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Circular RNAs as Novel Biomarkers

for Cardiovascular Diseases 13
Qiulian Zhou, Zhongrong Zhang, Yihua Bei,
Guoping Li, and Tianhui Wang

Abstract digestive diseases. Here we summarize recent


Cardiovascular diseases are among the most studies on circRNAs and compare the charac-
serious diseases, which are a leading cause of teristics of circRNAs with traditional bio-
death across the world. Early diagnosis and markers. Finally, we highlight the value of
prognosis prediction are keys for treatment circRNAs as potential biomarkers for cardio-
and reduction of death rates. Circular RNAs vascular diseases, including acute myocardial
(circRNAs) play a critical role in the physiol- infarction, heart failure, coronary artery dis-
ogy and pathology of biological system and ease, and hypertension. In conclusion, cir-
participate in the development of diseases. In cRNAs may be promising biomarkers for
addition, circRNAs are relative stable and cardiovascular diseases.
abundant. Therefore, many studies have sug-
gested that circRNAs could be used as bio- Keywords
markers for diseases, such as neurological Circular RNA · Biomarkers · Cardiovascular
diseases, cancers, immune diseases, and diseases

Q. Zhou Z. Zhang · Y. Bei · T. Wang (*)


Shanghai Applied Radiation Institute, School of Cardiac Regeneration and Ageing Lab, Institute of
Environmental and Chemical Engineering, Shanghai Cardiovascular Sciences, School of Life Science,
University, Shanghai, China Shanghai University, Shanghai, China
Cardiac Regeneration and Ageing Lab, Institute of Shanghai Key Laboratory of Bio-Energy Crops,
Cardiovascular Sciences, School of Life Science, School of Life Sciences, Shanghai University,
Shanghai University, Shanghai, China Shanghai, China
e-mail: wangth@shu.edu.cn
Shanghai Key Laboratory of Bio-Energy Crops,
School of Life Sciences, Shanghai University, G. Li
Shanghai, China Cardiovascular Division of the Massachusetts
General Hospital, Harvard Medical School,
Boston, MA, USA

© Springer Nature Singapore Pte Ltd. 2018 159


J. Xiao (ed.), Circular RNAs, Advances in Experimental Medicine and Biology 1087,
https://doi.org/10.1007/978-981-13-1426-1_13
160 Q. Zhou et al.

