review article circulating micrornas: a potential role in...

Hindawi Publishing CorporationDisease MarkersVolume 35 (2013) Issue 5 Pages 561ndash566httpdxdoiorg1011552013217948

Review ArticleCirculating microRNAs A Potential Role in Diagnosis andPrognosis of Acute Myocardial Infarction

Ali Sheikh Md Sayed1 Ke Xia12 Tian-Lun Yang1 and Jun Peng3

1 Cardiology Department Xiangya Hospital Central South University No 87 Xiangya Road Changsha 410078 China2 Cardiovascular Division Department of Medicine Center for Vascular Biology and Inflammation Brigham and Womenrsquos HospitalHarvard Medical School Boston MA 02108 USA

3Department of Pharmacology School of Pharmaceutical Sciences Central South University Changsha 410078 China

Correspondence should be addressed to Tian-Lun Yang tianluny163com and Jun Peng junpengcsueducn

Received 14 August 2013 Accepted 30 September 2013

Academic Editor Marco Peluso

Copyright copy 2013 Ali Sheikh Md Sayed et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Rapid and correct diagnosis of acute myocardial infarction (AMI) plays a crucial role in saving patientsrsquo life Although somebiomarkers (such as cardiac troponin and creatine kinase) are available for AMI diagnosis so far there is still a clinical need fornovel biomarkers which can reliably rule in or rule out AMI immediately on admission Circulating microRNAs (miRNAs) are apotential choice for novel biomarkers in AMI diagnosis and prognosis with high sensitivity and specificity Circulating microRNAsare endogenous miRNAs that are detectable in whole blood serum or plasma in a highly stable form Until now around 20circulating miRNAs were reported to be closely associated with AMI In this minireview we summarized recent available dataon the correlation between circulating miRNAs and AMI Some miRNAs such as miR-208 miR-499 miR-133 and miR-1 weregiven special attention since they may have a potential prospect in diagnosis and prognosis of AMI

1 Introduction

Coronary artery disease (CAD) is a very common healthproblem in the developed as well as developing countriesAcute myocardial infarction (AMI) is one of the leadingcauses for death worldwide and it is the single largest causeof death in the United States responsible for 1 out of every6 deaths [1 2] Rapid and correct diagnosis of AMI playsan important role in therapy and prognosis for this diseaseOver the past two decades huge progress has been made inthe diagnosis treatment and prognosis of AMI Particularlythe progress in biomarkers for AMI attracted a great deal ofattention Currently cardiac troponins are the most commonbiomarkers used for diagnosis of AMI in clinical practiceHowever there is still a clinical need for novel biomarkerwhich is able to reliably rule in or rule out AMI immediatelyon admissionMicroRNAs (miRNAs) seem to be a promisingcandidate of novel biomarker for early diagnosis of AMI [3]In the last few years the dysregulation of tissue expressionlevels of miRNAs has been directly linked to cardiac disease

In addition to expression changes in tissues more recentstudies have indicated that miRNAs are detectable in serumplasma urine and other body fluids in a highly stable formthat is protected from endogenous RNase activity [4] Alteredcirculating miRNA concentrations have been detected inpatients with AMI [5] acute coronary syndrome (ACS) [6]stable coronary artery disease [7] heart failure [8] coronaryatherosclerosis cardiac arrhythmia cardiomyopathy andcardiac hypertrophy [9]This review will focus on a potentialrole of circulating miRNAs in diagnosis of AMI as novelbiomarkers

2 Circulating miRNAs

miRNAs are endogenous small (sim22 nucleotides) single-stranded noncoding RNAs that regulate gene expression atthe posttranscriptional level by binding to the 31015840 untranslatedregions (UTRs) of their target mRNAs [10] When miRNAscompletely bind to their target mRNAs degradation of

562 Disease Markers

the target mRNAs will be initiated But when the miRNAsbind partially the translation of mRNA is repressed miR-NAs are involved in multiple biological processes such asproliferationmigration differentiation secretion excitationconduction cell cycle ageing and apoptosis by changingprotein expression of potential candidate targets [11]

miRNAs have recently been detected in serum or plasmawhich are referred to circulating miRNAs Despite intenseresearch the origin of circulating miRNAs remains largelyunknown A number of studies have reported that miRNAsare actively secreted in microvesicles or exosomes fromdifferent cell types [12] which are a likely source of circulatingmiRNAs So far around 121 miRNAs were identified inexosomes from mast cells [13] and sphingomyelinase 2(nSMase2) a rate-limiting enzyme for ceramide biosynthesiscontrols the secretion of exosomes to the extracellular matrix[14] In addition tomicrovesicles or exosomes microparticlesand lipoprotein complexes (such as high-density lipoproteincomplexes) are other possible sources of circulatingmiRNAs

