MicroRNAs in Organogenesis and Disease

8/13/2019 MicroRNAs in Organogenesis and Disease

1/13

698 Current Molecular Medicine 2008, 8, 698-710

1566-5240/08 $55.00+.00 2008 Benth am Science Publis hers Ltd .

MicroRNAs in Organogenesis and Disease

Naisana S. Asli, Mara E. Pitulescu and Michael Kessel*

Research Group Developmental Biology, Department of Molecular Cell Biology, Max-Planck-Institute forBiophysical Chemistry, 37077 Gttingen, Germany

Ab st rac t: Large numbers and quantities of different, small RNA molecules are present in the cytoplasm ofanimal and plant cells. One subclass of these molecules is represented by the noncoding microRNAs. Sincetheir discovery in the 1990s a multitude of basic information has accumulated, which has identified theirfunction in post-transcriptional control, either via degradation or translational inhibition of target mRNAs. Thisfunction is in most of the cases a finetuning of gene expression, working in parallel with transcriptionalregulatory processes. MicroRNA expression profiles are highly dynamic during embryonic development and inadulthood. Misexpression of microRNAs can perturb embryogenesis, organogenesis, tissue homeostasis andthe cell cycle. Evidence from gain- and loss-of function studies indicates roles for microRNAs inpathophysiologic states including cardiac hypertrophy, muscle dystrophy, hepatitis infection, diabetes,Parkinson syndrome, hematological malignancies and other types of cancer. In this review, we focus onstudies addressing the role of various microRNAs in heart, muscle, liver, pancreas, central nervous system,and hematopoiesis.

Keywords: microRNA, development, disease, cell cycle.

INTRODUCTIONMicroRNAs represent a previously overlooked class

of abundant, relatively small molecules, which hasbecome a major focus of research in the last years.From an exponentially growing number of studies theyemerged as important factors in a post-transcriptionalregulatory network, relevant in nearly all processes oflife.

By using high-throughput sequencing of small RNAlibraries from different organ systems and cell types,416 different human, 386 mouse and 325 rat microRNAgenes were identified [1]. These numbers match veryclosely the entries in miRBase, the microRNA registrydatabase (http://microrna.sanger.ac.uk/sequences).However, predicted numbers based on bioinformaticsand genomic sequences are often larger, withestimates as high as 1000 genes in the human genome[2].

The microRNA nomenclature follows guidelines ofmiRBase, which also decides on the acceptance ofnewly characterized microRNAs. MicroRNAs (alsocalled miRNAs) are named using a miR- prefix,followed by a unique identifying number. OrthologousmicroRNAs with the same sequence, but from differentspecies, receive the same numbers, and the species

name can be added as a prefix (e.g. hsa-miR-124).Identical microRNAs from different genomic loci anddifferent precursors within a given species areregarded as paralogous, and are differentiated by anadditional numbering (e.g. hsa-miR-1-1). ParalogousmicroRNAs which have a difference of one or two

*Address correspondence to this author at the Research GroupDevelopmental Biology, Department of Molecular Cell Biology, Max-Planck-Institute for Biophysical Chemistry, 37077 Gttingen,Germany; Tel: ++49-551-2011752; Fax: ++49-551-2011504;E-mail: [email protected]

bases, are differentiated by the addition of small letters(e.g. hsa-miR-20a). MicroRNAs which originate fromdifferent arms of the same stem loop structure, arefurther differentiated by the addition of 3p or 5paccording to which arm (3 or 5 respectively) they aredriven from (e.g. miR-17-5p) [3-6].

The expression of microRNAs involves initially themachinery used for the transcription of proteinencoding genes, and subsequently a highly conservedprocessing mechanism. RNA polymerase II generateslarge primary transcripts known as pri-microRNA whichcan have a length of more than 1kb (Fig. 1) [7-9].

These molecules pass through a first processing stepby the nuclear RNAse Drosha, which results in theformation of a hairpin molecule of around 70nucleotides, the pre-microRNA [9]. These RNAs enterthe next processing step in the cytoplasm, where theDicer RNAse cleaves the hairpin structure and yieldsa dsRNA of 21-24 base pairs, with the typical feature ofa 2 nucleotides overhang at the 3 end [9]. From thedouble stranded Dicer product, the strand with lesserthermodynamic stability at its 5 end is selectivelystabilized, whereas the complementary strandbecomes subsequently degraded. The stable, maturemicroRNA molecule of 21-24 nucleotide length is thenready to act as the guide strand in the silencingprocess [9] (Fig. 1). Mature microRNAs exert theirfunction within a ribonucleoprotein complex, themicroRNA-induced silencing complex (miRISC). ThemiRISC complex includes the mature microRNAtogether with a number of proteins of the Argonautefamily [10]. Within the miRISC complex the binding of amicroRNA to a mRNA occurs through base-pairingbetween the nucleotides 2-8 at the 5end of themicroRNA (seed region) and the complementarysequence in the 3UTR of the target mRNA. Theconsequences of this binding depend to a large extent


2/13

MicroRNAs in Organogenesis and Disease Current Molecular Medicine, 2008, Vol. 8, No. 8 699

on the degree of complementation between the twoRNA molecules.

The most common effect of the binding of miRNA tothe 3 UTR is to significantly affect translational

efficiency, resulting from non-perfect base pairing.MicroRNA-mediated inhibition of translation can occurvia two mechanisms: 1) At the translation initiationstep, by impairing the cap recognition process [10, 11]or 2) via premature termination of translation or co-translational protein degradation [11]. In addition totranslational repression of mRNAs by microRNA, thereis also evidence for microRNA-mediated destabilizationof target transcripts. This process involves thedeadenylation and subsequent decapping of a mRNAmolecule which results in mRNA degradation in specialcytoplasmic foci, known as P-bodies [11]. A RISC-

associated microRNA recognizes its target mRNA bybase pairing at a target sequence on the mRNA. The

Argonaute protein then interacts with a P-body protein,and is delivered to the P-bodies [11]. The target mRNAis then either decapped and degraded, or just stored ina translationally static state. Which mechanism is finallyadopted depends on an individual microRNA-mRNApair, and the specific sequence context [11]. A

translationally-stalled mRNA, can be reshuffled intoactive polysomes in special cases of stress or otherexternal stimuli [10].

Although most of the microRNAs recognize theirtargets through imperfect pairing, there are also someexamples of perfect complementarity between themiRNA guide strand and the mRNA target. Thisgenerates an A-form microRNA-mRNA double helix,which marks the duplex for the siRNA pathway, andeventually results in the degradation of the targetmRNA by the endonucleolytic capacity of the siRISCthrough the cleavage of the mRNA in the central part(positions 10 and 11) of the duplex [10].

The high conservation of microRNAs in differentspecies combined with highly dynamic expressionpatterns suggested that they might have regulatoryfunctions in gene expression [12, 13]. The firstexperimental evidence for a significant function duringembryonic development came from embryos, wherethe complete microRNA supplies were depletedthrough an inactivation of the pre-microRNA processingenzyme Dicer. Dicer knockout mice die at very earlystages of development, before gastrulation [14].Deletion of the zebrafish Dicer homologue also resultedin an early arrest of embryonic development, thoughthe embryos survived longer than murine dicermutants, possibly as a result of maternal contributions

[15-17]. The Dicer RNase is also involved in theprocessing of interfering RNA molecules other thanmicroRNAs. Therefore, to draw far reachingconclusions from the absence of a central molecule likeDicer should only be done with caution, since functionsin development and homeostasis not related tomicroRNAs could also be affected [14].