1 Introduction of Circular RNA consist of intronic sequences. Exon-intron circu-


lar RNAs consist of both exonic and intronic
Circular RNAs (circRNAs) are currently a sequences. They predominantly localize in the
research focus in the field of noncoding RNAs, nucleus and have association with the RNA poly-
including microRNAs (miRNAs), long noncod- merase II. Therefore, exon-intron circular RNAs
ing RNAs (lncRNAs), etc. [1]. Noncoding RNAs are regarded as transcriptional regulators. Three
are involved in the development of many diseases types of circRNAs share the same characteristics,
[2], including cardiovascular diseases. They are such as stable, abundant, and conservative.
recognized as regulators of gene expression.
However, in the past, circRNAs were ignored and
considered as transcriptional noise [3] or errant 3 Characteristics of circRNAs
by-products of splicing [4], owing to their unique
structure and low abundance [4]. Based on Desirable biomarkers are required to be stable
advances in bioinformatics and biotechnology, a against harsh conditions, for example, tempera-
large number of abundant and significant cir- ture variation, extreme pH, and thawing cycles
cRNAs were discovered and identified. The evi- [11, 12]. Biomarkers should have high stability in
dence is mounting that circRNAs play essential blood and other body fluids. The biggest natural
roles in various physiological and pathological enemy of RNA is RNA exonucleases (or RNase
pathways. R). CircRNAs are closed circular molecules con-
sist of covalently closed loop structure and there-
fore resistant to RNA exonucleases that solely
2 Biogenesis of circRNAs digests linear transcripts [13–17]. CircRNAs are
more stable compared to linear RNAs [18].
CircRNAs are closed circular molecules, which Furthermore, based on their stability in blood and
are distinguished from other linear RNA mole- other body fluids [19, 20], circRNAs are suitable
cules. First circRNA was found by electron biomarkers for disease diagnosis.
microscopy from RNA viruses [5]. Nowadays, Clinical trials must be properly based upon
more and more circRNAs are being identified by reliable earlier trials and sufficient preclinical
RNA sequencing and bioinformatics. Due to the studies. CircRNAs are conserved across mam-
closed loop characteristic of circRNAs, they can mals [21], including human, mouse, and rat [22,
be extracted from the ribosomal RNA and linear 23]. In hearts, RNA-Seq analysis found that
RNA molecules in total RNA. RNA sequencing plenty of circRNAs could be detected in mammal
can obtain more circRNAs from specific accumu- species: human (16427), mouse (9953), and rat
lation of circRNAs samples. The covalently (13086). Among them, about 30% (3171) were
closed loop structure of circRNAs, also called conserved between mouse and rat, and about
“back-splicing,” is formed by joining of 3′ splice 10% (1288) were conserved in all three species
site to 5′ splice site [6, 7]. There are three types of [24]. These circRNAs conserved in different spe-
circRNAs: exonic circular RNAs (ecircRNAs) cies become abundant resources of promising
[8], intronic circular RNAs (ciRNAs) [9], and candidates for biomarkers. In addition to the ben-
exon-intron circular RNAs (EIciRNAs) [10]. efits mentioned above, circRNAs have been dem-
Exonic circular RNAs have three formation onstrating specificity in expression [25].
models: lariat-driven circularization, intron Emerging evidence have confirmed thousands of
pairing-­driven circularization, and resplicing-­ the tissue-specific expression of circRNAs [26],
driven circularization. They form from exon such as the heart, brain, skin, prostate, thyroid,
back-splicing and only consist of exonic blood, or blood cell. Through biclustering algo-
sequences. The formation of circular intronic rithm, studies have already reported thousands of
RNAs mainly depends on consensus RNA motifs tissue-specific expressed circRNAs in human
and branchpoint. Circular intronic RNAs only [26]. Except for tissue-specific expression,
13  Circular RNAs as Novel Biomarkers for Cardiovascular Diseases 161

c­ircRNAs have specific expression in different as RNA-binding protein (RBP) sponges [33, 34].
diseases. In cardiac development and diseases, Thirdly, circRNAs can be a regulator of tran-
the expression of circRNAs is regulated [27, 28]. scription and alternative splicing that directly
Through whole transcriptome sequencing, 826 influence gene expression [35].
putative circRNAs have been identified in human For all those reasons and more, circRNAs
heart with dilated cardiomyopathy patient and have been reported to play important roles in
hypertrophic cardiomyopathy [27]. Through physiological and pathological processes [36–
microarray expression profile assay, 63 circRNAs 38], such as proliferation, apoptosis, and autoph-
were differentially expressed in mouse between agy. CircRNA circHIPK3 regulates the viability
heart failure and sham group [28]. and proliferation of endothelial cell viability by
In particular, circRNAs (3841) are found inhibiting the activity of miR-30a-3p [36].
unique in different composition and cell types of CircRNA TTBK2 regulates the cell proliferation
blood [14], such as plasma (51), serum (37), neu- and apoptosis of human glioma cells by regulat-
trophils (58), and platelets (2339). Circulating ing miR-217/HNF1β/Derlin-1 pathway [37].
blood markers have enormous potential to be In addition, circRNAs can play regulatory
applied to clinical practice. Human peripheral function in diseases, such as neurological dis-
whole blood is an easily accessible body fluid. eases [39, 40], cancers [41], immune diseases
Thousands of circRNAs have been detected in [42], aging [43], diabetes [44], digestive diseases
blood. Except for the blood, distinct circRNAs [45], reproductive system diseases, skin, muscles
have also been founded in other body fluids, such and bones diseases, and especially cardiovascular
as saliva [19]. On the other hand, circRNAs have diseases [46, 47]. CircRNA HRCR can function
been found enriched in exosomes [29, 30]. as an endogenous miR-223 sponge through
Exosomes are currently regarded as specific upregulation of ARC, which attenuate hypertro-
secretory membrane vesicles and are involved in phic responses [48]. CircRNA Cdr1as can inhibit
intercellular communication. Exosomes are also miR-7a through upregulating PARP and SP1,
thought to be the way of transferring miRNA in which induces myocardial apoptosis and aggra-
the circulatory system, which indirectly reflects vates myocardial infarction injuries [49].
the changes of miRNAs in diseases. Serum exo-­ Furthermore, circRNAs constitute promising
circRNAs may be involved in intercellular com- new biomarkers in multiple diseases [25, 50].
munication and can be used as indicators of CircRNA hsa_circ_0014130 is significantly
diseases development. Therefore, exo-circRNAs upregulated in non-small cell lung cancer
in the blood of patients are expected to be mark- (NSCLC) tissues that may be a new biomarker of
ers for early diagnosis. According to the charac- NSCLC [51]. CircRNA hsa_circ_0004277 is
teristics talked above, circRNAs are promising downregulated in mononuclear cells from bone
new biomarkers. marrow of acute myeloid leukemia (AML)
patients [52]. CircRNA hsa_circ_0001649 is
downregulated in hepatocellular carcinoma
4 Functions of circRNAs (HCC) tissues and is correlated with tumor size
and tumor embolus’s occurrence [53].
As most other noncoding RNAs, most of the cir- In addition to the above examples, circRNAs
cRNAs do not encode proteins. But circRNAs are have the potential to become biomarkers in many
involved in regulating the expression of genes cardiovascular diseases. Here we will briefly intro-
[31]. Firstly, circRNAs can adsorb some specific duce the characteristics of circRNAs by comparison
miRNAs that prevent mRNA translation, func- with the existing biomarkers of cardiovascular dis-
tioning as miRNA sponges [25, 32]. Secondly, eases, which expound the value of circRNAs. Then
circRNAs can bind to RNA-associated proteins we will briefly summarize recent findings on cir-
and form RNA-protein complexes that function cRNAs as biomarkers in cardiovascular diseases.
162 Q. Zhou et al.

