Role of microRNAs in cardiac hypertrophy, myocardial fibrosis and heart failure

REVIEW

Role of microRNAs in cardiac hypertrophy,myocardial

?brosis and heart failure$

De-li Dong,Bao-feng Yang n

Department of Pharmacology,Harbin Medical University,Biopharmaceutical Key Laboratory of Heilongjiang Province,

Harbin150086,China

Received27January2011;revised4March2011;accepted25March2011

KEY WORDS

microRNAs;

Cardiac hypertrophy;

Myocardial?brosis;

Heart failure

Abstract MicroRNAs(miRNAs)are endogenous small non-coding RNA molecules that post-

transcriptionally regulate gene expression.MiRNA expression and function in heart disease remain

to be determined but modulation of miRNA expression in vivo has revealed that miRNAs play an

important role in controlling heart function and structure.In fact,abnormal expression of miRNAs

may initiate and contribute to the progress of heart disease.Here,we summarize the literature

relating to the involvement of miRNAs in cardiac hypertrophy,myocardial?brosis and heart

failure.

&2011Institute of Materia Medica,Chinese Academy of Medical Sciences and Chinese Pharmaceutical

Association.Production and hosting by Elsevier B.V.All rights reserved.

Institute of Materia Medica,Chinese Academy of Medical Sciences

Chinese Pharmaceutical Association

https://www.sodocs.net/doc/091075253.html,/locate/apsb

https://www.sodocs.net/doc/091075253.html,

Acta Pharmaceutica Sinica B

2211-3835&2011Institute of Materia Medica,Chinese Academy of Medical Sciences and Chinese Pharmaceutical Association.Production and hosting by Elsevier B.V.All rights reserved.

Peer review under responsibility of Institute of Materia Medica,Chinese Academy of Medical Sciences and Chinese Pharmaceutical Association.

doi:10.1016/j.apsb.2011.04.010

$This work was supported by the China Postdoctoral Special Science Foundation,the Foundation for the Author of a National Excellent Doctoral Dissertation of P.R.China(2007B72),and the National Basic Research Program of China(2007CB512006).

n Corresponding author.Tel.:t8645186671354;fax:t8645186667511.

E-mail addresses:delidong2004@https://www.sodocs.net/doc/091075253.html,(De-li Dong),yangbf@https://www.sodocs.net/doc/091075253.html,(Bao-feng Yang)

Acta Pharmaceutica Sinica B2011;1(1):1–7

1.Introduction

MicroRNAs(miRNAs)are small22–24nucleotide non-coding RNA molecules that regulate gene expression by hybridization to30untranslated regions(30UTRs)of target messenger RNAs(mRNAs)resulting in their degradation or translational inhibition1.Collectively more than4000miRNA sequences exist in a wide range of species of which more than 700are encoded by the human genome2.Of the latter,some 150–200are expressed in the heart where they are dynamically regulated in response to cardiac disease3.

MiRNAs constitute a particularly abundant and funda-mental class of regulators of gene expression.Apart from their involvement in developmental aspects of higher organisms, they are now implicated in an increasing number of physio-logical processes.Initially,miRNAs were shown to play important roles in plant biology4,viral diseases5,develop-mental processes6and cancer7.More recently,several studies have demonstrated their importance in the regulation of cardiac development8and various cardiovascular diseases such as myocardial infarction9,cardiac hypertrophy10and heart failure11.

The heart responds to injury and hemodynamic overload by promoting myocyte hypertrophy,reexpressing a fetal gene program and remodeling the extracellular matrix.Speci?c miRNAs are disregulated in the diseased heart and,in mice, up-and down-regulation of miRNAs have been found to be necessary and suf?cient to explain diverse heart diseases12. Thus,normalizing miRNA expression in the heart represents a new approach to the pharmacotherapy of heart disease13–15.

In this paper,we review miRNA biology in general and the literature relating to the mechanisms of miRNA action in cardiac hypertrophy,myocardial?brosis and heart failure. Understanding how miRNAs regulate not only single genes but the whole gene network has enormous therapeutic implications.