The study of microRNAs, involves a variety oftechniques from high throughput screening methodslike small RNA library sequencings and microarrayexpression profilings, to finer methods of functionalanalysis. The latter, include loss of functionapproaches, such as application of antisenseoligonucleotides against microRNA sequences, orcomplete deletion of a microRNA coding sequence byconventional knockout strategies. Additionalapproaches include ectopic expression of themicroRNAs to further elucidate their downstreameffects. In the following paragraphs we will describeexemplary cases, where the function of microRNAswere experimentally validated. We will review evidencefor the role of microRNAs in development and disease,with a particular focus on heart, musculature, liver,pancreas, the nervous system, hematopoiesis, and thecell cycle.

Fig. (1). Mechanism of microRNA production and function.MicroRNAs are transcribed by RNA polymerase II,processed through two successive steps by RNAses Droshaand Dicer, and the mature microRNA attacks the targetmRNA within the RISC, inhibiting its translation, or leading toits degradation.


3/13

700 Current Molecular Medicine, 2008, Vol. 8, No. 8 Asli et al.

MicroRNAs in Heart and Muscle Development andDisease

Somatic and cardiac muscles are derived frommesodermal cells ingressing through the primitivestreak during gastrulation. These progenitors developinto still dividing myoblasts, before they exit from thecell cycle and fuse to form myotubes, which thenmature into muscle fibres. Cardiac and skeletalmuscles represent principally different tissues, whichdiffer in origin, migratory pathways, inductiverequirements and molecular determinants.

The complete loss of all microRNAs in zebrafishembryos due to a mutation of the Dicer gene ledamong other defects also to problems in heartdevelopment [15]. However, it was the analysis ofmouse embryos with a conditional inactivation of theDicer gene in the developing heart which clearlydefined the essential role of microRNAs incardiogenesis [18]. These mice died from cardiacfailure on day 12.5, presenting an underdevelopedventricular myocardium and pericardial edema.

Up- or downregulation of several microRNAsaccompanied pathological cardiac remodeling inrodents and humans [19-24]. Several candidates,including miR-1, miR-21, miR-133, miR-181, miR-195,miR-206 and miR-208 were more specifically studied ingain- or loss-of-function experiments (Fig. 2A ). Thecardiac-specific microRNA miR-208 is encoded by anintron of the alpha myosin heavy chain (MHC) gene[25]. Mice carrying a miR-208 mutation demonstrate itsinvolvement in cardiomyocyte hypertrophy, fibrosis,and expression of beta MHC in response to stress andhypothyroidism. miR-208 downregulates THRAP1, anegative regulator of beta MHC after birth [25]. Thus,the alpha MHC gene, in addition to encoding a majorcardiac contractile protein, regulates cardiac growthand gene expression in response to stress andhormonal signaling through miR-208.

Roles in cardiac growth were found for miR-195,miR-21, miR-133, and miR-1. Moderate overexpressionof miR-195 in the developing heart of mice inducedhypertrophy, and higher levels of expression led tocardiomyopathy with ventricular dilatation and wallthinning, finally resulting in heart failure [19]. MiR-21 isone of the microRNAs found increased in hypertrophy-induced mouse hearts and in angiotensin II orphenylephrine-induced cultured rat neonatalhypertrophic cardiomyocytes [20]. However, while

Cheng et al . observed that knocking-down miR-21interfered with the induction of hypertrophy in culturedcardiomyocytes [20], the opposite was found in thestudy by Tatsuguchi et al . [22]. These authorsdescribed that miR-21 over-expression reducedcardiomyocyte hypertrophy, while its inhibition inducedmyocyte hypertrophy. The reason for the observeddiscrepancies after functional modulation of miR-21levels is currently unclear.

Another microRNA which functions as a negativeregulator of cardiac hypertrophy is miR-133 [26]. The

miR-133 family consists of three members, encoded bytwo miR-133a and one miR-133b genes, all beingexpressed in both cardiac and skeletal muscle tissuesof adults [27, 28]. MiR-133 is down-regulated in threemurine models of cardiac hypertrophy, and in heartsfrom patients with hypertrophic cardiomyopathy andatrial dilatation [26]. Overexpression of miR-133 in bothneonatal and adult cultured cardiomyocytes reduced

hypertrophy, while suppression of endogenous miR-133 resulted in marked hypertrophy in the absence ofany hypertrophic stimulus. However, changes of miR-133 levels were not detected by expression profiling inthree different types of human cardiac disease,emphasizing the cautions necessary when concludingfrom one model to the other [24].

From Drosophila to higher vertebrates, miR-1 isexpressed in developing skeletal and heart muscle [18,29-36]. miR-1 and miR-133a are derived from the samebicistronic RNA, and have identical mature sequences[28]. Downregulation of miR-1 was observed in amouse model of thoracic aortic constriction-inducedcardiac hypertrophy [21]. The authors concluded thatlow levels of miR-1 might be important for the inductionof hypertrophy, releasing its growth-related targets frominhibitory influence. Previous studies had shown thatthe overexpression of miR-1 in the developing mouseheart lead to a decreased proliferation of myocytes,with a developmental arrest on day 13.5 [34]. Thisphenotype was due to a failure of ventricularcardiomyocyte expansion, caused by a decrease of themiR-1 target Hand2, a cardiac transcription factorexpressed early in heart development and required forventricular growth [18, 34, 37]. A further study on theloss of miR-1-2 function in mice confirmed its role inrepressing the proliferation of cardiomyocytes [18].

Adult mice lacking miR-1-2 had hyperplas tic hearts withstill proliferating cardiomyocytes, while a normal adultmouse heart has only terminally differentiatedcardiomyocytes. In mice, miR-1 expression is regulatedby the transcription factors MyoD, Mef2 and SRF [34].This reminds of the situation in Drosophila, where miR-1 expression in the presumptive and early mesoderm isregulated by the transcription factors Twist, Mef2, andSRF [34-36]. Severely affected Drosophila miR-1mutant embryos showed abnormal cardiac and musclepatterning, their heart progenitor cells were in excessand failed to differentiate into distinct lineages [36]. Anoverexpression of miR-1 in cardiac mesoderm ofDrosophila had an opposite effect. Precursors

differentiated prematurely, so that fewer progenitorswere observed [36]. Another loss of function miR-1study in Drosophila showed that it is not necessary fornormal specification and patterning of the somaticmuscle, but is essential for maintaining thedevelopment and integrity of body wall muscle duringphases of rapid muscle growth [35].

Besides their function in regulating cardiachypertrophy, miR-1 and miR-133 play roles inmodulating heart electrical conduction [18, 38-40]. miR-1-2 mouse mutants had a decreased heart rate andproblems in ventricular repolarization [18]. 50% of the


4/13


Fig. (2). Exemplary microRNAs and their field of function.


5/13


miR-1-2 knockout embryos died during embryonicdevelopment due to defects in their ventricular septum,and 15% died 2-3 months after birth. The ones thatsurvived to adulthood died frequently by sudden death.

After myocardial infarction, the surviving heartsbecame hypertrophic and underwent an electricalremodeling process favoring arrhythmia [38]. miR-1 levels were increased in rats with experimental

myocardic infarcts, in rat myocardium that hadundergone ischemia and reperfusion, and in individualsaffected by coronary artery disease [39]. MiR-1 is aproarrhythmic and arrhythmogenic factor directlytargeting the cardiac gap junction channel connexin 43,and the K + channel subunit Kir2.1. Knocking-down miR-1 in myocardial infarction normalized the expression ofthese targets and reduced arrhythmias. The direct co-silencing of miR-1 with the two targets inducedarrhythmias, indicating that the role of miR-1 inelectrical remodeling and the proarrhythmic effect wereat least partially due to downregulation of these ionchannels proteins. MiR-133 plays an important role inQT prolongation, a disorder of the hearts electrical

conduction system, and the associated arrhythmias indiabetic hearts [40]. This syndrome is furtherassociated with episodic ventricular arrhythmias andsudden death. MiR-133 targets a subunit of a cardiacK+ channel that conducts one of the cardiacrepolarization currents [40]. This protein was repressedin rabbit hearts of a diabetes mellitus model wheremiR-133 and its upstream regulator, the SerumResponse Factor (SRF), were increased. Inhibition orsilencing of SRF decreased miR-133 levels andincreased the cardiac repolarization current density inventricular myocytes.