Microparticles are larger than exosomes of plasma mem-brane origin contain miRNAs and actively control cell com-munication process [15] High-density lipoprotein (HDL) hasbeen reported to transport endogenous miRNAs and deliverthem to recipient cells with functional targeting capabilitiesbesides its basic role as a delivery vehicle for excess cellularcholesterol Interestingly the human HDL-miRNA profilefrom normal subjects is significantly different than familialhypercholesterolemia subjects The most abundant miRNAsassociated with HDL from normal subjects were hsa-miR-135a hsa-miR-188-5p and hsa-miR-877 whereas the mostabundant familial hypercholesterolemia HDL-miRNAs werehsa-miR-223 hsa-miR-105 and hsa-miR-106a Thereforehuman HDL not only transports endogenous miRNAs butalso can display different functions under different conditionsand act as potentialmediators in gene regulation and intercel-lular communication The detection of circulating miRNAsin the plasma and serum indicates that miRNAs may fulfillbiological functions outside the cell and act as potentialbiomarkers for diseases [16]The human genome is estimatedto encode up to 1000miRNAs and over 100miRNAs in serumwere identified from healthy subjects [17]

3 Stability and Isolation ofCirculating microRNAs

Human miRNAs isolated from plasma are highly stablein boiling water in solution with very high or low pHPlasma miRNA levels remain stable when it is subjected toprolonged room temperature incubation or freeze-thawedmultiple times It has been reported that endogenous plasmamiRNAs exist in a form that is resistant to plasma RNaseactivity [18] Compared to the endogenous plasma miR-NAs rapid degradation was observed within minutes whensynthetic miRNAs (corresponding to caenorhabditis elegansmiRNAs cel-miR-39 cel-miR-54 and cel-miR-238) wereadded into human plasma Inactivating the RNase activ-ity before the addition the exogenous miRNAs escapeddegradation Therefore exogenous plasma miRNAs are not

stable and are susceptible to rapid degradation whereasnative circulating miRNAs are stable and resistant to RNaseactivity [19] There are multiple factors that account for theprotection of circulating miRNAs against RNase-dependentdegradation For example circulating miRNAs are not freein blood which are normally included in microvesicles orexosomes Furthermore the circulating miRNAs can formthe protein-miRNA complexes with some special proteins(such as nucleolar RNA-binding protein nucleophosmin 1)to resist the degradation [20]

Although circulatingmicroRNAs are stable reproducibleisolation of cell-free miRNAs with high purity is a techni-cal challenge for multiple reasons First plasma or serumcontains very low amounts of RNA The yield of RNAfrom small volume of plasma or serum (usually less than1mL) falls below the limit of accurate quality control byregular methods Second high concentration of proteins inplasma or serum could potentially interfere with the samplepreparation and the detection assay Thus avoiding proteincontamination and hemolysis (lysis of red blood cells) inserumplasma samples are important challenges to overcomeFinally some of the larger small RNA species frequentlyused as reference genes (such as U6 RNA cel-miR-39) tonormalize the data of qPCR are present in extremely lowconcentrations in serumor plasmaTherefore great caremustbe taken when choosing controls (miR-156 is an option)for normalization Fortunately with circulating microRNAswell recognized for potential diagnostic biomarkers in a widerange of diseases and biological processes more commercialkits for high quality of circulating microRNAs isolation areavailable in the market

4 Circulating miRNAs of Cardiac Origin andAcute Myocardial Infarction

Rapid and correct diagnosis is crucial to treatment andprognosis of AMI Up to date cardiac troponins and creatinekinase-MBare themost commonly used biomarkers forAMIbut their clinical value is limited in many cases Secreted bycardiac cells and accumulated in bloodmiRNAs are expectedto reflect cardiac injury in response to cardiovascular riskfactors and various pathological conditions Thus the circu-lating cardiac-specific or -enriched miRNAs (such as miR-208 miR-499 and miR-133) may provide unique biomarkersfor diagnostic and therapeutic interventions of AMI [21]

41 miR-208 miR-208 is a cardiac-enriched microRNA andfound at much higher levels during cardiac tissue injury Itis also involved in stress hormone or electric stimulation incardiac cells [22 23] During cardiac development miR-208exerts a regulatory role in the production of themyosin heavychain [24] It has been reported that plasma concentrationof circulating miR-208 levels was significantly increased fol-lowing isoproterenol-induced ratmodel ofmyocardial injuryBut this phenomenon was not observed after renal injuryindicating that circulating miR-208 is specific for cardiacinjury There was a good correlation between the changes

Disease Markers 563

in plasma miR-208 and cardiac troponin I (cTnI) a classicalbiomarker of AMI [25]