5 Biomarkers miR-133, miR-423-5p, miR-126) [66–68].


of Cardiovascular Diseases However, only a few of them have been validated,
and they are necessary to prove by large-scale
Cardiovascular diseases threaten human health clinical studies [69]. Traditional biomarkers are
and have become one of the leading diseases also limited by low stability [70]. Therefore, cir-
which causes death in the world [54]. Nowadays, cRNAs, which are stable, conservative, and spe-
more and more risk factors are aggravating the cific [71, 72], might open a novel avenue.
development and progression of cardiovascular
diseases. Early diagnosis and prognosis predic-
tion are keys for treatment and reducing mortal- 6  ircular RNAs as Biomarkers
C
ity. There are multiple diagnostic techniques for in Cardiovascular Disease
cardiovascular diseases, such as blood pressure
monitoring, dynamic electrocardiogram, cardio- 6.1 Acute Myocardial Infarction
vascular ultrasound, magnetic resonance imag-
ing, electronic computed tomography, and so on. Acute myocardial infarction is a myocardial
These diagnostic techniques require lots of necrosis caused by acute and persistent ischemia.
resources and precise instruments. What’s more, Acute myocardial infarction can be complicated
these diagnostics still cannot ensure the accurate with arrhythmia, shock, or heart failure, which is
diagnosis of the occurrence and development of often life-threatening. It tends to occur in patients
cardiovascular diseases. with coronary atherosclerosis stenosis and coro-
Some blood markers are commonly regarded nary spasm. Acute myocardial infarction carries a
as sensitive biomarkers for cardiovascular dis- high mortality rate and become a global chal-
eases, such as atrial natriuretic peptide (ANP), lenge [73, 74]. Reperfusion is the most common
creatine kinase (CK), myocardial band isoen- treatment strategy for myocardial infarction
zyme (CK-MB), and cardiac troponin (cTn) [55]. patient. This treatment strategy will cause myo-
Brain natriuretic peptide (BNP) and N-terminal cardial ischemia-reperfusion injury leading to
portion of its prohormone (NT-proBNP) are also left ventricular remodeling and even heart failure.
used as biomarkers for cardiovascular diseases There are some biomarkers for clinical assess-
[56]. But there are problems difficult to solve. ment of the severity, course, and prognosis of
Firstly, the detection time is uncertain, since acute myocardial infarction. Creatine kinase(CK)
markers keep changing over time and exhibit a and cardiac troponin(cTn) are markers for myo-
relative “delayed” release time [57]. Secondly, cardial injury [75–77]. In skeletal muscles and
disease degree and patient’s background are var- heart muscles, creatinine kinase facilitates the
ied, while these biomarkers are low in specificity transfer of energy phosphates between outside
[58]. Thirdly, there are still many kinds of cardio- and inside mitochondria. The increased concen-
vascular diseases without proper marker, such as tration of creatine kinase in serum indicates myo-
coronary artery disease. As a result, there remain cardial damage, such as myocardial infarction or
many challenges in the field of cardiovascular ischemia. But it can also be caused by vigorous
diseases diagnosis. exercise or other kinds of tissue damage [78].
There is growing evidence that noncoding Cardiac troponin I and T untimely are released
RNAs have the potential to become biomarkers into plasma when actin and myosin filaments
in many cardiovascular diseases. Circulating occur in pathologic degeneration in the heart.
miRNAs are suggested as suitable biomarkers for Thus, cardiac troponin can be a marker in myo-
cardiovascular diseases [59–61], including acute cardial infarction. But the abnormal release of
myocardial infarction (miR-1, miR-133, miR-­ cardiac troponin can also occur in myocarditis
208, and miR-499) [62, 63]; acute coronary syn- [75, 76], pulmonary embolism with acute right
drome (miR-208a, miR-34a, miR-133a, heart overload [79], and even heart failure [80].
miR-499) [64, 65]; and heart failure (miR-499, Therefore, new biomarkers are required.
13  Circular RNAs as Novel Biomarkers for Cardiovascular Diseases 163