The multiple functions of miR-1 and miR-133 furtherinclude the regulation of cardiac cell fate through themodulation of apoptosis, possibly targeting the heatshock and caspase proteins [41]. Induced by oxidativestress, miR-1 and miR-133 produced opposing effectson apoptosis, with miR-1 being pro-apoptotic and miR-133 being anti-apoptotic. Therefore, the miR-1/miR-133ratio is very important for cardiac cell fate regulation.Several studies focused on the role of microRNAs inthe acquisition of a myogenic fate. They identified miR-1, miR-133, miR-181 and miR-206 as functionalregulators of the fine balance between cell proliferationand differentiation (Fig. 2B ). MiR-181 is poorlyexpressed in terminally differentiated muscle, but isstrongly upregulated in differentiated, regenerated

muscle fibers of a muscle injury mouse model. MiR-181directly targets Hoxa11 mRNA, which encodes asuppressor of MyoD, a trigger of the muscledifferentiation program. However, neither upregulationof miR-181, nor downregulation of Hoxa11 wassufficient to trigger terminal differentiation ofproliferating myoblasts [42]. The murine myoblast-derived cell line C2C12 is frequently used as a modelsystem for myogenesis. The overexpression of theclosely related microRNAs miR-1 or miR-206 in C2C12cells decreased proliferation, and increasedmyogenesis through the regulation of targets including

a histone deacetylase (HDAC4), a subunit of DNApolymerase (Pola1), and of a gap-junction protein(Connexin43) [28, 43-45]. Even in HeLa cells, miR-1 iscapable of promoting myogenesis [46]. Although miR-133 derives from the same bicistron as miR-1, it stillhas an opposite effect on myogenesis, promotingrather than inhibiting myoblast proliferation, anddecreasing myogenesis [28]. Its effect was attributed to

a direct downregulation of the muscle transcriptionfactor SRF. These findings indicate a negativeregulatory loop, where tissue-specific miR-1 expressionis regulated by SRF, whose expression is regulated bymiR-133 [28] (Fig. 3A ).

In conclusion, a full range of miroRNAs isexpressed in heart and/or skeletal muscle, and theirmisregulation alone or in association affects cell fate,regulates the balance between cell proliferation anddifferentiation, and induces or influences heart diseasestates, such as hypertrophy, fibrosis, electricalremodeling and arrhythmia (Fig. 2A, B ).

MicroRNA Functio ns in the LiverThe formation of the foregut pocket marks the

beginning of endodermal organogenesis, leading notonly to an elaborated intestinal system, but also to thedevelopment of adjacent endodermal organs, like lung,liver, and pancreas. The hepatic primordium forms inthe cardiac region, and becomes obvious by anincrease of proliferation and cell migration in a cord-likefashion into the mesenchyme of the septumtransversum. Hepatic ducts begin to form in thegrowing liver parenchyme, and finally a common ductconnects the rapidly developing, distinct organ to thegut tube. While many embryonic signals and inductiveinteractions during liver development had beencharacterized, an involvement of microRNAs has notbeen shown so far, but appears most likely. Theavailable data rather focus on the adult liver, and inparticular the role of miR-122, a liver-specificmicroRNA which was cloned from mouse, rat andhuman liver tissues, as well as from several hepatic celllines [29, 47]. The potential importance of miR-122 indiseases like hepatitis fuelled the interest in thismicroRNA. Expression studies revealed its relation tothe replication competence of the Hepatitis C virus(HCV), and showed that the cellular abundance of miR-122 often correlated with the replication ability of theHCV RNA [47]. A sequence comparison of the HCV

genome and the miR-122 microRNA suggested thattwo potential target sequences were located in the 5and 3 UTRs of the HCV coding core [47]. Mutationalanalysis of the two putative targets showed, that the5UTR, but not the 3UTR is an efficient targetsequence for miR-122, necessary for the accumulationof viral RNA [47]. Furthermore, the analysis of HCVRNA in miR-122 transfected cells revealed that theeffect on HCV is rather at the level of the genomic RNAreplication, than of translation. Therefore, it wasconcluded that miR-122 affects the replication of theHCV genome upon binding to its 5UTR, and probably


6/13


changing the secondary structure of the viral genomeby making it replication competent [47]. The sequenceof miR-122 is an integral constituent of the non-codinghcr RNA, a 166 nucleotide molecule formerly identifiedin liver tumors, different stages of embryonic liver, andliver cell lines [48]. The computational analysis of thehcr secondary structure predicted a stem loopconfiguration, with the miR-122 sequence in one arm.

Together these data confirmed that the non-coding hcris the unprocessed form of miR-122, which showsdifferential expression in different types of hepatictumors [48]. Computational target predictions for miR-122 predicted that the 3UTR of a mRNA encoding thecationic amino acid transporter (CAT)-1 was a target.This was further confirmed by reporter analysis. Furtherevidence was obtained by following the expression ofmiR-122 and CAT-1 in different stages of embryonicand adult livers, and in several cell lines, showing aconverse correlation [48]. A systemic ablation of miR-122 in mouse liver by intraperitoneal injection of the 2-O-methoxyethyl (2OME) phosphorothioate antisenseoligonucleotides, showed a further role of miR-122 in

lipid metabolism [49]. Mice injected with the miR-122antisense oligonucleotides, showed a normal plasmalevel of glucose, but a reduction of cholesterol andtriglycerides, comparing to the control animals [49].Further microarray analysis of liver gene expression inthese mice, showed a high fluctuation of genesinvolved in lipid metabolism, which was furtherconfirmed by ex-vivo studies showing a reduction offatty acid synthesis and an increase of fatty acidoxidation [49].

All in all, the results from these functional studieselucidate the function of a microRNA in the replicationcompetence of the HCV genome, and more generally,a role in the lipid metabolism of the liver.

MicroRNAs and Pancreas Development

Pancreas development begins early in vertebrateembryogenesis with dorsal and ventral evaginationsfrom the endodermal epithelium of the foregut. The twopancreatic rudiments grow together to form a singlemass of cells, which then becomes structured into acharacteristic pattern of lobules, separated bymesenchyme and drained by a duct system.Cytodifferentiation then gives rise to exocrine andendocrine cell types. The latter develop in smallclusters, the islets of Langerhans and secrete

hormones, most importantly insulin, glucagon andsomatostatin.

The proper development and function of thepancreas is a highly regulated and tuned process,which involves an extensive array of transcriptionfactors and tight regulatory pathways. MicroRNAexpression profiles show a considerable degree ofdynamics from the early bud stages to the formation ofthe definitive pancreas [50]. Among the manymicroRNAs which are suggested either by high-throughput RNA profiling or computational methods tobe involved in pancreas development or function, only

a few were functionally validated. The mainconclusions on the role of microRNAs in pancreasmorphogenesis stem from loss-of-function experimentsin zebrafish embryos, where morpholinooligonucleotides against the precursor or the maturemicroRNA sequence of interest were applied, whichimpaired the microRNA function [51].