411 miR-208a Throughmicroarray analysis miR-208a wasabsolutely identified to be expressed in the human heartmiR-208a was undetectable in plasma of healthy individualsor non-AMI patients (such as patients with acute kidneyinjury chronic renal failure stroke or trauma) but it waseasily detected in 909 AMI patients and in 100 AMIpatients within 4 h of the onset of chest pain while cTnIwas not yet affected suggesting that miRNAs may leak intothe bloodstream at an earlier stage of myocardial injury(the biological peak of troponins is sim18 h after AMI) Itis reasonable to speculate that circulating miR-208a hasadvantages over cTnI (or cTnT) in distinguishing pure AMIfrom patients with renal disease stroke and trauma [5]

Interestingly the elevation of circulating miR-208a inAMI could be persistent in adult mouse but not in humanA possible explanation for this phenomenon might be dueto the different miRNA expression pattern between humanand mice miR-208a is encoded within an intron of 120572-cardiacmuscle myosin heavy chain gene (Myh6) while miR-208b aclose familymember of miR-208 is encoded within an intronof 120573-cardiac muscle myosin heavy chain gene (Myh7) Thereexists high degree of homology between miR-208a and miR-208b inmice but not in human It is likely that the persistentlyelevated plasma level of miR-208a after AMI might be theresult of miR-208b interference [23]

412 miR-208b In case of acute ST segment elevationmyocardial infarction (STEMI) patients the levels of plasmamiR-208b increased 3000-fold compared to healthy controlswithin 12 h after infarction Peak values of miR-208b werewell correlated with peak troponin I (cTnI) and the ejectionfraction indicating a possible role for circulating miR-208bas a biomarker in diagnosis of STEMI or in predictionof long-term complications [26] In AMI patients plasmamiR-208b was increased 1600-fold compared with healthysubjects The receiver operating characteristic (ROC) curveanalysis revealed an area under the ROCcurve (AUC) of 094suggesting that miR-208b was an ideal biomarker for AMIThe changes in plasma level ofmiR-208bwere consistent withplasma troponin-T indicating that miR-208b was releasedfrom injured cardiomyocytes like troponin-T [27] Howeverit is not all of the studies that supported the role ofmiR-208 asa biomarker for AMI Adachi et al reported that circulatingmiR-208b level appeared to be very low in AMI patients andit was a useful plasma biomarker for AMI diagnosis [28]Another study has recently demonstrated that circulatingmiR-208b might be used as a biomarker for AMI but it didnot show any advantages over cTNT for AMI diagnosis [29]

42 miR-499 The gene encoded miR-499 is embeddedwithin a ventricular-specific myosin heavy chain gene whichis almost exclusively expressed in the heart [30] Plasma levelof miR-499 was obviously increased in all patients with AMIbut it was below the limit of detection for acute coronary syn-dromes congestive heart failure and for all healthy controls

These results indicated that circulating miR-499 might be auseful biomarker for the diagnosis of AMI [28] It has beendemonstrated that plasma level of miR-499 increased 250-fold or 100-fold in patients with acute STEMI or AMI com-paredwith healthy controls [26] ROCcurve analysis revealedan area under the curve of 092 which is correlated withcirculating cTnT In AMI patients with chest pain lt3 h miR-499 was positive in 93 of patients while the positive ratefor hs-cTnT was 88 suggesting that miR-499 is a sensitiveand novel biomarker for the diagnosis of AMI [31] Howeverresults of ROC curve analysis from Li et alrsquos study showed noadvantage for plasma miR-499 over cTNT in AMI diagnosis

Circulating miR-499-5p a family member of miR-499was increased more than 80-fold in geriatric patients withacute non-ST elevation myocardial infarction (NSTEMI)compared with healthy subjects The ROC curve analysisshowed that diagnostic accuracy of miR-499-5p was higherthan that of TNT (AUC of miR-499-5p versus cTnT versushs-cTnT 086 versus 068 versus 070)Thus circulatingmiR-499-5p is a potential biomarker and its accuracy is superiorto conventional cTnT for the diagnosis of acute NSTEMI inthe elderly patients [32] Recently a study demonstrated thatmiR-499-5p was higher in MI patients and was negativelycorrelated with the value of left ventricular ejection fractionstrongly suggesting that circulating miR-499-5p might beused for the evaluation of mortality risk [33]

43 miR-133 In a rat AMI model plasma level of miR-133a was increased at 1ndash3 h peaked at 3ndash12 h and decreasedat 12ndash24 h after coronary artery ligation In AMI patientsmiR-133 plasma level was substantially higher compared withhealthy controls A positive correlation was also reportedbetween the elevated plasmamiR-133 and cTNIThese resultsprovided evidence that miR-133 is a powerful biomarker forthe diagnosis of AMI [5] However there were some differentreports on circulating miR-133 In a report no significantdifference in plasma level ofmiR-133 had been found betweenAMI patients and healthy subjects [34] Circulating miR-133 also showed no significant changes in AMI patientswith tachyarrhythmia or bradyarrhythmia compared to thosein patients without arrhythmias Plasma levels of miR-133aand miR-133b family members of miR-133 were reportedto be upregulated in acute STEMI patients compared withhealthy subjects which was peaked at about 156 minutesafter occurrence of infarction consistent with time course ofchange in cTNI level In contrast the level of circulatingmiR-133a was not significantly changed in mice subjected to acutehind-limb ischemia [35]