Recently, a study observed that a circRNA myocardial structure and function. Heart failure
called MICRA (myocardial infarction-associated is a major cause of morbidity and mortality
circular RNA) is absent in peripheral blood cells, worldwide [91–93], even though its molecular
plasma, and serum samples [81, 82]. The particu- mechanisms become more and more clear and a
lar circRNA (MICRA) is formed by 874 nucleo- large amount of pharmacological interventions
tides, which is mainly derived from zinc finger come to light. In current guidelines, the plasma
protein 609 (ZNF609) gene exon 1 [82, 83]. levels of a natriuretic peptide (such as brain natri-
MICRA is associated with development of heart uretic peptide, BNP, and N-terminal pro brain
failure after myocardial infarction and can be natriuretic peptide, NT-proBNP) are important
used to predict left ventricular remodeling after diagnostic indicators of acute heart failure [94].
acute myocardial infarction by multivariable anal- Most of biomarkers for the assessment of heart
yses (0.83[0.69–1.01]; p  =  0.066) [81]. Lower failure are widely acknowledged and used. But
level of MICRA classifies patients into decreased there is still limitation that circulating levels of
ejection fraction groups; and high level of MICRA BNP will significantly change with the develop-
is able to predict a preserved ejection fraction at ment of acute myocardial infarction [95].
4 months. When MICRA is included, a multivari- Therefore, these biomarkers cannot be used in
able clinical model, Akaike Information Criteria, the management of heart failure patients, who
becomes more accurate in prediction of preserved have a complicated disease history.
ejection fraction at 4 months. CircRNA MICRA An increasing number of RNA including miR-
is potential to become a novel biomarker in the NAs [19, 96] and lncRNAs [83, 97, 98] have been
future prognostication strategies of acute myocar- found with enormous potential as biomarkers for
dial infarction [81]. heart failure. LncRNA LIPCAR was found
In another study, circRNA MICRA was found downregulated early after myocardial infarction
lower in 642 myocardial infarction patients com- and meanwhile upregulated in plasma of 788
pared to 86 healthy volunteers [82]. Besides, heart failure patients [97]. LncRNA NRON and
MICRA was significantly lower in patients who LncRNA MHRT were found upregulated in
have a low ejection fraction. In particular, both plasma of 104 heart failure patients [99].
univariate and multivariate analyses proved that CircRNAs have great potential to act as bio-
circRNA MICRA may be a strong predictor for marker for myocardial infarction and heart fail-
prognoses of myocardial infarction after ure. By comparing the transcriptome sequencing
3–4  months. Collectively, circRNA MICRA is through bioinformatic analysis, 1163 circRNAs
suggested to be able to inform the risk of devel- were found changed in hypertrophied myocardial
oping left ventricular dysfunction for myocardial tissues grouping with or without heart failure
infarction patients. [28]. Through analyzing by quantitative PCR, 29
upregulated and 34 downregulated circRNAs
was validated [28]. Thus, these circRNAs may be
6.2 Heart Failure potential biomarkers for heart failure. Besides, a
circRNA MICRA was downregulated in
Heart failure is a disorder of the systolic function decreased ejection fraction groups (heart failure).
and/or diastolic function of the heart [84–86]. This circRNA might become a biomarker for
Heart failure is not an independent disease, but a both myocardial infarction and heart failure.
terminal stage in the development of heart dis-
eases [87, 88]. Almost all kinds of cardiovascular
diseases can eventually lead to the occurrence of 6.3 Coronary Artery Disease
heart failure, such as myocardial infarction, car-
diomyopathy, hemodynamic overload, myocar- Coronary artery disease, a big threat to heart
dial injury (any cause), and inflammation [89, health, is the main cause of coronary heart
90]. All these diseases can lead to alterations of ­disease, cerebral infarction, and peripheral vas-
164 Q. Zhou et al.