A morphlino knockdown approach was used tostudy the function of miR-375, which was originallyreported as a microRNA specifically expressed in thepancreatic islet and the pituitary gland [33], and waslater isolated from pancreatic beta cells [52]. The lossof miR-375 function in zebrafish embryos, did not resultin any defect of the pituitary gland as demonstrated bythe expression of pit1 as an early marker for thepituitary gland formation [51]. The pancreatic islets,however, showed a scattered pattern of islet-1expression which was also followed by the expressionof further endocrine pancreatic markers, such asglucagon and somatostatin [51]. The results suggestthat miR-375 is involved in the maintenance ofpancreatic integrity, but the exact mechanism and thedownstream pathway still remains to be understood.The study of miR-375 in pancreatic beta cells furthershowed a direct effect on insulin secretion in thesecells [52]. The overexpression of miR-375 in the murinepancreatic beta cell line MIN6 resulted in a decrease ofglucose-induced insulin secretion. The functionalknockdown of miR-375 on the other hand, using 2-O-methyl antisense oligonucleotides, had a converseeffect, and increased the insulin secretion uponinduction of these cells with glucose. The function ofmiR-375 was not resulting from impaired signalingpathways, but rather from a direct effect on the insulinexocytosis [52]. Among the different targets of miR-375, myotrophin, an ankyrin repeat-rich cytoplasmicprotein which promotes insulin secretion, was validatedas a functional mediator of miR-375 in the pancreaticbeta cells [52]. According to these studies,overexpression of miR-375 in MIN-6 cells impaired theglucose-induced insulin secretion by downregulatingmyotrophin and subsequently resulting in a defect ininsulin exocytosis [52-54].

The insulin secretory pathway is further regulatedby miR-9, a microRNA expressed in neurons,pancreatic beta cells and the rat pancreatic beta cellline INS-1E [55]. The overexpression of miR-9 in INS-1E cells resulted in a decrease of insulin exocytosis,which was further proved to result from a defect in the

secretory signaling pathway [55]. In the subsequentcheck for misregulated downstream effectorsconnected to the insulin secretion signaling cascade,Granuphilin/Slp4, a Rab GTPase effector associatedwith beta cell secretory granules, was shown to bedramatically upregulated. As microRNAs are known todownregulate their target mRNAs, the study wasfurther followed to find a putative miR-9 target whichwould be acting upstream of the Granuphilin/SLP gene,finally resulting in the identification of the transcriptionfactor Onecut-2 which is associated with theGranuphilin promoter, and represses its transcription.


7/13


8/13


markers. The genes encoding miR-124, miR-9 andmiR-132 are targets of the transcriptional repressorREST in non-neural cells, similar to many otherneuronal genes [65, 66]. When progenitors mature intoneurons, REST relieves miR-124, which then becomesinvolved in the inactivation of non-neural genes,including REST itself and its cofactors MeCP2 andCoREST, which have binding sites for miR-124 in their

3 UTRs. These findings indicate a double negativefeedback loop at the transition from non-neural toneural gene expression (Fig. 3B ).

The first direct studies of microRNAs in the centralnervous system of vertebrate embryos were performedby electroporation of the neural tube in chick embryos.Cao et al . observed that neither inhibition noroverexpression of miR-124 altered the acquisition ofneuronal fate, indicating that miR-124 did not act as aprimary determinant of neural differentiation [67]. Incontrast, Visvanathan et al . claim to have observed astimulation of neuronal differentiation by miR-124,which is antagonized by the target SCP1 [68]. Theprimary function of miR-124 seemed to be the inhibitionof target genes such as SCP1, lamin, and integrin,which need to be downregulated in order to proceedfrom a neural progenitor cell to a differentiated neuron.

Among the many targets of miR-124 is also the globalrepressor of differential splicing PTBP1 [69]. Byrepressing PBTP1, an alternatively spliced mRNAencoding PTBP2 becomes favored. This transition isimportant for the proceeding from a neural progenitor toa differentiated neuron. Thus, the regulation by aneural specific microRNA becomes connected to asuperimposed regulatory level, to alternative splicing.

Similar to miR-124, miR-134 is confined to braincells [70]. It was found localized in synaptic sites of

dendrites of cultured hippocampal neurons.Overexpression of miR-134 decreased the dendriticspine volume, whereas a knockdown increased thevolume slightly. As a molecular target of miR-134, themRNA encoding LIM domain containing kinase Limk1was identified, which inhibits Limk1 translation not inthe cell body, but locally within dendrites. DendriticRNAs are normally transported to synaptic sites,remain dormant, and are translated only uponstimulation by factors such as BDNF. The findingssuggest a miR-134 associated silencing complex,which needs to be inactivated in order to allow Limk1translation, thereby contributing to synaptic plasticity[70] .

The neuron enriched microRNA miR-132, wasidentified in a genome-wide screen as a target of thecAMP-response element binding protein (CREB) [71].Expression of miR-132 in cortical neurons inducedneurite outgrowth. Conversely, inhibition of miR-132function attenuated neuronal outgrowth. The studyestablished a neurogenetic pathway from an extrinsicneurotrophin, to the transcription factor CREB, to miR-132 and to the GTPase-activating protein p250GAP.

MiR-133b was identified in a subtractive screensearching for microRNAs enriched in dopaminergic

neurons [72, 73]. It is enriched in normal adult humanand rodent midbrain samples, and deficient in samplesstemming from Parkinson patients, as well as inmidbrains from murine dopamine deficiency models.The influence of miR-133b in midbrain neurons wasanalyzed in embryonic stem cell derived culturesystems, or in primary midbrain cells. Overexpressionusing a lentivirus vector did not affect early markers of

dopaminergic neuron development. However, a latemarker indicating mature dopaminergic neurons wassuppressed. Knocking down miR-133b on the otherhand, induced markers for dopaminergic neuronformation. A concrete target for miR-133b lies in the3UTR of the Pitx3 gene, a necessary transcriptionfactor for the development of dopaminergic neurons.These findings suggests a negative feedback circuit:Pitx3 activates midbrain-specific neuronal geneexpression, including miR-133b, which then in turnpost-transcriptionally represses its inducer Pitx3 (Fig.3C ).

Taken together these examples demonstrate thatneural specific microRNAs play significant roles in thelast step of differentiation towards a mature neuron(Fig. 2D ). They are part of an environment, whichrepresses non-neural molecules at a relatively late stepin development. Effects on earlier steps are much lessobvious, and may either not occur at all, or may turnout to be much more difficult to measure. However, itseems conceivable that also for early intermediates ofa developmental pathway specific functionalmicroRNAs exist.

MicroRNAs in Hematopoiesis

During hematopoiesis pluripotent, self-renewingstem cells give rise to the different blood cell lineages.These include the erythroid cells, the lymphoid B and Tcells, and the myeloid cells, including granulocytes,megakaryocytes and macrophages. Aberrations ofregulatory pathways in hematopoiesis can result insevere disease states, such as leukemias, lymphomasor myelomas. Recently, it was demonstrated that alsomicroRNAs are misregulated in various hematopoieticdiseases or relevant experimental model systems [74,75]. As shown before for many protein encoding genes,also the genomic loci encoding microRNAs were foundto be altered by translocations or deletions [76].

Erythrocyte differentiation normally coincides withthe downregulation of a number of microRNAs,

including miR-24, miR-221 and miR-222 [77]. Theforced expression of miR-24 in K562 erythroleukemiccells impairs the activin-induced maturation of erythroidcells, and represses the accumulation of hemoglobin inthese cells [77]. This effect results from thedownregulation of Activin type I receptor ALK4, whichis targeted by miR-24 [77]. The differentiation of cordblood CD34+ cells associates with an increase in thereceptor protein Kit, a critical factor for erythropoiesis.The two microRNAs miR-221 and miR-222 target themRNA for Kit, and therefore a pro-erythropoietic,undifferentiated state is maintained [78, 79]. The


9/13


downregulation of the two microRNAs and theconsequent induction of the Kit receptor is sufficient torelease the inhibition of erythropoietic differentiation[79].

MiR-181 is expressed in thymus and bone marrowwith an expression increasing during B-lymphocytematuration [76]. Ectopic expression of miR-181 inhematopoietic progenitor cells, doubled the amount ofB-lymphocytes, with little change in the number of T-cells, indicating a specific role of this microRNA in B-lymphocyte differentiation [76]. Also miR-142 and miR-223 have a preferential expression in hematopoietictissues [76]. In contrast to miR-181, however, theectopic expression of these two microRNAs, drovehematopoietic differentiation towards T-lymphocyteformation, indicating a preferential role of thesemicroRNAs in the maturation of T-cells [76].