44 miR-1 The plasma level of miR-1 was reported tomarkedly increased in rats with AMI or in AMI patientswhich was only mildly increased in patients with non-AMIcoronary heart disease or patients with other cardiovasculardiseases [5] In a rat model of AMI induced by coronaryligation serummiR-1 level was rapidly increased around 200-fold after AMIwith a peak at 6 h and themiR-1 level returnedto basal levels at 3 days after AMI which displayed a strongpositive correlation with myocardial infarct size In a clinical

564 Disease Markers

Table 1 Summary of circulating miRNAs and AMI

miRNAs Diseases Species Upregulationdownregulation Correlation with cardiac troponin ReferencemiR-208a AMI Human Up Positive [5]miR-208a AMI Rat Up [23]

miR-208bAcute STEMI Human Up Positive [26]

AMI Human Up [27]AMI Human Up Positive [29]

miR-499

Acute STEMI Human Up Positive [26]AMI Human Up Positive [31]

NSTEMI Human Up Positive [32]AMI Human Up [29]AMI Human Up [33]

miR-133AMI Rat Up [5]AMI Human UP [35]AMI Human Up Positive [38]

miR-1

AMI Rat Up [5]AMI Human rat Up [36]AMI Human Up Positive [34]AMI Human Up [26]AMI Human Up [29]

miR-328 AMI Human Up Positive [43]miR-1291 AMI Human Up [36]miR-21 AMI Human Up [44]miR-30 miR-195 AMI Human Up [45]miR-let-7b AMI Human Down [45]miR-378 AMI Rat Down [46]miR-126 AMI Human Down [40]AMI acute myocardial infarction STEMI ST segment elevation myocardial infarction NSTEMI non-ST segment elevation myocardial infarction

study the level of circulating miR-1 was increased nearly 100-fold in AMI patients compared with healthy subjects andshowed a positive correlation with serum creatine kinase-MB (CK-MB) level In another separate clinical study plasmamiR-1 level was significantly higher in AMI patients com-pared with non-AMI subjects and the level was returnedto normal on discharge following medication Interestinglythe increased plasma miR-1 was not associated with agegender blood pressure diabetesmellitus or other establishedbiomarkers for AMI including cTnI and CK-MB Moreoverin a necrosis model of cultured cardiac cells miR-1 was foundto be released into the culture medium and was stable atleast for 24 h Collectively these results strongly support thatcirculating miR-1 might be a novel sensitive biomarker forAMI diagnosis [36]

5 Circulating miRNAs of Noncardiac Originand Acute Myocardial Infarction

In theory the change of circulating miRNAs after AMI isdue to the injured heart which releases miRNAs into theblood However there is evidence to show that many changesin circulating miRNAs may not originate from the injuredheart In a recent study whole-genome miRNA expressionin peripheral total blood samples from AMI patients was

analyzed and 121miRNAswere reported to be dysregulated incomparison to healthy controls Among them miR-30c andmiR-145 levels were found to be correlated with infarct sizemiR-1291 and miR-663b were highly sensitive and specificfor the discrimination of cases from controls and a uniquesignature of 20miRNAs was shown to predict AMI with evenhigher power (specificity 96 sensitivity 90 and accuracy93) [37] These results indicate that miRNA signaturesderived from peripheral blood cells could be also used asnovel sensitive biomarkers for the diagnosis of AMI

miR-126 is specifically and highly expressed in humanendothelial cells It has a potential role in angiogenesis and inregulation of endothelial homeostasis and vascular integrity[38 39] Circulating plasma miR-126 was significantly down-regulated in AMI patients compared with healthy subjectsThe plasma concentration of miR-126 also showed a goodcorrelation with the plasma concentration of cTnI in AMI[40] Circulating levels of miR-126 were decreased duringtranscoronary passage in patients with evidence of myocar-dial injury compared with noncoronary artery disease [41]Recently a prospective study has reported that circulatingmiR-126 has a positive association with fatal myocardialinfarction [42]

So far more andmore circulatingmiRNAs cardiac originor noncardiac origin were reported to be potential biomark-ers for AMI Table 1 is a summary of the correlation between