2.MiRNA biogenesis and biological function

MiRNAs are encoded in the genomes of plants and animals. They are initially transcribed by RNA polymerase II or,in a few cases,RNA polymerase III as long primary miRNAs (pri-miRNAs)containing a65-nucleotide stem loop16. Pri-miRNAs fold into hairpin structures containing imperfectly base-paired stems and are processed by the endonuclease Drosha and its cofactor DiGeorge syndrome critical region8 (dgcr8)into60–100nt hairpins known as pre-miRNAs17,18. Pre-miRNAs are then exported to the cytoplasm by Exportin-5 and subsequently processed by the cytoplasmic RNaseIII, Dicer,which also resides in a multiprotein complex19.

Cleavage of the pre-miRNA by Dicer produces mature miRNAs,which are incorporated into the RNA-induced silencing complex(RISC).This then recognizes speci?c targets and induces post-transcriptional gene silencing through bind-ing of the mature miRNA to the30untranslated region of its target mRNA through exact and partial complementarity20. Depending on the overall degree of complementarity,gene silencing occurs through either inhibition of translation or degradation of its target mRNAs.Interestingly,in rare cases, miRNAs have also been reported to stimulate mRNA transla-tion21,a?nding which has sparked controversy and led to demands for more research into the mechanisms by which miRNAs regulate gene expression22.

3.MiRNAs and cardiac hypertrophy

Cardiac hypertrophy is an important compensatory mechan-ism of the heart in response to diverse pathophysiological stimuli.Initially,the response aims to normalize wall stress and preserve contractile performance but chronically it pro-duces hypertrophy and may eventually lead to heart failure. Although various pathways provide coordinated control of the hypertrophic process,little is known about their under-lying molecular mechanisms.Exposure of the heart to some stressors may lead to cardiac remodeling with a change in the gene expression pro?le and a detrimental outcome.The involvement of miRNAs in this pathological process is now recognized and increasing evidence shows that miRNAs are key modulators of cardiovascular development and function and the process of cardiac hypertrophy.

Number of miRNAs are found to be regulated during cardiac hypertrophy and two,miR-1and miR-133,play a key role in inhibiting it.MiR-1and miR-133are included in the same bicistronic unit and are speci?cally expressed in skeletal muscle and cardiac myocytes8.Embryonic overexpression of miR-1in vivo results in thin-walled ventricles,whereas miR-1 knockout mice display chambers with thickened walls23.MiR-1is down-regulated at the onset of pressure overload on the heart that appears to be suf?cient to account for the changes in gene expression underlying the initiation and progression of cardiac hypertrophy24.MiR-1down-regulates calcium–calmodulin signaling through the calcineurin/NFAT pathway and negatively regulates the expression of Mef2a and Gata4to inhibit cardiomyocyte growth25.The cytoskeleton regulatory protein,twin?lin-1,is a novel target of miR-1and reduction of miR-1by hypertrophic stimuli up-regulates twin?lin-1which, in turn,evokes hypertrophy through regulation of the cardiac cytoskeleton26.

MiR-133has a critical role in determining cardiomyocyte hypertrophy;its overexpression inhibits hypertrophy whereas its suppression induces hypertrophy both in vitro and in vivo10. Recently,Dong et al.27found miR-133expression was down-regulated,and calcineurin activity enhanced in both in vivo and in vitro models of cardiac hypertrophy27.In addition,they found that using cyclosporine A to inhibit calcineurin pre-vented the down-regulation of miR-133in cardiac hypertrophy. These results indicate that miR-133and calcineurin are reci-procally repressed in cardiac hypertrophy.Moreover,another study indicated that miR-133a plays a role in diabetes-induced cardiomyocyte hypertrophy;down-regulation alters gene expression and mediates glucose-induced cardiomyocyte hyper-trophy through SGK1and IGFR128.However,van Rooij et al.29found that miR-133did not produce any of the mor-phological changes in cardiomyocytes associated with hyper-trophic growth29.

Several other miRNAs have been found to be pro-hyper-trophic including miR-208,21,18b,195,199,23,24,27and9. MiR-208is cardiac speci?c and is shown to be necessary for cardiomyocyte hypertrophy,?brosis and expression of b-MHC in response to stress and hypothyroidism30.Mice subjected to targeted deletion of miR-208failed to undergo stress-induced cardiac remodeling,hypertrophic growth and

De-li Dong,Bao-feng Yang

2

b-MHC up-regulation;whereas,overexpression of miR-208 was shown to induce cardiac hypertrophy.During cardiac hypertrophy,a reduction in the expression of a-MHC resulted in diminished transcription of miR-208and subsequently to loss of its negative effects on thyroid hormone receptor associated protein1(THRAP1)and to a blunted response to pressure overload.