cular disease. Coronary artery disease with high peripheral blood, including 12 upregulated and
morbidity and mortality pose a threat to human 10 downregulated. Hsa_circ_0124644 was sig-
health and cause burden to economies [98]. There nificantly upregulated in coronary artery disease
are some treatments for coronary artery disease, group, and this circRNA was of high sensitivity
such as medications, percutaneous coronary (0.867) and specificity (0.767) for coronary
intervention (PCI), and coronary artery bypass artery disease [108]. Therefore, circRNAs are
graft surgery (CABG) [100–102]. Minimizing prospective diagnostic biomarkers in coronary
the time between plaque rupture and treatment is artery disease.
critical for reducing the morbidity and mortality
[103]. But the diagnosis and prognosis of coro-
nary artery disease is almost blank at present 6.4 Hypertension
[104, 105].
Early detection of coronary artery disease has High blood pressure (hypertension) refers to sys-
the potential to increase survival rates and to temic arterial blood pressure (systolic pressure)
improve the life quality of patients. MiR-221 and and/or diastolic blood pressure increase as the
miR-222 have been reported reduced in carotid main characteristics (systolic blood pressure,
plaques after an acute ischemic cerebrovascular 140  mmHg or higher; diastolic blood pressure,
syndrome [106]. CircR-284 possesses a miR-221 90 mmHg or higher). Hypertension can be asso-
and miR-222 binding site. Therefore, circR-284 ciated with the function of the heart, brain, kid-
may act as a regulator on miR-221/miR-222 ney, and other organs or physical damage to the
activity. A validation study found that miR-221 is clinical syndrome. Hypertension is the most
significantly decreased in the urgent carotid ath- common chronic disease and the most important
erosclerotic plaques, compared with the asymp- risk factor for cardiovascular disease [109–111].
tomatic control. But perhaps even more Profiling and bioinformatics identified 13
interesting is that circR-284 has been reported to downregulated and 46 upregulated circRNAs in
be significantly increased in the serum of urgent hypertension patients [112]. Among them, four
carotid atherosclerotic plaque patient [103]. circRNAs (hsa-circ-0000437, hsa-circ-0008139,
Combining the expression of serum circR-284 hsa-circ-0005870, and hsa-circ-0040809) showed
and miR-221, the ratio of circR-284 and miR-221 significant difference in hypertensive patients. By
was uniquely increased in the urgent group further validation, hsa-circ-0005870 was con-
(P  <  0.001); and this ratio was sensitive (0.76) firmed significantly downregulated in plasma
and specific (0.88) for detecting plaque rupture between 49 hypertension patients (systolic blood
and stroke [103]. Based on these results, the ratio pressure over 150/90 mmHg) and 49 healthy (sys-
of circR-284 and miR-221 has the potential to tolic blood pressure lower than 140/90 mmHg). In
become a diagnostic biomarker in cardiovascular addition, hsa-circ-0005870 has also been found
disease for prediction of the risk of plaque related to many biological processes, such as cel-
rupture. lular response to stress. Therefore, this circRNA
Identified by microarray analysis, there were may be a novel biomarker for hypertension.
2036 circRNAs in blood differentially expressed In human umbilical vein endothelial cells,
between coronary artery disease and control, 7388 circRNAs have been identified. Under
including 376 upregulated and 1660 downregu- hypoxia in endothelial cells, circRNAs cZNF292,
lated ones [107]. Among them, a circRNA (hsa-­ cAFF1, and cDENND4C were found upregu-
circRNA11783-­ 2) was significantly lated, whereas cTHSD1 was reduced. Among
downregulated in coronary artery disease group them, cZNF292 is the highest expressed with
but also in type 2 diabetes group [107]. In a sepa- proangiogenic activities. These circRNA may be
rate set of experiments, RNA microarray showed new therapeutic targets and endogenous bio-
22 circRNAs were differentially expressed in markers [113].
13  Circular RNAs as Novel Biomarkers for Cardiovascular Diseases 165

