MicroRNA Biomarkers for Coronary Artery Disease?
Curr Atheroscler Rep
MicroRNA Biomarkers for Coronary Artery Disease?
Dorothee Kaudewitz 0
Anna Zampetaki 0
Manuel Mayr 0
0 King's British Heart Foundation Centre, King's College London , 125 Coldharbour Lane, London SE59NU , UK
MicroRNA (miRNA, miR) measurements in patients with coronary heart disease are hampered by the confounding effects of medication commonly used in cardiovascular patients such as statins, antiplatelet drugs, and heparin administration. Statins reduce the circulating levels of liverderived miR-122. Antiplatelet medication attenuates the release of platelet-derived miRNAs. Heparin inhibits the polymerase chain reactions, in particular the amplification of the exogenous Caenorhabditis elegans spike-in control, thereby resulting in an artefactual rise of endogenous miRNAs. As these limitations have not been previously recognised, a reevaluation of the current miRNA literature, in particular of case-control studies in patients with cardiovascular disease or coronary interventions, is required.
MicroRNA; Biomarker; Coronary heart disease; Cardiovascular; Myocardial infarction
This article is part of the Topical Collection on Genetics
Before the completion of the Human Genome Project, the
genome was expected to contain at least 100,000
proteincoding genes. DNA sequencing, however, revealed that only
about 21,000 such genes can be found within the
approximately 3 billion DNA bases. Although approximately 76 %
of the human genome is transcribed, less than 3 % encodes for
proteins . The remaining 97 % were thought to be ‘junk’
DNA as these sequences are not translated into protein and
were not known to encode relevant information with the
exception of few non-coding RNAs such as transfer or
ribosomal RNAs. This assumption was based on the central dogma of
molecular biology postulated by Francis Crick in 1958, which
states that all relevant biological information flows
unidirectionally from DNA to mRNA to protein [2–4]. As the number
of protein-coding genes was unexpectedly low and similar to
much simpler organisms, it was not immediately clear how the
high degree of human complexity could derive from such a
small protein repertoire. Additionally, the proteomes of higher
organisms have been shown to be relatively stable, with
humans and mice sharing 99 % of their protein-coding genes.
Though mechanisms like alternative splicing can increase the
variation of the protein repertoire, today, the complexity of the
regulatory network of non-coding RNAs, i.e. the control
architecture of the system, is thought to be the main source of
diversity [5, 6]. This is supported by the fact that while there is only a
small increase in the number of protein-coding genes in
humans as compared to the nearly 19,000 protein-coding genes
in the nematode Caenorhabditis elegans, the ratio of
nonprotein coding to protein-coding sequences is almost 17-fold
higher in humans [6, 7]. MicroRNAs (miRNAs) represent one
subgroup of non-coding RNAs. More than 2000 miRNAs are
encoded in the human genome, but only about 400 miRNAs
can be found in C. elegans (http://www.mirbase.org/). The
miRNA system is also highly evolutionarily conserved: 196
miRNA families are conserved among mammals and 34
miRNA families from C. elegans are conserved in humans
with few examples of secondary loss and very low levels of
nucleotide substitutions to the primary sequence [8, 9].
Epigenetic control mechanisms, including miRNAs that
temporarily modulate gene expression, allow the cell to
respond quickly to environmental changes as messenger
RNA (mRNA) molecules can be targeted for degradation or
blocked from translation into proteins .
Biogenesis and Function of miRNAs
miRNAs are one of the largest gene families . Due to
their mode of operation, they have the potential to target
approximately 60 % of human genes and thereby influence
many biological pathways [12, 13]. miRNAs are expressed
in a temporal and tissue-specific manner , e.g.
miR208a can only be found in cardiomyocytes  and the
miRNA content in different cells can vary from 1 to more
than 30,000 copies . miRNAs play an important role
in the regulation of embryonic development as well as in
adult life with distinct expression profiles in every cell type
at each developmental stage . They show dynamic and
site-specific expression patterns during embryogenesis and
suppression leads to death during early gestation [18, 19].