MicroRNAs are also shown to be involved in thedifferentiation of hematopoietic progenitors to themyeloid cell lineages. When induced towards themonocyte/macrophage fate, the level of miR-424

increases in cord blood CD34+ cells [80].Concomitantly, the transcription factor PU.1, requiredfor the terminal maturation steps of monocyte/macrophage cells, becomes upregulated. The PU.1protein interacts physically with the promoter of themiR-424 gene, and enhances its transcription. MiR-424itself then targets the mRNA of NFI-A, a factor whichneeds to be downregulated during monocyte/macrophage maturation [80]. Through these regulatoryinteractions a molecular circuitry becomes establishedwith miR-424 as its central component, controlling theterminal differentiation of the monocyte/macrophagelineage (Fig. 3D ) [80]. The level of miR-223 increasesduring granulocytic differentiation [81]. Studies of the

miR-223 promoter revealed overlapping binding sites ofthe transcription factors NFI-A and C/EBP . Inundifferentiated cells, the NFI-A protein maintains a lowlevel of miR-223 expression by binding to its promoter.During granulocyte differentiation however, the level ofC/EBP as a mediator of differentiation processesincreases, competes eventually for the binding of NFI-Ato the miR-223 promoter, and acts as a strongeractivator of microRNA transcription [81]. The presenceof a target site for miR-223 in the 3UTR of the NFI-AmRNA, enhances the effect of miR-223 on granulocyticdifferentiation (Fig. 3E ) [81].

Expression profiling and the functionalcharacterizations of microRNAs in differenthematopoietic lineages indicate their relevance innormal as well as malignant hematopoiesis (Fig. 2E ).The available medical possibilities in the treatment ofleukemias or lymphomas make it conceivable, thathematopoietic microRNAs qualify as targets or agentsin therapy one day

MicroRNAs in t he Cell Cycle and Cancer

Nature has developed multiple mechanisms ofcoupling cell proliferation with apoptosis in order to

Fig. (3). Feedback regulation between microRNAs andtranscriptional regulators.

A . Promotion of muscle fate through a feedback loop,repressing the translation of myoblast proteins afterdifferentiation [28, 34, 40 , 44, 45].B. Balancing between a non-neural and a neuronal fatethrough a double negative feedback loop repressing inneuronal cells the translation of the REST complexmembers, which themselves would maintain a non-neuralfate [65, 66, 68].C. Double negative feedback inhibition involved in thedifferentiation of a neural progenitor to a mature neuron [73].

D. Differentiation of promyelocytes intomonocyte/macrophages upon downregulation of NFI-A [80].E. The balance of NFI-A and C/EBP- in the differentiation ofundifferentiated cord blood cells into granulocytes. C/EBP-competes NFI-A in binding the microRNA promoter andresults in a higher microRNA expression [81].F . Feedback inhibition of E2F transcription factors bymicroRNAs after depletion of c-myc from B-cells. ThemicroRNA genes are under positive control in the presenceof c-myc [85, 86, 89, 91].For a detailed discussion please see the main text.


10/13


control aberrant proliferation and to avoid cancerformation. One cause for cancer formation andprogression is the misregulation of this link. Obviously,the involvement of microRNAs in the regulation of thecell cycle and apoptotic pathways, and their functionalroles in tissue homeostasis is of considerable interest.In the last years a number of microRNAs with highlydynamic and complex patterns in cancer tissues were

identified by expression profiling, suggesting functionsin tumor formation through the regulation of factorslinked to cell cycle control and apoptosis [82-84].

A group of transcription factors related in manyrespects to cell cycle regulation is the E2F family. E2Ftranscription factors are regulated by severalmicroRNAs, including miR-17-5p, miR-20, miR-34a,miR-221 and miR-222 [85-89]. MicroRNAs from themiR-17-92 cluster, namely miR-17-5p and miR-20a,influence the translation of E2F1 mRNA [85], whilemiR-20a targets in addition E2F2 and E2F3 [86]. Thismodulation occurs through binding sites in the 3'-untranslated region of the respective mRNAs. Viceversa, E2F1-3 directly regulates the transcription of themiR-17-92 cluster, suggesting an auto regulatoryfeedback loop between E2F factors and miRNAs (Fig.3F ). A further component in this regulatory network isc-Myc, a transcription factor known to regulate cellproliferation, growth and apoptosis [90]. C-Myc istranscriptionally induced by E2F1, and itself directlyactivates the transcription of the miR-17-92 cluster [85,86, 91]. Since miR-17-5p and miR-20 preventuncontrolled reciprocal activation of c-Myc and E2F1products, they are involved in the control of c-Mycmediated cellular proliferation. MicroRNAs from themiR-17-92 cluster have an oncogenic activity whenoverexpressed with c-myc in a mouse model of humanB-cell lymphoma [92]. In addition, miR-20aoverexpression decreased apoptosis in a prostatecancer cell line, and its inhibition resulted in anincrease of cell death after DNA damage cell treatment[86].

E2F3 is not only targeted by miR-20, but also bymiR-34a, a microRNA which is downregulated inneuroblastoma derived cell lines and primaryneuroblastoma tumors, a tumor type accounting for15% of cancer related childhood deaths [87]. MiR-34aamounts increased in parallel with decreased E2F3protein levels, when differentiation of neuroblastomawas induced by all-trans retinoic acid. Proliferation ofneuroblastoma cells became repressed upon

overexpression of miR-34 by inducing a caspase-dependent apoptotic pathway.

Another well-known cell cycle regulator, the p27 Kip1 protein, was found to be subject to control bymicroRNAs. Drosophila germline stem cells with animpaired dicer-1 activity were delayed at the transitionfrom G1 to S, due to an increase of Dacapo, a homologof human p27 Kip1 [93]. Also in human glioblastoma cellsthe downregulation of Dicer led to a decrease of cellproliferation involving p27 Kip1 [89]. MiR-221 and miR-222, two homologous microRNAs, were shown to

regulate p27 Kip1 [88, 89]. These two microRNAs arepresent at high levels in tumor tissues, such as primaryglioblastomas, papillary thyroid carcinoma andpancreas tumors [92, 94-96]. In many glioblastomacancers low levels of p27 Kip1 were observed [88].Suppression of miR-221 and miR-222 levels in severalcancer cell lines with antagomirs led to a proliferationarrest and increase of p27 Kip1 levels [88]. Thus, miR-

221 and miR-222 seem to function as oncogenes bycontrolling cell cycle progression through inhibition ofp27 Kip1.

The occurrence of chronic lymphocytic leukemia(CLL) is often correlated with the upregulation of theanti-apoptotic factor BCL2 [97]. On the other hand, thetwo microRNAs miR-15a and miR-16-1 are significantlydownregulated in many CLL cells, often due to analtered or deleted genomic locus [97]. This inversecorrelation suggested BCL2 as a target of these twomicroRNAs. Experimental testing proved that bothmicroRNAs were able to repress the translation ofBCL2, resulting finally in the activation of the APAF-1-caspase 9-PARP apoptotic pathway [97]. Comparativeanalysis of microRNA pools in normal and p53-mutantembryonic fibroblasts of mice identified a correlation ofthe expression levels of miR-34a, miR-34b, and miR-34c with the p53 status [98]. Moreover, theoverexpression of miR-34 could mimic the downstreamfunctions of p53 and its canonical effector p21 inactivating the DNA damage response pathway, inapoptosis, and in cell cycle arrest [98, 99]. Chromatinimmunoprecipitation showed a direct interaction of p53and the miR-34 promoter, leading to the conclusionthat p53 is in fact acting upstream of miR-34. A screenfor miR-34 targets revealed many cell cycle regulationgenes thus further strengthening the evidence for afunction of miR-34 in the regulation of the cell cycle asa downstream effector of p53 [98, 99].

