 2008;451:1125–9. [Link] 53. Schulte C, Molz S, Appelbaum S, et al. miRNA-197 and
Biophys Acta 2016;1859:71–81. [Link] org/10.1038/nature06607; PMID: 18278031. miRNA-223 predict cardiovascular death in a cohort of
bbagrm.2015.08.007; PMID: 26303205. 35. Nagalingam RS, Sundaresan NR, Gupta MP, et al. A cardiac- patients with symptomatic coronary artery disease. PLoS One
16. Wang K, Zhang S, Weber J, et al. Export of microRNAs and enriched microRNA, miR-378, blocks cardiac hypertrophy by 2015;10:e0145930. [Link]
microRNA-protective protein by mammalian cells. Nucleic targeting Ras signaling. J Biol Chem 2013;288:11216–32. pone.0145930; PMID: 26720041.
Acids Res 2010;38:7248–59. [Link] [Link] PMID: 23447532. 54. Rupprecht HJ, Blankenberg S, Bickel C, et al. Impact of viral
gkq601; PMID: 20615901. 36. Care A, Catalucci D, Felicetti F, et al. MicroRNA-133 controls and bacterial infectious burden on long-term prognosis in
17. Diehl P, Fricke A, Sander L, et al. Microparticles: major cardiac hypertrophy. Nat Med 2007;13:613–8. [Link] patients with coronary artery disease. Circulation
transport vehicles for distinct microRNAs in circulation. org/10.1038/nm1582; PMID: 17468766. 2001;104:25–31. [Link]
Cardiovasc Res 2012;93:633–44. [Link] 37. Corsten MF, Dennert R, Jochems S, et al. Circulating PMID: 11435333.
cvs007; PMID: 22258631. microRNA-208b and microRNA-499 reflect myocardial 55. Karakas M, Schulte C, Appelbaum S, et al. Circulating
18. Vickers KC, Remaley AT. Lipid-based carriers of microRNAs damage in cardiovascular disease. Circ Cardiovasc Genet microRNAs strongly predict cardiovascular death in patients
and intercellular communication. Curr Opin Lipidol 2010;3:499–506. [Link] with coronary artery disease-results from the large
2012;23:91–7. [Link] CIRCGENETICS.110.957415; PMID: 20921333. AtheroGene study. Eur Heart J 2017;38:516–23. [Link]
MOL.0b013e328350a425; PMID: 22418571. 38. Wittchen HU, Glaesmer H, Marz W, et al. Cardiovascular risk org/10.1093/eurheartj/ehw250; PMID: 27357355.
19. Weber JA, Baxter DH, Zhang S, et al. The microRNA factors in primary care: methods and baseline prevalence 56. Zeller T, Keller T, Ojeda F, et al. Assessment of microRNAs in
spectrum in 12 body fluids. Clin Chem 2010;56:1733–41. rates – the DETECT program. Curr Med Res Opin patients with unstable angina pectoris. Eur Heart J
[Link] 2005;21:619–30. [Link] 2014;35:2106–14. [Link]
PMID: 20847327. PMID: 15899112. PMID: 24727883.

EUROPEAN CARDIOLOGY REVIEW

[Link]
 MicroRNAs as Novel Risk Biomarkers for CAD

57. Jansen F, Yang X, Proebsting S, et al. MicroRNA expression patients in the EURO-ASPIRE III survey. Cor Vasa fundamentals. Cardiovasc Res 2011;89:710–21. [Link]
in circulating microvesicles predicts cardiovascular events in 2008;50:156-62. [Link] org/10.1093/cvr/cvq350; PMID: 21051420.
patients with coronary artery disease. J Am Heart Assoc 62. Mariscalco G, Klersy C, Zanobini M, et al. Atrial fibrillation 67. Rizvi F, Mirza M, Olet S, et al. Noninvasive biomarker-based
2014;3:e001249. [Link] after isolated coronary surgery affects late survival. risk stratification for development of new onset atrial
PMID: 25349183. Circulation 2008;118:1612–8. [Link] fibrillation after coronary artery bypass surgery. Int J Cardiol
58. Urbich C, Kuehbacher A, Dimmeler S. Role of microRNAs in CIRCULATIONAHA.108.777789; PMID: 18824644. 2020;307:55–62. [Link]
vascular diseases, inflammation, and angiogenesis. 63. Mathew JP, Fontes ML, Tudor IC, et al. A multicenter risk PMID: 31952855.
Cardiovasc Res 2008;79:581–8. [Link] index for atrial fibrillation after cardiac surgery. JAMA 68. Dawson K, Wakili R, Ordog B, et al. MicroRNA29: a
cvn156; PMID: 18550634. 2004;291:1720-9. [Link] mechanistic contributor and potential biomarker in atrial
59. Tang QJ, Lei HP, Wu H, et al. Plasma miR-142 predicts major PMID: 15082699. fibrillation. Circulation 2013;127:1466–75, 75e1–28. https://
adverse cardiovascular events as an intermediate biomarker 64. Harling L, Lambert J, Ashrafian H, et al. Elevated serum [Link]/10.1161/CIRCULATIONAHA.112.001207;
of dual antiplatelet therapy. Acta Pharmacol Sin 2019;40:208– microRNA 483-5p levels may predict patients at risk of post- PMID: 23459615.
15. [Link] operative atrial fibrillation. Eur J Cardiothorac Surg 69. Felekkis K, Papaneophytou C. Challenges in using
PMID: 29891858. 2017;51:73–8. [Link] circulating micro-RNAs as biomarkers for cardiovascular
60. Mayer O Jr, Seidlerova J, Cerna V, et al. The low expression PMID: 27422887. diseases. Int J Mol Sci 2020;21:561. [Link]
of circulating microRNA-19a represents an additional 65. Feldman A, Moreira DAR, Gun C, et al. Analysis of circulating ijms21020561; PMID: 31952319.
mortality risk in stable patients with vascular disease. Int J miR-1, miR-23a, and miR-26a in atrial fibrillation patients 70. Chen Y, Gelfond JA, McManus LM, Shireman PK.
Cardiol 2019;289:101–6. [Link] undergoing coronary bypass artery grafting surgery. Ann Reproducibility of quantitative RT-PCR array in miRNA
ijcard.2019.05.008; PMID: 31085080. Hum Genet 2017;81:99–105. [Link] expression profiling and comparison with microarray
61. Mayer O Jr, Šimon J, Galovcová M, et al. The quality of PMID: 28422282. analysis. BMC Genomics 2009;10:407. [Link]
secondary prevention of coronary heart disease in Czech 66. Wang Z, Lu Y, Yang B. MicroRNAs and atrial fibrillation: new org/10.1186/1471-2164-10-407; PMID: 19715577.

EUROPEAN CARDIOLOGY REVIEW

[Link]