The first miRNA was identified in 1993 during the study of
C. elegans mutants that exhibited abnormal developmental
timing. The gene responsible for this phenotype, lin-4, did
not code for a protein but for two small RNAs with
complementary sequences to the 3′-untranslated region of the
lin-14 mRNA. Lin-4 had been shown previously to
negatively regulate the protein level of LIN-14, creating a
temporal decrease in LIN-14 during postembryonic
development that regulates the execution of stage-specific larval
programmes [2, 20, 21]. A similar mechanism of RNA
silencing had been known in plants since the beginning of
the 1990s , but as lin-4 was not detected in other
species, this new mechanism was believed to be a process
occurring only in nematodes. However, in the year 2000,
let-7, that also coordinates developmental timing in C.
elegans, was discovered. Let-7 codes for a 21-nucleotide
non-coding RNA transcript that negatively regulates the
mRNA of lin-41 through complementary Watson-Crick
base pairing at the 3′-UTR and therefore influences gene
expression in a way similar to lin-4 . Unlike lin-4, the
sequence of let-7 was found to be conserved in a wide
range of species including vertebrates, making the
biological significance of this finding apparent [4, 23]. Since
then, over 25,000 miRNAs have been identified from more
than 190 different species including algae, plants,
nematodes, protozoa, viruses or vertebrates .
MiRNA genes are located in different parts of the human
genome and hence show a great variation in their
transcriptional regulation and expression patterns . The majority
of human miRNAs are co-expressed with their host gene
within intronic sequences of protein-coding genes, while
others are transcribed independent of coding genes .
More than 40 % of human miRNAs appear in clusters and
are transcribed together, forming a transcript that contains
multiple miRNA sequences . After transcription by
mainly RNA polymerase II , the primary miRNA
(primiRNA) is processed by the RNase III Drosha, into a
stemloop precursor of ~70 nucleotides, the so called pre-miRNA
 (Fig. 1). As Drosha by itself cannot bind pri-miRNAs
sufficiently, it interacts with the cofactor DGCR8, forming
the microprocessor complex . The pre-miRNA is then
exported to the cytoplasm in a Ran-GTP dependent manner
by Exportin-5 that specifically binds the pre-miRNA .
Once it has reached the cytoplasm, the pre-miRNA is cleaved
by the RNaseIII Dicer together with the cofactor TRBP [30,
31] and PACT  into a duplex, consisting of two miRNA
strands . After transcription, the individual miRNAs can
be additionally regulated by adenosine deaminases that
convert adenosine to inosine and thereby influence the
hybridisation of miRNAs to their targets . The RNA
duplex is subsequently loaded onto an Argonaute protein,
together forming the RNA-induced silencing complex (RISC).
After the duplex has been unwound, one of the strands is
released and in most cases degraded. The other strand guides
the RISC to miRNA response elements (MRE) of the target
gene that are complementary to its sequence [4, 8]. MiRNAs
mainly target mRNAs, but also have the potential to bind to a
wide variety of other molecules including tRNAs or rRNAs
. Each miRNA locus produces two mature miRNAs, the
‘guide’ strand with a prevalence of 96–99 % and the
‘passenger’ miRNA* strand . miRNA strand selection depends on
different factors including the thermodynamic stability at the
ends of the miRNA:miRNA* duplex [36, 37]. Though the
miRNA* strand is usually degraded, in some cases, both
strands can be functional  with either overlapping or
different target sites . miRNA*-sequences are likely to have
functional relevance in small RNA regulatory networks, as
they are highly evolutionary conserved with e.g. more than
Fig. 1 Schematic diagram
of miRNA biogenesis and
proposed mechanisms for
miRNA function. MiRNAs
can affect protein expression
by inducing an Ago-mediated
cleavage of the mRNA, or
destabilisation and degradation
of mRNA by deadenylation,
translational inhibition and
sequestration of mRNAs
in P-bodies. Adapted
from [4, 75, 76]
40 % of miRNA*-sequences resisting nucleotide divergence
across Drosophila evolution . Given that both the
‘mature’ and the ‘passenger’ strands can be functional, the
miRNA/miRNA* nomenclature has now been retired and
instead, the two sequences are referred to as 5-p or 3-p strand
of the respective miRNA.