It is interesting to speculate on whether modulation of miR-208in vivo is a potentially useful therapeutic strategy in cardiac hypertrophy.In support of this,Callis et al.31reported that overexpression of miR-208a in mouse heart was suf?cient to induce hypertrophic growth resulting in pronounced repression of the miR-208regulatory targets,THRAP1and myostatin,the negative regulators of muscle growth and hypertrophy31.

Tatsuguchi et al.32reported that inhibition of endogenous miR-21or miR-18b augmented hypertrophic growth whereas introduction of functional miR-21or miR-18b into cardio-myocytes repressed it32.Despite this,the effect of miR-21on myocyte hypertrophy remains controversial.Cheng et al.33 found that miR-21was strikingly up-regulated both in hypertrophic mouse hearts and cultured neonatal hyper-trophic cardiomyocytes and that modulating miR-21expres-sion via antisense-mediated depletion produced a signi?cantly negative effect on cardiomyocyte hypertrophy33.Recently, Patrick et al.34reported that miR-21was not essential for pathological cardiac remodeling based on the fact that miR-21-null mice did not display improved tolerance to a variety of cardiac stressors compared to their wild-type littermates and that inhibition of miR-21failed to block stress-induced cardiac remodeling34.

Overexpression of miR-195during cardiac hypertrophy results in pathological cardiac growth and heart failure in transgenic mice29.MiR-199a is predominantly expressed in cardiomyocytes where it maintains cell size and may play a role in the regulation of cardiac hypertrophy.It is also con?rmed to target hypoxia-inducible factor1alpha35.Previously,it was shown that miR-23a,27a and24-2are up-regulated during cardiac hypertrophy36 and that knockdown of miR-23a attenuates hypertrophy37. Finally,miR-9appears to regulate cardiac hypertrophy by suppressing myocardin expression38.

4.MiRNAs and cardiac?brosis

Cardiac?brosis is an established morphological feature of structural myocardial remodeling that occurs in several car-diac diseases including myocardial infarction,dilated hyper-trophic cardiomyopathies and heart failure.The cellular basis of?brosis is the adverse accumulation of collagens and other extracellular matrix(ECM)proteins that increase the risk for adverse cardiovascular events such as ventricular dysfunction and arrhythmias.The etiology of the?brogenic cardiac phenotype is still being elucidated but a small number of studies have already demonstrated that altered expression of several miRNAs in myocardial?brosis is associated with ischemia or mechanical overload.

MiR-29acts as a regulator of cardiac?brosis and represents a potential therapeutic target for tissue?brosis in general39. The miR-29family targets a series of mRNAs that encode proteins involved in?brosis,including multiple collagens,?brillins and elastin.Down-regulation of miR-29is therefore predicted to repress the expression of some mRNAs and enhance the?brotic response.Indeed,such down-regulation with anti-miRs in vitro and in vivo induces the expression of collagens whereas overexpression of miR-29in?broblasts reduces it39.Down-regulation of miR-149and up-regulation of miR-21,214and223were shown to accompany down-regulation of miR-2939,but the functional consequences of these changes are unknown.

MiR-21contributes to myocardial remodeling through regulating the ERK-MAP kinase signaling pathway in cardiac ?broblasts during cardiac ischemia/reperfusion40or in the later stages of heart failure41.In vivo silencing of miR-21by a speci?c antagomir suppresses pathological ERK-MAP kinase signaling and prevents cardiac dysfunction in a mouse pressure-overload-induced disease model.MiR-21regulates ?broblast survival and growth factor secretion that ultimately control the extent of interstitial?brosis and cardiac hyper-trophy41.Overall,these?ndings indicate that miR-21,like miR-29,contributes to myocardial remodeling by affecting cardiac?broblasts.Down-regulation of miR-21could there-fore be a bene?cial approach to block?broblast proliferation in heart disease and thereby inhibit secondary cardiac remodeling.