Together, these results suggest that microRNAs areimportant for maintaining the balance between cellularproliferation and apoptosis, and thus can function asboth tumor promoters or suppressors, depending oncomplex regulatory networks (Fig. 2F ).

OUTLOOK

The studies discussed in this review support thesteadily growing evidence that microRNAs function askey players of post-transcriptional regulation. Mostphysiological processes seem to involve this level ofregulation, and appear to require microRNAs for theexact final decision on the states of gene expression. Inparticular, microRNAs seem to increase therobustness, speed, and stability in response to dynamicchanges. Fig. ( 2 ) lists the microRNA studies which arediscussed in the current review. The body of suchfunctional studies is continuously growing in a realavalanche of publications. However, if the emergingpicture is true that microRNAs are involved rather in thefine tuning of many targets, than in very specific on oroff decisions, it may turn out to become increasinglydifficult to obtain a systematic understanding of


11/13


microRNA functions. Already now, the importance ofup-to date high-throughput methods and broadscreening approaches are the way of choice to tacklesuch questions.

Currently, the field of microRNA research is mostlyfocused on basic research in many aspects of the lifesciences, including developmental and cell biology.However, the many disease models in various animalsystems indicate already that the time is ripe to transferthe knowledge into clinical research, and then possiblyalso to clinical applications [100]. The possibility toaddress many targets simultaneously within a givencontext may eventually turn out to be an explicitadvantage, as compared to highly specific techniques.MicroRNAs may finally represent ideal tools for a so-called one-hit-multiple-target approach [100]. Thesmall size of the active molecules will allow theintroduction of chemical modifications, and thus themodulations of microRNA functions, finally providingprecisely tailored, agonistic or antagonistic moleculeswith defined cellular, and therefore also medicalconsequences. The development of methods for asystemic delivery or applications of microRNA willcertainly represent a major hurdle to take beforepharmacological applications come into reach [100].Numerous patents have been filed in recent years in anattempt to ensure later commercial exploitations of thebasic findings. A growing number of biotech companiesare being founded to initiate the next steps inmicroRNA applications. Taken together, there can beno doubt that the importance of microRNAs inresearch, clinic, and business will increase in the nextyears. The expectations are high.

ACKNOWL EDGEMENTS

We thank Dr. Samuel M. Young for critical readingof the manuscript and Hartmut Sebesse for graphics.

AB BREVIA TIONS

CAT = Cationic Amino acid TransporterCLL = Chronic Lymphocytic LeukemiaCREB protein = cAMP-response element binding

proteinHCV = Hepatitis C VirusMHC = Myosin Heavy ChainSRF = Serum Response Factor

REFERENCES

[1] Landgraf, P., Rusu, M., Sheridan, R., Sewer, A., Iovino, N., Aravin, A., Pfeffer, S ., Rice, A., Kamphorst, A.O., La ndthaler,M., Lin, C., Socci, N.D., Hermida, L., Fulci, V., Chiaretti, S.,Foa, R., Schliwka, J., Fuchs, U., Novosel, A., Muller, R.U.,Schermer, B., Bissels, U., Inman, J., Phan, Q., Chien, M.,Weir, D.B., Choksi, R., De Vita, G., Frezzetti, D., Trompeter,H.I., Hornung, V., Teng, G., Hartmann, G., Palkovits, M., DiLauro, R., Wernet, P., Macino, G., Rogler, C.E., Nagle, J.W.,Ju, J., Papavasiliou, F.N., Benzing, T., Lichter, P., Tam, W.,Brownstein, M.J., Bosio, A., Borkhardt, A., Russo, J.J.,

Sander, C., Zavolan, M., and Tuschl, T. (2007) Cell , 1291401-1414.

[2] Griffiths-Jones, S. (2007) Annu Rev. Genomics. Hum.Genet. , 8 , 279-298.

[3] Ambros, V., Bartel, B., Bartel, D.P., Burge, C.B., Carrington,J.C., Chen, X., Dreyfuss, G., Eddy, S.R., Griffiths-Jones, S.,Marshall, M., Matzke, M., Ruvkun, G., and Tuschl, T. (2003)RNA , 9 , 277-279.

[4] Griffiths-Jones, S. (2004) Nucleic Acids Res. , 32 , D109-111.[5] Griffiths-Jones, S. (2006) Methods. Mol. Biol. , 342 , 129-138.[6] Griffiths-Jones, S., Grocock, R.J., van Dongen, S., Bateman,

A., and Enright, A.J. (2006) Nucleic Acids Res. , 34 , D140-144.

[7] Cai, X., Hagedorn, C.H., and Cullen, B.R. (2004) RNA , 101957-1966.

[8] Lee, Y., Kim, M., Han, J., Yeom, K.H., Lee, S., Baek, S.H.,and Kim, V.N. (2004) EMBO J. , 23 , 4051-4060.

[9] Zeng, Y. (2006) Oncogene , 25 , 6156-6162.[10] Pillai, R.S., Bhattacharyya, S.N., and Filipowicz, W. (2007)

Trends. Cell Biol. , 17 , 118-126.[11] Nilsen, T.W. (2007) Trends Genet. , 23 , 243-249.[12] Ason, B., Darnell, D.K., Wittbrodt, B., Berezikov, E.,

Kloosterman, W.P., Wittbrodt, J., Antin, P.B., and Plasterk,R.H. (2006) Proc. Natl. Acad. Sci. USA , 103 , 14385-14389.

[13] Kloosterman, W.P., and Plasterk, R.H. (2006) Dev. Cell , 11441-450.

[14] Bernstein, E., Kim, S.Y., Carmell, M.A., Murchison, E.P., Alcorn, H., Li, M.Z., Mills, A.A., Elledge, S.J ., Anderson, K.V.,and Hannon, G.J. (2003) Nat. Genet. , 35 , 215-217.

[15] Giraldez, A.J., Cinalli, R.M., Glasner, M.E., Enright, A.J.,Thomson, J.M., Baskerville, S., Hammond, S.M., Bartel,D.P., and Schier, A.F. (2005) Science , 308 , 833-838.

[16] Wienholds, E., Koudijs, M.J., van Eeden, F.J., Cuppen, E.,and Plasterk, R.H. (2003) Nat. Genet. , 35 , 217-218.

[17] Ying, S.Y., and Lin, S.L. (2005) Biochem. Biophys Res.Commun. , 335 , 1-4.

[18] Zhao, Y., Ransom, J.F., Li, A., Vedantham, V., von Drehle,M., Muth, A.N., Tsuchihashi, T., McManus, M.T., Schwartz,R.J., and Srivastava, D. (2007) Cell , 129 , 303-317.

[19] van Rooij, E., Sutherland, L.B., Liu, N., Williams, A.H.,McAnally, J., Gerard, R.D., Richardson, J.A. and Olson, E.N.(2006) Proc. Natl. Acad. Sci. USA , 103 , 18255-18260.

[20] Cheng, Y., Ji, R., Yue, J., Yang, J., Liu, X., Chen, H., Dean,

D.B., and Zhang, C. (2007) Am. J. Pathol. , 170 , 1831-1840.[21] Sayed, D., Hong, C., Chen, I.Y., Lypowy, J., and Abdellatif,M. (2007) Circ. Res. , 100 , 416-424.

[22] Tatsuguchi, M., Seok, H.Y., Callis, T.E., Thomson, J.M.,Chen, J.F., Newman, M., Rojas, M., Hammond, S.M., andWang, D.Z. (2007) J. Mol. Cell. Cardiol., 42 , 1137-1141.

[23] Thum, T., Galuppo, P., Wolf, C., Fiedler, J., Kneitz, S., vanLaake, L.W., Doevendans, P.A., Mummery, C.L., Borlak, J.,Haverich, A., Gross, C., Engelhardt, S., Ertl, G., andBauersachs, J. (2007) Circulation , 116 , 258-267.