Disease Markers 565

circulating miRNAs and AMI from the recent literatures Asdisplayed in Table 1 at least 13 miRNAs were reported aspotential biomarkers for AMI Among them cardiac-specificor -enriched miRNAs (such as miR-208 miR-499 miR-133and miR-1) attracted more attention [25 27 28 34] SincemiR-208 andmiR-499were only detected in heart whilemiR-133 and miR-1 were highly expressed in both skeletal andcardiac muscle in theory miR-208 andmiR-499 should havethe advantages (such as more specific or sensitive) over miR-133 and miR-1 in AMI diagnosis Actually in Wang et alrsquosstudy they examined the plasma levels of miR-208 miR-499 miR-133 and miR-1 in AMI patients and rat model ofAMI After careful comparison and analysis of the resultsthey concluded that among these four miRNAs miR-208 isa more reliable biomarker for AMI diagnosis [5] However inDevaux et alrsquos study they provided evidence that circulatingmiR-499 appeared clearly superior to circulatingmiR-208 fordetecting cardiac damage [31] Thus it needs further studiesto determine which one (miR-208 or miR-499) is the mostpotential biomarker for AMI diagnosis

6 Conclusion

In summary circulatingmiRNAs are resistant to endogenousribonuclease activity and can be present in human plasmaor serum in a remarkably stable form It is believed thatcirculatingmiRNAs provide a novel class of minimal invasivebiomarkers forAMIdiagnosis Until now around 20miRNAswere reported as potential biomarkers for AMI Howevermost of the findings were based on relatively small sampleswhich lead to many divergences between different reportsTherefore it is necessary to conduct independent and largecohort studies to identify those circulating miRNAs with realvalue in AMI diagnosis So far most of the studies focuson the role of circulating miRNAs as biomarkers for AMIdiagnosis The information regarding the prognostic impactof circulatingmiRNAs inAMI is still largely lacking Actuallyas ideal biomarkers it is also important to be useful in AMIprognosis Recently we have found that plasma levels ofmiR-208bmiR-499miR-149 andmiR-424were significantlyelevated in AMI Among them miR-208b miR-499 andmiR-424 were rapidly decreased after percutaneous coronaryintervention (PCI) (unpublished data) suggesting that miR-208bmiR-499 andmiR-424might be potential candidates asbiomarkers forAMIprognosis It is a possible future directionto seek specific circulating miRNAs with potential value inAMI diagnosis as well as prognosis

Conflict of Interests

All authors declare that there is no conflict of interests

Acknowledgments

The authors would like to thank Dr Umme Salma forrevision of the paper This work was supported by Special-ized Research Fund for the Doctoral Program of HigherEducation of China (no 200805331178 to K Xia) NuoMei

Foundation of China Branch International AtherosclerosisSociety (no NMF 2008-003 to T L Yang) CGICC Foun-dation of Chinese Medical Association (no 08010008 to KXia) Hunan Provincial Natural Science Foundation of China(no 13JJ2008 to J Peng) and Doctoral Fund of Ministry ofEducation of China (no 20120162110056 to J Peng)

References

[1] H D White and D P Chew ldquoAcute myocardial infarctionrdquoTheLancet vol 372 no 9638 pp 570ndash584 2008

[2] V L Roger A S Go D M Lloyd-Jones et al ldquoHeart diseaseand stroke statisticsmdash2011 update a report from the Americanheart associationrdquoCirculation vol 123 no 4 pp e18ndashe209 2011

[3] B Lindahl ldquoAcute coronary syndromemdashthe present andfuture role of biomarkersrdquo Clinical Chemistry and LaboratoryMedicine vol 23 no 3 pp 1ndash8 2013

[4] S S C Chim T K F Shing E CWHung et al ldquoDetection andcharacterization of placental microRNAs in maternal plasmardquoClinical Chemistry vol 54 no 3 pp 482ndash490 2008

[5] G KWang J Q Zhu J T Zhang et al ldquoCirculatingmicroRNAa novel potential biomarker for early diagnosis of acutemyocar-dial infarction in humansrdquo European Heart Journal vol 31 no6 pp 659ndash666 2010

[6] C Widera S K Gupta J M Lorenzen et al ldquoDiagnostic andprognostic impact of six circulating microRNAs in acute coro-nary syndromerdquo Journal of Molecular and Cellular Cardiologyvol 51 no 5 pp 872ndash875 2011

[7] S Fichtlscherer S de Rosa H Fox et al ldquoCirculating microR-NAs in patients with coronary artery diseaserdquo CirculationResearch vol 107 no 5 pp 677ndash684 2010

[8] A J Tijsen E E Creemers P DMoerland et al ldquoMiR423-5p asa circulating biomarker for heart failurerdquo Circulation Researchvol 106 no 6 pp 1035ndash1039 2010

[9] Z W Pan Y J Lu and B F Yang ldquoMicroRNAs a novel class ofpotential therapeutic targets for cardiovascular diseasesrdquo ActaPharmacologica Sinica vol 31 no 1 pp 1ndash9 2010