The potential importance of miR-133in cardiac?brosis has been underlined by reports that miR-133-a1and miR-133-a2 knockout mice develop severe?brosis and heart failure42and that miR-133and miR-590are down-regulated in a canine model of nicotine-induced atrial interstitial?brosis43,44. Duisters et al.45reported that knockdown of miR-133or miR-30increased the level of connective tissue growth factor(CTGF) and that overexpression of miR-133or miR-30decreased CTGF levels accompanied by decreased production of collagens45.They showed that CTGF was a direct target of these miRNAs because they directly interacted with the30untranslated region of CTGF. Another report showed that transgenic expression of miR-133a prevented TAC-associated miR-133a down-regulation and improved myocardial?brosis and diastolic function without affecting the extent of hypertrophy46.Interestingly,mechanistic studies in the canine model and in cultured atrial?broblasts showed that the protective actions of miR-133and miR-590are mediated through targeting TGF-b1and TGF-b receptor type II,respectively44.

In addition to cardiac?brosis,miRNAs are involved in the ?brotic process in other organs such as lung,kidney and liver47.However,much research remains to be done to identify which miRNAs have a direct role in the development of ?brosis,and which have altered expression secondary to cardiac?brosis.Characterization of individual miRNAs or miRNA expression pro?les that are speci?cally associated with myocardial?brosis will hopefully allow the development of diagnostic tools and innovative therapies for?brogenic cardiac diseases.

5.MiRNAs and heart failure

As stated earlier,the heart responds to cardiac injury or hemodynamic overload by activating a variety of intracellular signaling pathways that provoke myocyte hypertrophy, re-expression of embryonic genes and remodeling of the extracellular matrix48.When the heart transits from adaptive to maladaptive hypertrophy,heart failure occurs.The patho-logical mechanism of heart failure is a result of the

Role of microRNAs in heart disease3

concomitant cross-talk between various deleterious and com-pensatory signaling pathways,the balance between which ultimately determines the pathological progression.Despite signi?cant advances in the identi?cation of genes and signaling pathways,the overall complexity of cardiac hypertrophic remodeling suggests the involvement of additional global regulatory signaling networks.A growing body of evidence suggests that some miRNAs are involved in the process of heart failure.

Cardiomyocyte-speci?c deletion of a gene or endonuclease, such as dgcr8and Dicer,which are required for microRNA biogenesis and processing,leads to a dramatic fall in the level of mature miRNAs and to left ventricular malfunction progressing to a dilated cardiomyopathy and premature lethality49,50.Sucharov et al.51demonstrated that the expres-sion of subsets of miRNAs was differentially regulated in different heart failure models51.They found that inhibition of miR-100,which was up-regulated in the failing heart,pre-vented b-adrenoceptor(b-AR)mediated down-regulation of the adult gene component of the fetal gene program.They also suggested that overexpression of miR-133b attenuated b-AR mediated changes in gene expression.van Rooij et al.29 showed that overexpression of individual stress-inducible miRNAs was suf?cient to induce hypertrophic growth in isolated cardiac myocytes and to provoke a dilated cardio-myopathy in transgenic mice,suggesting that individual miRNAs are suf?cient to provoke heart failure29.In their other study30,they found that deletion of miR-208,a cardiac-speci?c miRNA encoded by an intron in the gene that encodes the a-myosin chain,protected against cardiac myocyte hyper-trophy,up-regulated b-myosin heavy chain and produced myocardial?brosis in response to thyroid signaling and hemodynamic overload.They also showed that animals de?cient in miR-208did not ramp up cardiac expression of b-myosin heavy chain in response to thyroid signaling.

MiR-199b is a direct calcineurin/NFAT target gene the expression of which is increased in mouse and human heart failure.miR-199b targets nuclear NFAT kinase dual-speci?-city tyrosine-(Y)-phosphorylation regulated kinase1a (Dyrk1a)in a process that constitutes a pathogenic feed forward mechanism affecting calcineurin-responsive gene expression52.In vivo inhibition of miR-199b by a speci?c antagomir normalized Dyrk1a expression,reduced nuclear NFAT activity and caused marked inhibition and even reversal of hypertrophy and?brosis in mouse models of heart failure.Naga Prasad et al.53examined the alterations of miRNA expression in human heart failure samples in order to elucidate the global regulation of the signaling networks by a unique miRNA pattern in end-stage human heart failure53. They showed that eight miRNAs(miR-1,214,29b,342,7, 125,378and181)were signi?cantly altered in dilated cardi-omyopathy compared with non-failing controls.They also identi?ed two novel miRNAs(miR-7and378)that were down-regulated in end-stage heart failure.Tijsen et al.54 identi?ed a number of circulating miRNAs as putative biomarkers of heart failure and showed that miR423-5p in particular distinguished heart failure cases from non-heart failure cases54.