[24] Ikeda, S., Kong, S.W., Lu, J., Bisping, E., Zhang, H., Allen,P.D., Golub, T.R., Pieske, B., and Pu, W.T. (2007) Physiol.Genomics. , 31 (3), 367-373.

[25] van Rooij, E., Sutherland , L.B., Qi, X., Richardson, J.A., Hill,J., and Olson, E.N. (2007) Science , 316 , 575-579.

[26] Care, A., Catalucci, D., Felicetti, F., Bonci, D., Addario, A.,Gallo, P., Bang, M.L., Segnalini, P., Gu, Y., Dalton, N.D.,Elia, L., Latronico, M.V., Hoydal, M., Autore, C., Russo, M.A.,Dorn, G.W., 2nd, Ellingsen, O., Ruiz-Lozano, P., Peterson,K.L., Croce, C.M., Peschle, C., and Condorelli, G. (2007)Nat. Med. , 13 , 613-618.

[27] van Rooij, E. and Olson, E.N. (2007) J. Clin. Invest. , 1172369-2376.

[28] Chen, J.F., Mandel, E.M., Thomson, J.M., Wu, Q., Callis,T.E., Hammond, S.M., Conlon, F.L., and Wang, D.Z. (2006)Nat. Genet ., 38 , 228-233.

[29] Lagos-Quintana, M., Rauhut, R., Yalcin, A., Meyer, J.,Lendeckel, W., and Tuschl, T. (2002) Curr. Biol. , 12 , 735-739.

[30] Lee, R.C., and Ambros, V. (2001) Science , 294 , 862-864.


12/13


[31] Sempere, L.F., Freemantle, S., Pitha-Rowe, I., Moss, E.,Dmitrovsky, E., and Ambros, V. (2004) Genome Biol. , 5 ,R13.

[32] Mansfield, J.H., Harfe, B.D., Nissen, R., Obenauer, J.,Srineel, J., Chaudhuri, A., Farzan-Kashani, R., Zuker, M.,Pasquinelli, A.E., Ruvkun, G., Sharp, P.A., Tabin, C.J., andMcManus, M.T. (2004) Nat. Genet ., 36 , 1079-1083.

[33] Wienholds, E., Kloosterman, W.P., Miska, E., Alvarez-Saavedra, E., Berezikov, E., de Bruijn, E., Horvitz, H.R.,Kauppinen, S., and Plasterk, R.H. (2005) Science , 309 , 310-311.

[34] Zhao, Y., Samal, E., and Srivastava, D. (2005) Nature , 436 ,214-220.

[35] Sokol, N.S., and Ambros, V. (2005) Genes Dev. , 19 , 2343-2354.

[36] Kwon, C., Han, Z., Olson, E.N., and Srivastav a, D. (2005)Proc. Natl. Acad. Sci. USA , 102 , 18986-18991.

[37] Srivastava, D., Thomas, T., Lin, Q., Kirby, M.L., Brown, D.,and Olson, E.N. (1997) Nat. Genet , 16 , 154-160.

[38] Anderson, M.E., and Mohler, P.J. (2007) Nat. Med. , 13 , 410-411.

[39] Yang, B., Lin, H., Xiao, J., Lu, Y., Luo, X., Li, B., Zhang, Y.,Xu, C., Bai, Y., Wang, H., Chen, G., and Wang, Z. (2007)Nat. Med. , 13 , 486-491.

[40] Xiao, J., Luo, X., Lin, H., Zhang, Y., Lu, Y., Wang, N., Yang,B., and Wang, Z. (2007) J. Biol. Chem. , 282 , 12363-12367.

[41] Xu, C., Lu, Y., Pan, Z., Chu, W., Luo, X., Lin, H., Xiao, J.,Shan, H., Wang, Z., and Yang, B. (2007) J. Cell. Sci., 120 ,3045-3052.

[42] Naguibneva, I., Ameyar-Zazoua, M., Polesskaya, A., Ait-Si- Ali, S., Groisman, R., Souidi, M., Cuvellier, S., and Harel-Bellan, A. (2006) Nat. Cell. Biol. , 8 , 278-284.

[43] Lu, J., McKinsey, T.A., Zhang, C.L., and Olson, E.N. (2000)Mol. Cell. , 6 , 233-244.

[44] Kim, H.K., Lee, Y.S., Sivaprasad, U., Malhotra, A., and Dutta, A. (2006) J. Cell. Biol. , 174 , 677-687.

[45] Anderson, C., Catoe, H., and Werner, R. (2006) Nucleic Acids Res. , 34 , 5863-5871.

[46] Lim, L.P., Lau, N.C., Garrett-Engele, P., Grimson, A.,Schelter, J.M., Castle, J., Bartel, D.P., Linsley, P.S., andJohnson, J.M. (2005) Nature , 433 , 769-773.

[47] Jopling, C.L., Yi, M., Lancaster, A.M., Lemon, S.M., andSarnow, P. (2005) Science , 309 , 1577-1581.

[48] Chang, J., Nicolas, E., Marks, D., Sander, C., Lerro, A.,Buendia, M.A., Xu, C., Mason, W.S., Moloshok, T., Bort, R.,Zaret, K.S., and Taylor, J.M. (2004) RNA Biol. , 1 , 106-113.

[49] Esau, C., Davis, S., Murray, S.F., Yu, X.X., Pandey, S.K.,Pear, M., Watts, L., Booten, S.L., Graham, M., McKay, R.,Subramaniam, A., Propp, S., Lollo, B.A., Freier, S., Bennett,C.F., Bhanot, S., and Monia, B.P. (2006) Cell Metab. , 3 , 87-98.

[50] Joglekar, M.V., Parekh, V.S., and Hardikar, A.A. (2007)Trends Endocrinol. Metab. , 18 , 393-400.

[51] Kloosterman, W.P., Lagendijk, A.K., Ketting, R.F., Moulton,J.D., and Plasterk, R.H. (2007) PLoS Biol. , 5 , e203.

[52] Poy, M.N., Eliasson, L., Krutzfeldt, J., Kuwajima, S., Ma, X.,Macdonald, P.E., Pfeffer, S., Tuschl, T., Rajewsky, N.,Rorsman, P., and Stoffel, M. (2004) Nature , 432 , 226-230.

[53] Mello, C.C., and Czech, M.P. (2004) Nat. Med. , 10 , 1297-1298.

[54] Gauthier, B.R., and Wollheim, C.B. (2006) Nat. Med. , 12 , 36-38.

[55] Plaisance, V., Abderrahmani, A., Perret-Menoud, V.,Jacquemin, P., Lemaigre, F., and R egazzi, R. (2006) J. Biol.Chem. , 281 , 26932-26942.

[56] Baroukh, N., Ravier, M.A., Loder, M.K., Hill, E.V., Bounacer, A., Scharfmann, R., Rutter, G.A., and Van Obberg hen, E.(2007) J. Biol. Chem. , 282 , 19575-19588.

[57] Yatoh, S., Dodge, R., Akashi, T., Omer, A., Sharma, A.,Weir, G.C., and Bonner-Weir, S. (2007) Diabetes , 56 , 1802-1809.

[58] Joglekar, M.V., Parekh, V.S., Mehta, S., Bhonde, R.R., andHardikar, A.A. (2007) Dev. Biol. , 311 , 603-612.

[59] Cuellar, T.L., and McManus, M.T. (2005) J. Endocrinol. , 187327-332.

[60] Kolfschoten, I.G., and Regazzi, R. (2007) Nat. Clin. Pract.Endocrinol. Metab., 3 , 827-834.

[61] Johnston, R.J., and Hobert, O. (2003) Nature , 426 , 845-849.[62] Johnston, R.J., Jr., Chang, S., Etchberger, J.F., Ortiz, C.O.,

and Hobert, O. (2005) Proc. Natl. Acad. Sci. USA , 10212449-12454.