[10] D Baek J Villen C Shin F D Camargo S P Gygi and D PBartel ldquoThe impact of microRNAs on protein outputrdquo Naturevol 455 no 7209 pp 64ndash71 2008

[11] E M Small and E N Olson ldquoPervasive roles of microRNAs incardiovascular biologyrdquoNature vol 469 no 7330 pp 336ndash3422011

[12] J Skog T Wurdinger S van Rijn et al ldquoGlioblastoma micro-vesicles transport RNA and proteins that promote tumourgrowth and provide diagnostic biomarkersrdquoNature Cell Biologyvol 10 no 12 pp 1470ndash1476 2008

[13] G Camussi M C Deregibus S Bruno V Cantaluppi and LBiancone ldquoExosomesmicrovesicles as a mechanism of cell-to-cell communicationrdquo Kidney International vol 78 no 9 pp838ndash848 2010

[14] N Kosaka H Iguchi Y Yoshioka F Takeshita Y Matsuki andT Ochiya ldquoSecretory mechanisms and intercellular transfer ofmicroRNAs in living cellsrdquoThe Journal of Biological Chemistryvol 285 no 23 pp 17442ndash17452 2010

[15] M PHunterN Ismail X Zhang et al ldquoDetection ofmicroRNAexpression in human peripheral blood microvesiclesrdquo PLoSONE vol 3 no 11 Article ID e3694 2008

[16] S Gilad E Meiri Y Yogev et al ldquoSerum microRNAs arepromising novel biomarkersrdquo PLoS ONE vol 3 no 9 ArticleID e3148 2008

566 Disease Markers

[17] X Chen Y Ba L Ma et al ldquoCharacterization of microRNAs inserum a novel class of biomarkers for diagnosis of cancer andother diseasesrdquoCell Research vol 18 no 10 pp 997ndash1006 2008

[18] A Turchinovich L Weiz A Langheinz and B BurwinkelldquoCharacterization of extracellular circulating microRNArdquoNucleic Acids Research vol 39 no 16 pp 7223ndash7233 2011

[19] N B Y Tsui E K O Ng and Y M D Lo ldquoStability ofendogenous and added RNA in blood specimens serum andplasmardquo Clinical Chemistry vol 48 no 10 pp 1647ndash1653 2002

[20] K Wang S Zhang J Weber D Baxter and D J GalasldquoExport of microRNAs and microRNA-protective protein bymammalian cellsrdquo Nucleic Acids Research vol 38 no 20 pp7248ndash7259 2010

[21] K B Margulies ldquoMicroRNAs as novel myocardial biomarkersrdquoClinical Chemistry vol 55 no 11 pp 1897ndash1899 2009

[22] E van Rooij L B Sutherland X Qi J A Richardson J Hilland E N Olson ldquoControl of stress-dependent cardiac growthand gene expression by amicroRNArdquo Science vol 316 no 5824pp 575ndash579 2007

[23] T E Callis K Pandya H Y Seok et al ldquoMicroRNA-208a isa regulator of cardiac hypertrophy and conduction in micerdquoJournal of Clinical Investigation vol 119 no 9 pp 2772ndash27862009

[24] A P Malizia and D Z Wang ldquoMicroRNAs in cardiomyocytedevelopmentrdquo Wiley Interdisciplinary Reviews Systems Biologyand Medicine vol 3 no 2 pp 183ndash190 2011

[25] X Ji R Takahashi Y Hiura G Hirokawa Y Fukushima andN Iwai ldquoPlasma miR-208 as a biomarker of myocardial injuryrdquoClinical Chemistry vol 55 no 11 pp 1944ndash1949 2009

[26] O Gidlof P Andersson J van der Pals M Gotberg and DErlinge ldquoCardiospecificmicroRNA plasma levels correlate withtroponin and cardiac function in patients with ST elevationmyocardial infarction are selectively dependent on renal elim-ination and can be detected in urine samplesrdquo Cardiology vol118 no 4 pp 217ndash226 2011

[27] M F Corsten R Dennert S Jochems et al ldquoCirculat-ing MicroRNA-208b and MicroRNA-499 reflect myocardialdamage in cardiovascular diseaserdquo Circulation CardiovascularGenetics vol 3 no 6 pp 499ndash506 2010

[28] T Adachi M Nakanishi Y Otsuka et al ldquoPlasma microRNA499 as a biomarker of acute myocardial infarctionrdquo ClinicalChemistry vol 56 no 7 pp 1183ndash1185 2010

[29] Y Q Li M F Zhang H YWen et al ldquoComparing the diagnos-tic values of circulating microRNAs and cardiac troponin T inpatients with acute myocardial infarctionrdquoClinics vol 68 no 1pp 75ndash80 2013

[30] J T C Shieh Y Huang J Gilmore and D Srivastava ldquoElevatedmiR-499 levels blunt the cardiac stress responserdquo PLoS ONEvol 6 no 5 Article ID e19481 2011