Transfection of isolated adult rat cardiomyocytes with a set of fetal miRNAs(miR-21,129and212)resulted in hyper-trophic morphological changes in neonatal cardiomyocytes similar to those observed in the failing heart but transfection of any one of the set produced only minor effects11.However, many reports indicate that associations exist between single miRNAs and heart failure and indeed that the pathological changes in heart failure may require an alteration of the miRNA environment.

6.MiRNAs-based strategies for diagnostics and therapeutics MiRNA expression has been shown to play a role in the growth,development,function and stress responsivenesses of the heart as well as in heart disease.The possibility of exploiting miRNAs as diagnostic markers or therapeutic tools has much to recommend it because of their speci?city to their targets in a particular cellular pathway.

MiRNAs show different expression patterns in the normal and diseased heart and some data indicate that miRNA expression may represent an ef?cient diagnostic marker of heart disease11,29.Identi?cation of abnormal miRNA levels in tissue or plasma could certainly aid in early diagnosis and, in fact,there is increasing evidence that circulating miRNAs in various body?uids are potential biomarkers of disease diagnosis55–57.For example,certain miRNAs have been proposed for screening colorectal cancer58and for monitoring pregnancy59.Recently,many miRNAs have been reported to be potential biomarkers for cardiovascular disease and, in particular,miR-208,miR-1and miR-499have been shown to indicate the diagnosis and progression of myocardial infarction60–62.In the case of miR-208,its plasma concentration increased in isoproterenol-induced myocardial injury and was well correlated with the plasma concentration of cTnI,the classical marker of myocardial injury60.For miR-1,the plasma level was signi?cantly higher in patients with acute myocardial infarction(AMI)compared with non-AMI subjects and the level dropped to normal on discharge following medication61.For miR-499,plasma concentrations were signi?cantly increased in patients with acute myocardial infarction but were below the detection limit in other patient groups62.

Other circulating miRNAs have been proposed as sensitive and informative biomarkers for the diagnosis of heart failure. MiR423-5p is speci?cally enriched in the blood of heart failure patients but not in healthy people indicating that certain miRNAs are not only diagnostic predictors of heart failure but also a means of distinguishing clinical heart failure from other diseases54.The plasma concentration of miR-126has also been proposed as a biomarker of heart failure63.Moreover, miRNAs are more sensitive than mRNAs in detecting end-stage heart failure64.These and other studies reveal the potential of miRNAs in assessing the risk of certain kinds of disease and in evaluating the ef?cacy of treatment.The development of miRNAs as biomarkers for cardiovascular diseases is still in its infancy and,no doubt,will remain an active?eld for many years yet.

Identi?cation of speci?c miRNAs and target genes that contribute to adult cardiac pathology is likely to provide new therapeutic targets.In the classical course of drug discovery,it takes many years to identify such targets,devise and execute accurate screening methods,and eventually develop the molecules affecting those targets as therapeutic agents. However,miRNAs are not likely to require such a prolonged process since they can be ef?ciently manipulated to tune gene regulation and are more amenable to incorporation in drug

De-li Dong,Bao-feng Yang

4

delivery systems because of their small size65,66.Furthermore, miRNAs are predicted to have multiple mRNA targets67, some of which appear to work in concert to control a common pathway or biological function.However,this can also be seen as a major disadvantage because of its propensity to cause side effects.Although there are several approaches to change miRNA levels in vivo and in vitro as exempli?ed in numerous gain-of-function and loss-of-function studies,a detailed under-standing of miRNA biology and function pertaining to the heart remains some way off.

Exogenous miRNAs,either synthetic or constructed in virus vectors,have been used to restore the decreased levels that accompany certain cardiovascular diseases and thereby retard the associated pathological process24,41.Generally, these miRNAs are double-stranded and have the same sequence as endogenous miRNAs.One technique to increase the cellular level of a speci?c miRNA is through the use of a miRNA mimic,which utilizes synthetic nucleic acids to bind to target mRNAs and elicit post-transcriptional regulatory effects.The double-stranded structure enables ef?cient recog-nition and loading into RISC to elicit miRNA action.This strategy can potentially repress the target gene at the post-transcriptional level with minimal effects on the mRNA level. These constructs are analogous to siRNA molecules and have been successfully utilized in vitro.Ef?cacy in vivo remains to be demonstrated.