6.5 Chronic Thromboembolic early stages of cardiovascular diseases, the diag-


Pulmonary Hypertension nosis of diseases is important for treatment and
prognosis. Traditional nonspecific diagnosis can-
Chronic thromboembolic pulmonary hyperten- not fulfill the needs. A novel biomarker demands
sion (CTEPH) is harmful to human health. (1) noninvasiveness and safety, (2) high sensitiv-
Pulmonary hypertension is caused by obstruction ity and specificity, and (3) stability and
of pulmonary arteries and subsequent vascular abundance.
remodeling [114–116]. Early detection of pulmo- CircRNAs are suitable biomarkers for disease
nary hypertension has the potential to increase diagnosis. Firstly, circRNAs are closed circular
physiological function of heart and improve molecules that are resistant to RNA exonucleases
prognosis [117, 118]. In the blood samples of or RNase R and hence have stability in blood and
chronic thromboembolic pulmonary hyperten- other body fluids. Secondly, many circRNAs are
sion patient, 351 circRNAs showed significant reported to be conserved across mammals.
difference, including 122 upregulations and 229 Especially, circRNAs have high sensitivity and
downregulations [119]. Among them, circRNA specificity. CircRNAs can be source for search-
hsa_circ_0002062 can regulate 761 miRNAs, ing new therapeutic targets and candidates in dis-
and circRNA hsa_circ_0022342 can regulate 453 ease diagnosis [123]. Multiple studies have
miRNAs. Through miRNAs regulation, these cir- suggested that circRNAs may become potential
cRNAs may be involved in regulating the path- biomarkers for many cardiovascular diseases,
ways in chronic thromboembolic pulmonary such as heart failure, acute myocardial infarction,
hypertension [119–121], such as ErbB signaling coronary artery disease, and hypertension
pathway. Therefore, these unique circRNAs (Table 13.1).
which are changed and involved in chronic There needs to be more research on explora-
thromboembolic pulmonary hypertension may tion of circRNAs as potential biomarkers. In
be novel biomarkers for this disease. experimental design, patients’ physiological con-
ditions need to be more explicit, including crite-
ria such as ages, genders, colors, diet habits, and
7 Future Perspectives so on. There are some details we should consider:
(1) What kind of sample can be collected, blood
Cardiovascular diseases are not only manifold sample, serum, or plasma? (2) What can be used
but involute. Due to the influence of heredity for normalization, Cel-mirR-39, GAPDH, or
[122], diet, living habit, and external environ- SF3a1? (3) Which test method can be use, SYBR
ment, different people have different age of onset, or TaqMan real-time quantitative?
and some people do not have obvious symptoms It is important to make strict screening crite-
in their whole life. Nowadays, many people live a ria. Although many studies assume that circRNAs
stressful, chronically overloaded, unbalanced life have high stability, there are some conditions that
that cause rising morbidity and mortality. In the need to be investigated, such as sample collection

Table 13.1  Circulating circular RNAs as biomarkers in cardiovascular diseases


Diseases CircRNAs Regulation Normalization Sample Ref
Acute myocardial infarction MICRA Down SF3a1 Blood [81, 82]
Heart failure MICRA Down SF3a1 Blood [81, 82]
Coronary artery disease CircR-284/miR-221 Up – Serum [103]
hsa_circ_0124644 Up GAPDH Blood [108]
Hypertension Hsa-­circ-­0005870 Down GAPDH Plasma [112]
Chronic thromboembolic pulmonary hsa_circ_0002062 Down – Blood [119]
hypertension hsa_circ_0022342 Down – Blood [119]
166 Q. Zhou et al.

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