Once they have bound to their target mRNA, miRNAs are
able to regulate gene expression in different and sometimes
opposite ways depending on factors such as the degree of
complementarity with the target mRNA. Near-perfect
complementarity of miRNAs and their targets which mainly occurs in
plants leads to direct cleavage of the target mRNA . In
animals, however, target recognition in most cases does not
require perfect complementarity but mainly depends on
pairing to the ‘miRNA seed’, the nucleotides 2–8 of the 5′
portion of the miRNA . The short seed match and
incomplete base pairing enable the miRNA to target different RNA
molecules while a single target gene can contain multiple
conserved regions of complementarity [41, 42]. The predominant
mechanism by which miRNAs reduce protein output is by
triggering deadenylation of the target mRNA, which makes
t h e m R N A m o r e s u s c e p t i b l e t o d e g r a d a t i o n [ 4 3 ] .
Additionally, miRNAs can inhibit eukaryotic initiation factors
 or interfere with translational elongation . In some
cases, the miRNA response has been reported to switch from
inhibition of gene expression to enhancement, thereby e.g.
inducing up-regulation of target mRNAs on cell cycle arrest
and repressing translation in proliferating cells [4, 46]. These
characteristics create a complex regulatory control network
that changes in relation to age, developmental or
pathophysiological state of the cell, and involves multiple cooperative
effects on a large number of targets enabling miRNAs to
control various pathways at different levels .
The majority of miRNAs are located intracellularly. In 2007,
however, miRNAs were found in exosomes, in which they
were delivered to other cells allowing gene-based
communication between cells . Sequence motifs present in
miRNAs can thereby enable specific interaction and loading
into exosomes . This transfer of RNA through exosomes
might enable local and systemic intercellular exchange of
biological information . In the following years, miRNAs
were detected in most extracellular biological fluids including
serum, plasma, saliva and urine where they showed distinct
compositions . MiRNAs can be released into the blood
circulation by various mechanisms, including active secretion,
apoptosis or necrosis. These miRNAs circulate in different
types of vesicles, such as apoptotic bodies, microvesicles
(100–1000 nm), exosomes (50–100 nm) and lipoproteins
. Many extracellular miRNAs in circulation, however,
are also independent of vesicles and are associated with
RNA-binding proteins like Argonaute 2 protein, a part of the
RNA-induced silencing complex . Human miRNAs
isolated from plasma are highly stable in boiling water and
resistant to very high or low pH, prolonged room temperature
incubation or repeated freeze-thawing . Compared to
endogenous plasma miRNAs, synthetic miRNAs are rapidly
degraded when added to human plasma unless the RNase
activity was inactivated beforehand . Therefore, though
miRNAs are susceptible to degradation, circulating miRNAs
are resistant to RNase activity as they are secreted in a
complex with other molecules such as Argonaute proteins or
lipoproteins or in membrane-derived vesicles .
Deregulated levels of circulating miRNAs have been linked
to different disease states . During cellular stress or
pathophysiological conditions such as hypoxia, miRNAs can provide
an efficient way of gene regulation to allow the cells to adopt
and recover . As they are disease-specifically modulated
and easily accessible, circulating miRNAs are potential
bloodbased biomarkers, useful for diagnostic application e.g. in
screening programmes and for monitoring of treatment
response or outcome prediction [4, 51]. Based on their biology,
circulating miRNAs may have a high level of sensitivity and
specificity allowing early and reliable detection of pathological
states . Arguably, miRNAs offer some advantages over the
most commonly used biomarkers : miRNAs are often
expressed in a tissue-, development- or disease-specific manner
and the circulating levels of miRNAs are reproducible and
consistent among individuals of the same species [51, 57].