Ef?cient reduction in the level of speci?c miRNAs associated with a particular disease should be therapeutically advanta-geous.Inhibiting miRNAs expression can be achieved using antisense inhibitor oligonucleotides(AMO)designed to fully complement their target miRNAs in order to degrade them. Besides being potentially therapeutic,the application of AMOs is a popular method for studying miRNA function.Further-more,antagomirs based on cholesterol conjugated oligonucleo-tides have been used to facilitate cellular uptake and resolve the delivery problem faced by AMOs.Antagomirs which target a speci?c miRNA or disrupt the binding between an miRNA and its target represent a potentially effective way to inactivate pathological miRNAs.The?rst successful mammalian in vivo study using such an antagomir aimed to inhibit a liver-speci?c miR-12266.Further studies have since attempted to modify cellular uptake using high-density lipoproteins68,20-O-methoxy-ethyl phosphorothioate65,and locked-nucleic-acid-modi?ed oli-gonucleotides69that were subsequently tested in non-human primates70.With respect to the heart,in vivo inhibition of miR-133and miR-29with antagomirs in mice has implicated their participation in cardiac hypertrophy and cardiac?brosis, respectively10,39.Antagomirs are powerful tools to silence speci?c miRNAs in vivo and may represent a therapeutic strategy for silencing miRNAs in disease66.

Another way to interfere with miRNA–mRNA interactions is through competitive inhibition of miRNA using‘‘miRNA sponges’’.These can be expressed in cells as transcripts of strong promoters containing multiple,tandem binding sites to a related https://www.sodocs.net/doc/091075253.html,parable to sponges,miRNA erasers use only two copies of a perfectly complementary antisense sequence of a miRNA72.Recently,this approach has been tested in Drosophila in an attempt to understand the factors that contribute to the spatiotemporal speci?city of miRNAs73.

Research into the role of miRNAs in human heart disease holds great promise for future therapeutic applications.How-ever,developing miRNAs into therapeutic agents faces signi?cant challenges associated with their drug delivery and duration of action.Although local delivery to the heart through direct injection or via a catheter or coated stent would avoid these problems,its clinical feasibility remains to be determined.

7.Conclusions

MiRNAs have emerged as a novel class of key regulators in a variety of cardiovascular diseases including cardiac hyper-trophy,?brosis and heart failure.We summarize the current understanding of microRNAs in cardiac hypertrophy,myo-cardial?brosis and heart failure in Fig.1.MicroRNAs represent potential pharmacological targets since down-or up-regulation of a particular miRNA is enough to cause a speci?c cardiovascular disease and correcting their aberrant expression can reverse the pathological process.Ongoing efforts to identify the targets of speci?c miRNAs involved

in Figure1Current understanding of the role of microRNAs in cardiac hypertrophy,myocardial?brosis and heart failure. Thrap1:thyroid hormone receptor associated protein1;Cdc42: cell division cycle42;Nelf-A:negative elongation factor A; WHSC2:Wolf–Hirschhorn syndrome candidate2;IGFR1:insu-lin-like growth factor receptor1;SGK1:serum-and glucose-regulated kinase;RasGAP:Ras GTPase-activating protein;Cdk9: cyclin-dependent kinase9;Rheb:Ras homolog enriched in brain; MuFR1:the muscle speci?c ring?nger protein1;HIF1:hypoxia-inducible factor1;CTGF:connective tissue growth factor;PTEN: tensin homolog;MMP-2:matrix metalloprotease-2;Spry1:spro-uty homolog1;a-MHC:alpha-myosin heavy chain;Dyrk1a: dual-speci?city tyrosine-(Y)-phosphorylation regulated kinase1a.

Role of microRNAs in heart disease5

the cardiovascular system will increase our understanding of the molecular mechanisms underlying disease processes.How-ever,the fact that a single miRNA can control hundreds of distinct target genes and potentially affect many cellular pathways means that achieving a full understanding of miRNA-mediated molecular networks in the heart is a signi?cant challenge.Thus the routine application of miRNAs in the clinic remains a rather distant goal.

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