[31] Y Devaux M Vausort E Goretti et al ldquoUse of circulatingmicroRNAs to diagnose acute myocardial infarctionrdquo ClinicalChemistry vol 58 no 3 pp 559ndash567 2012

[32] F Olivieri R Antonicelli M Lorenzi et al ldquoDiagnostic poten-tial of circulating miR-499-5p in elderly patients with acutenon ST-elevation myocardial infarctionrdquo International Journalof Cardiology vol 167 no 2 pp 531ndash536 2013

[33] O Gidlof J G Smith K Miyazu et al ldquoCirculating cardio-enriched microRNAs are associated with long-term prognosisfollowing myocardial infarctionrdquo BMC Cardiovascular Disor-ders vol 13 article 12 2013

[34] J Ai R Zhang Y Li et al ldquoCirculating microRNA-1 as apotential novel biomarker for acute myocardial infarctionrdquoBiochemical and Biophysical Research Communications vol 391no 1 pp 73ndash77 2010

[35] Y DrsquoAlessandra P Devanna F Limana et al ldquoCirculatingmicroRNAs are new and sensitive biomarkers of myocardialinfarctionrdquo European Heart Journal vol 31 no 22 pp 2765ndash2773 2010

[36] Y Cheng N Tan J Yang et al ldquoA translational study of cir-culating cell-free microRNA-1 in acute myocardial infarctionrdquoClinical Science vol 119 no 2 pp 87ndash95 2010

[37] B Meder A Keller B Vogel et al ldquoMicroRNA signatures intotal peripheral blood as novel biomarkers for acute myocardialinfarctionrdquo Basic Research in Cardiology vol 106 no 1 pp 13ndash23 2011

[38] C van Solingen L Seghers R Bijkerk et al ldquoAntagomir-mediated silencing of endothelial cell specific microRNA-126impairs ischemia-induced angiogenesisrdquo Journal of Cellular andMolecular Medicine vol 13 no 8 pp 1577ndash1585 2009

[39] P Mocharla S Briand G Giannotti et al ldquoAngiomiR-126expression and secretion from circulating CD34+ and CD14+PBMCs role for proangiogenic effects and alterations in type 2diabeticsrdquo Blood vol 121 no 1 pp 226ndash236 2013

[40] G Long F Wang Q Duan et al ldquoHuman circulatingmicroRNA-1 and microRNA-126 as potential novel indicatorsfor acutemyocardial infarctionrdquo International Journal of Biolog-ical Sciences vol 8 no 6 pp 811ndash818 2012

[41] S de Rosa S Fichtlscherer R Lehmann B Assmus SDimmeler and A M Zeiher ldquoTranscoronary concentrationgradients of circulating MicroRNAsrdquo Circulation vol 124 no18 pp 1936ndash1944 2011

[42] A Zampetaki P Willeit L Tilling et al ldquoProspective studyon circulating MicroRNAs and risk of myocardial infarctionrdquoJournal of the American College of Cardiology vol 60 no 4 pp290ndash299 2012

[43] R Wang N Li Y Zhang Y Ran and J Pu ldquoCirculating microRNAs are promising novel biomarkers of acute myocardialinfarctionrdquo InternalMedicine vol 50 no 17 pp 1789ndash1795 2011

[44] M R Zile S M Mehurg J E Arroyo R E Stroud SM DeSantis and F G Spinale ldquoRelationship between thetemporal profile of plasma microRNA and left ventricularremodeling in patients aftermyocardial infarctionrdquoCirculationCardiovascular Genetics vol 4 no 6 pp 614ndash619 2011

[45] G Long FWangQDuan et al ldquoCirculatingmiR-30amiR-195and let-7b associated with acute myocardial infarctionrdquo PLoSONE vol 7 no 12 Article ID e50926 2012

[46] J Fang XW Song J Tian et al ldquoOverexpression ofmicroRNA-378 attenuates ischemia-induced apoptosis by inhibitingcaspase-3 expression in cardiac myocytesrdquo Apoptosis vol 17no 4 pp 410ndash423 2012

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562 Disease Markers

the target mRNAs will be initiated But when the miRNAsbind partially the translation of mRNA is repressed miR-NAs are involved in multiple biological processes such asproliferationmigration differentiation secretion excitationconduction cell cycle ageing and apoptosis by changingprotein expression of potential candidate targets [11]

miRNAs have recently been detected in serum or plasmawhich are referred to circulating miRNAs Despite intenseresearch the origin of circulating miRNAs remains largelyunknown A number of studies have reported that miRNAsare actively secreted in microvesicles or exosomes fromdifferent cell types [12] which are a likely source of circulatingmiRNAs So far around 121 miRNAs were identified inexosomes from mast cells [13] and sphingomyelinase 2(nSMase2) a rate-limiting enzyme for ceramide biosynthesiscontrols the secretion of exosomes to the extracellular matrix[14] In addition tomicrovesicles or exosomes microparticlesand lipoprotein complexes (such as high-density lipoproteincomplexes) are other possible sources of circulatingmiRNAs