Compared to numerous serum proteins, including various
processing variants, and posttranslationally modified proteins,
there are far fewer known miRNA species, making it possible
to obtain a comprehensive profile. In addition, due to their
small size and chemical composition, miRNAs are less
complex than most other biological molecules and more stable in
plasma than mRNAs . Also, miRNAs can be quantified
cost-effectively using real-time polymerase chain reaction and
a profile can be obtained by next-generation sequencing or
MiRNAs as Biomarkers for Coronary Heart Disease
An example for the potential use of miRNAs as biomarkers is
the detection of myocardial infarction, where they might
complement the existing biomarkers, such as cardiac troponins.
There is still a need for novel biomarkers, as troponins fail
to rule out myocardial infarction immediately on admission
and are not reliable in certain groups of patients [51, 53].
Another major limitation is their lack of specificity, as
unspecific elevation of troponin levels can be caused by
nonischemic conditions such as heart failure and renal disease
. MiRNAs that are specifically expressed in the heart
muscle, like miR-208a, which is involved in the regulation of
myosin heavy chain production during cardiac development
, have the potential to improve diagnosis of myocardial
infarction. In a study with 33 consecutive AMI and 30
Selected studies on circulating miRNAs and coronary heart disease (CHD)
Description of study population
CVD cardiovascular disease, CAD coronary artery disease, ACS acute coronary syndrome, HOCM hypertrophic cardiomyopathy, TASH transcoronary
ablation of septal hypertrophy, MV microvesicles, MP microparticles, CK creatine kinase, cTnI cardiac troponin I, cTnT cardiac troponin T
Finn et al.  Patients with significant CHD vs patients
with only CHD risk factors vs healthy
Liebetrau et al.  Patients with HOCM, before/after TASH
De Rosa et al.  Patients with stable CAD vs patients with
ACS vs patients without CAD
Fichtlscherer et al.  Patients with CAD vs healthy controls
Orlemans et al. 
21 vs 20 vs 27
33 vs 16 vs 17
Fig. 2 Normalisation of miRNA measurements. Platelet-poor EDTA
plasma was collected from patients undergoing percutaneous coronary
intervention (PCI, n = 20) at four time points: before heparin
administration (TP-pre), 5 min after heparin administration but just
prior to stent deployment (TP-0 min), and 30 and 360 min after stent
deployment (TP, 30 min and TP, 360 min). Additional samples were
obtained from patients undergoing cardiac catheterisation for diagnostic
purposes (Dx) with (n=7) and without (n=10) heparin administration.
Note the discrepancies at TP 0 min and TP 30 min with the
conventional normalisation of miRNA measurements using an
exogenous C.elegans spike-in control (Cel-miR 39, left panel)
compared to the normalisation using the average cycle threshold (Ct)
value of a panel of endogenous miRNAs (right panel). Reproduced
with permission from [71•]
AMI patients that presented with chest pain, miR-208a
remained undetectable in plasma of non-AMI patients
including patients with chronic renal failure or trauma, but it was
initially detected in 90.9 % of AMI patients and in 100 % of
AMI patients within 4 h of the onset of chest pain, even in
patients where cardiac troponin I (cTnI) levels were not yet
affected . This earlier miRNA peak might be caused by a
faster release of miRNAs from damaged cardiomyocytes, as
miRNAs are mainly bound to protein complexes in the cytosol
while most of the cTnI is bound to myofibrils . An
overview of miRNA studies on coronary heart disease and
myocardial infarction has been published elsewhere [61, 62]. A
major shortcoming of the current literature is the lack of large
cohort studies (Table 1). Few prospective studies on coronary
heart disease have been published to date . Instead,
circulating miRNAs have been measured in numerous small case–
control studies without adequate consideration of the effects
of comorbidities  and medication [61, 62].
Fig. 3 Confounding of miRNA
measurements by medication.
Antiplatelet medication and
statins reduce the endogenous
levels of platelet- and
liverderived miRNAs, respectively.
Heparin predominantly affects
the exogenous spike-in
controls commonly used
for normalisation. Yet, most
case–control studies on miRNA
biomarkers for coronary
heart disease did not adjust
or adequately control for
the effects of medication
Anti-platelet Drugs, Statins
Reduced Circulating miRNAs
Statins, for example, reduce the circulating levels of the
liver-derived miRNA, miR-122 [65•]. Our study by Willeit
and Zampetaki et al. [66••] identified circulating platelet
miRNAs that are responsive to antiplatelet therapy. In healthy
volunteers, prolonged platelet inhibition over 4 weeks affected
the levels of plasma miRNAs and resulted in a reduction of
several miRNAs, including miR-126 (P < 0.001), miR-150
(P = 0.003), miR-191 (P = 0.004), and miR-223 (P = 0.016).
Similar results were obtained in patients with symptomatic
carotid atherosclerosis (n=33) who were on 75-mg aspirin (ASA)
at baseline. After initiation of dual antiplatelet therapy with
either dipyridamole or clopidogrel, miRNA changes were
observed after 48 h. An effect of ASA on miR-126 plasma levels
was also demonstrated by de Boer et al. [67•]. Although the
miRNA content of platelets is low compared with other cells,
platelets contribute substantially to the circulating miRNA
pool. Antiplatelet therapy was a likely confounding factor in
previous case–control studies reporting a loss of miRNAs in
patients with coronary artery disease [62, 68].
Similarly, heparin, used in interventional cardiology, is
another potentially confounding factor that may influence
miRNA measurements due to its known interference with
polymerase chain reactions [69, 70]. In the study by Kaudewitz
et al. [71•], platelet-poor plasma was obtained from patients
undergoing cardiac catheterisation for diagnostic coronary
angiography, or for percutaneous coronary intervention, both
before and after heparin administration. Heparin had pronounced
effects on the assessment of the exogenous C. elegans spike-in
control (decrease by approx. 3 cycles), which disappeared 6 h
after the heparin bolus. Measurements of endogenous miRNAs
were less sensitive to heparin medication. Similar findings were
reported by Boeckel and colleagues [72•]. Kaudewitz et al.
[71•] suggested a potential solution for measuring miRNAs
more accurately in heparinized patients: normalisation of
individual miRNAs with the average cycle threshold value of all
miRNAs provided a suitable alternative to normalisation with
exogenous C. elegans spike-in control in this setting (Fig. 2).
Thus, both the timing of blood sampling relative to heparin
dosing and the normalisation procedure are critical for reliable
miRNA measurements in patients receiving intravenous
heparin. Otherwise, the rise in circulating miRNAs post myocardial
infarction can be misinterpreted as novel biomarkers for
myocardial injury, whereas the elevation in patients compared to
controls may, at least in part, be attributable to the
heparininduced suppression of the exogenous C. elegans normalisation
control commonly used in miRNA measurements .
In summary, medication is an important confounding factor
when investigating the relation of circulating miRNAs with
coronary heart disease (Fig. 3). Some effects may also apply to
measurements in full blood . Future studies will need to
address these shortcomings of the early literature on miRNA
biomarkers and overcome confounding factors of miRNA
measurements to assess the clinical utility of miRNA
biomarkers in coronary heart disease.
Acknowledgments DK was supported by a scholarship sponsored by
the ‘Studienstiftung des deutschen Volkes’. AZ is an Intermediate Fellow
of the British Heart Foundation (FS/13/2/29574). MM is a Senior
Research Fellow of the British Heart Foundation (FS/13/2/29892). This
work was supported by a special Project Grant of the British Heart
Foundation (SP/12/5/29574), the Juvenile Diabetes Research Foundation
(172011-658), Diabetes UK (12/0004530), the Fondation Leducq
(MIRVAD; 13 CVD 02) and the National Institute of Health Research
Biomedical Research Center based at Guy’s and St Thomas’ National
Health Service Foundation Trust and King’s College London in
partnership with King’s College Hospital.
Compliance with Ethical Standards
Conflict of Interest Dorothee Kaudewitz declares no conflict of
Anna Zampetaki and Manuel Mayr declare patent applications filed
through King’s College London on miRNA biomarkers (issued and
Human and Animal Rights and Informed Consent This article does
not contain any studies with human or animal subjects performed by any
of the authors.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted
use, distribution, and reproduction in any medium, provided you
give appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were made.
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