Microparticles are larger than exosomes of plasma mem-brane origin contain miRNAs and actively control cell com-munication process [15] High-density lipoprotein (HDL) hasbeen reported to transport endogenous miRNAs and deliverthem to recipient cells with functional targeting capabilitiesbesides its basic role as a delivery vehicle for excess cellularcholesterol Interestingly the human HDL-miRNA profilefrom normal subjects is significantly different than familialhypercholesterolemia subjects The most abundant miRNAsassociated with HDL from normal subjects were hsa-miR-135a hsa-miR-188-5p and hsa-miR-877 whereas the mostabundant familial hypercholesterolemia HDL-miRNAs werehsa-miR-223 hsa-miR-105 and hsa-miR-106a Thereforehuman HDL not only transports endogenous miRNAs butalso can display different functions under different conditionsand act as potentialmediators in gene regulation and intercel-lular communication The detection of circulating miRNAsin the plasma and serum indicates that miRNAs may fulfillbiological functions outside the cell and act as potentialbiomarkers for diseases [16]The human genome is estimatedto encode up to 1000miRNAs and over 100miRNAs in serumwere identified from healthy subjects [17]

3 Stability and Isolation ofCirculating microRNAs

Human miRNAs isolated from plasma are highly stablein boiling water in solution with very high or low pHPlasma miRNA levels remain stable when it is subjected toprolonged room temperature incubation or freeze-thawedmultiple times It has been reported that endogenous plasmamiRNAs exist in a form that is resistant to plasma RNaseactivity [18] Compared to the endogenous plasma miR-NAs rapid degradation was observed within minutes whensynthetic miRNAs (corresponding to caenorhabditis elegansmiRNAs cel-miR-39 cel-miR-54 and cel-miR-238) wereadded into human plasma Inactivating the RNase activ-ity before the addition the exogenous miRNAs escapeddegradation Therefore exogenous plasma miRNAs are not

stable and are susceptible to rapid degradation whereasnative circulating miRNAs are stable and resistant to RNaseactivity [19] There are multiple factors that account for theprotection of circulating miRNAs against RNase-dependentdegradation For example circulating miRNAs are not freein blood which are normally included in microvesicles orexosomes Furthermore the circulating miRNAs can formthe protein-miRNA complexes with some special proteins(such as nucleolar RNA-binding protein nucleophosmin 1)to resist the degradation [20]

Although circulatingmicroRNAs are stable reproducibleisolation of cell-free miRNAs with high purity is a techni-cal challenge for multiple reasons First plasma or serumcontains very low amounts of RNA The yield of RNAfrom small volume of plasma or serum (usually less than1mL) falls below the limit of accurate quality control byregular methods Second high concentration of proteins inplasma or serum could potentially interfere with the samplepreparation and the detection assay Thus avoiding proteincontamination and hemolysis (lysis of red blood cells) inserumplasma samples are important challenges to overcomeFinally some of the larger small RNA species frequentlyused as reference genes (such as U6 RNA cel-miR-39) tonormalize the data of qPCR are present in extremely lowconcentrations in serumor plasmaTherefore great caremustbe taken when choosing controls (miR-156 is an option)for normalization Fortunately with circulating microRNAswell recognized for potential diagnostic biomarkers in a widerange of diseases and biological processes more commercialkits for high quality of circulating microRNAs isolation areavailable in the market

4 Circulating miRNAs of Cardiac Origin andAcute Myocardial Infarction

Rapid and correct diagnosis is crucial to treatment andprognosis of AMI Up to date cardiac troponins and creatinekinase-MBare themost commonly used biomarkers forAMIbut their clinical value is limited in many cases Secreted bycardiac cells and accumulated in bloodmiRNAs are expectedto reflect cardiac injury in response to cardiovascular riskfactors and various pathological conditions Thus the circu-lating cardiac-specific or -enriched miRNAs (such as miR-208 miR-499 and miR-133) may provide unique biomarkersfor diagnostic and therapeutic interventions of AMI [21]

41 miR-208 miR-208 is a cardiac-enriched microRNA andfound at much higher levels during cardiac tissue injury Itis also involved in stress hormone or electric stimulation incardiac cells [22 23] During cardiac development miR-208exerts a regulatory role in the production of themyosin heavychain [24] It has been reported that plasma concentrationof circulating miR-208 levels was significantly increased fol-lowing isoproterenol-induced ratmodel ofmyocardial injuryBut this phenomenon was not observed after renal injuryindicating that circulating miR-208 is specific for cardiacinjury There was a good correlation between the changes

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