[63] Giraldez, A.J., Mishima, Y., Rihel, J., Grocock, R.J., VanDongen, S., Inoue, K., Enright, A.J., and Schier, A.F. (2006)Science, 312 , 75-79.

[64] Krichevsky, A.M., Sonntag, K.C., Isacson, O., and Kosik,K.S. (2006) Stem Cells , 24 , 857-864.

[65] Wu, J., and Xie, X. (2006) Genome Biol. , 7 , R85.[66] Conaco, C., Otto, S., Han, J.J., and Mandel, G. (2006) Proc.

Natl. Acad. Sci. USA , 103 , 2422-2427.[67] Cao, X., Pfaff, S.L., and Gage, F.H. (2007) Genes Dev. , 21

531-536.[68] Visvanathan, J., Lee, S., Lee, B., Lee, J.W., and Lee, S.K.

(2007) Genes Dev. , 21 , 744-749.[69] Makeyev, E.V., Zhang, J., Carrasc o, M.A., and Maniatis, T.

(2007) Mol. Cell, 27 , 435-448.[70] Schratt, G.M., Tuebing, F., Nigh, E.A., Kane, C.G., Sabatini,

M.E., Kiebler, M., and Greenberg, M.E. (2006) Nature , 439283-289.

[71] Vo, N., Klein, M.E., Varlamova, O., Keller, D.M., Yamamoto,T., Goodman, R.H., and Impey, S. (2005) Proc. Natl. Acad.Sci. USA , 102 , 16426-16431.

[72] Hebert, S.S., and De Strooper, B. (2007) Science , 3171179-1180.

[73] Kim, J., Inoue, K., Ishii, J., Vanti, W.B., Voronov, S.V.,Murchison, E., Hannon, G., and Abeliovich, A. (2007)Science , 317 , 1220-1224.

[74] Kluiver, J., Kroesen, B.J., Poppema, S., and van den Berg, A. (2006) Leukemia , 20 , 1931-1936.

[75] Lawrie, C.H. (2007) Br. J. Haematol. , 137 , 503-512.[76] Chen, C.Z., Li, L., Lodish, H.F., and Bartel, D.P. (2004)

Science , 303 , 83-86.[77] Wang, Q., Huang, Z., Xue, H., Jin, C., Ju, X.L., Han, J.D.,

and Chen, Y.G. (2008) Blood , 111 , 588-595.[78] Georgantas, R.W., 3rd, Hildreth, R., Morisot, S., Alder, J.,

Liu, C.G., Heimfeld, S., Calin, G.A., Croce, C.M., and Civin,C.I. (2007) Proc. Natl. Acad. Sci. USA , 104 , 2750-2755.

[79] Felli, N., Fontana, L., Pelosi, E., Botta, R., Bonci, D.,Facchiano, F., Liuzzi, F., Lulli, V., Morsilli, O., Santoro, S.,Valtieri, M., Calin, G.A., Liu, C.G., Sorrentino, A., Croce,C.M., and Peschle, C. (2005) Proc. Natl. Acad. Sci. USA102 , 18081-18086.

[80] Rosa, A., Ballarino, M., Sorrentino, A., Sthandier, O., De Angelis, F.G., Marchioni, M., Masella, B., Guarini, A., Fatica, A., Pesch le, C., and Bozzoni, I. (2 007) Proc. Natl. Acad. Sci.USA , 104 , 19849-19854.

[81] Fazi, F., Rosa, A., Fatica, A., Gelmetti, V., De Marchis, M.L.,Nervi, C., and Bozzoni, I. ( 2005) Cell , 123 , 819-831.

[82] Chen, C.Z. (2005) N. Engl. J. Med. , 353 , 1768-1771.[83] Garzon, R., Fabbri, M., Cimmino, A., Calin, G.A., and Croce,

C.M. (2006) Trends. Mol. Med. , 12 , 580-587.[84] Cho, W.C. (2007) Mol. Cancer , 6 , 60.[85] O'Donnell, K.A., Wentzel, E.A., Zeller, K.I., Dang, C.V., and

Mendell, J.T. (2005) Nature , 435 , 839-843.[86] Sylvestre, Y., De Guire, V., Querido, E., Mukhopadhyay,

U.K., Bourdeau, V., Major, F., Ferbeyre, G., and Chartrand,P. (2007) J. Biol. Chem. , 282 , 2135-2143.

[87] Welch, C., Chen, Y., and Stallings, R.L. (2007) Oncogene26 , 5017-5022.

[88] le Sage, C., Nagel, R., Egan, D.A., Schrier, M., Mesman, E.,Mangiola, A., Anile, C., Maira, G., Mercatelli, N., Ciafre, S.A.,Farace, M.G., and Agami, R. (2007) Embo. J. , 26 , 3699-3708.

[89] Gillies, J.K., and Lorimer, I.A. (2007) Cell Cycle , 6 , 2005-2009.

[90] Tsantoulis, P.K., and Gorgoulis, V.G. (2005) Eur. J. Cancer 41 , 2403-2414.


13/13


[91] Matsumura, I., Tanaka, H., and Kanakura, Y. (2003) CellCycle , 2 , 333-338.

[92] He, L., Thomson, J.M., Hemann, M.T., Hernando-Monge, E.,Mu, D., Goodson, S., Powers, S., Cordon-Cardo, C., Lowe,S.W., Hannon, G.J., and Hammond, S.M. (2005) Nature ,435 , 828-833.

[93] Hatfield, S.D., Shcherbata, H.R., Fischer, K.A., Nakahara, K.,Carthew, R.W., and Ruohola-Baker, H. (2005) Nature , 435 ,974-978.

[94] Ciafre, S.A., Galardi, S., Mangiola, A., Ferracin, M., Liu,C.G., Sabatino, G., Negrini, M., Maira, G., Croce, C.M., andFarace, M.G. (2005) Biochem. Biophys Res. Commun. , 334 ,1351-1358.

[95] Pallante, P., Visone, R., Ferracin, M., Ferraro, A., Berlingieri ,M.T., Troncone, G., Chiappetta, G., Liu, C.G., Santoro, M.,Negrini, M., Croce, C.M., and Fusco, A. (2006) Endocr.Relat. Cancer , 13 , 497-508.

[96] Lee, E.J., Gusev, Y., Jiang, J., Nuovo, G.J., Lerner, M.R.,Frankel, W.L., Morgan, D.L., Postier, R.G., Brackett, D.J.,

and Schmittgen, T.D. (2007) Expression profiling identifiesmicroRNA signature in pancreatic cancer. Int. J. Cancer 120 , 1046-1054.

[97] Cimmino, A., Calin, G.A., Fabbri, M., Iorio, M.V., Ferracin, M.,Shimizu, M., Wojcik, S.E., Aqeilan, R.I., Zupo, S., Dono, M.,Rassenti, L., Alder, H., Volinia, S., Liu, C.G., Kipps, T.J.,Negrini, M., and Croce, C.M. (2005) Proc. Natl. Acad. Sci.USA , 102 , 13944-13949.

[98] He, L., He, X., Lim, L.P., de Stanchina, E., Xuan, Z., Liang,Y., Xue, W., Zender, L., Magnus, J., Ridzon, D., Jackson,

A.L., Linsley, P.S., Chen, C., Lowe, S.W., Cleary, M.A., andHannon, G.J. (2007) Nature , 447 , 1130-1134.

[99] Bommer, G.T., Gerin, I., Feng, Y., Kaczorowski, A.J., Kuick,R., Love, R.E., Zhai, Y., Giordano, T.J., Qin, Z.S., Moore,B.B., MacDougald, O.A., Cho, K.R., and Fearon, E.R. (2007)Curr. Biol. , 17 , 1298-1307.

[100] Mack, G.S. (2007) Nat. Biotechnol. , 25 , 631-638.

Received: January 31, 2008 Revised: April 18, 2008 Accepted: May 01, 2008

Documents

MicroRNAs in Organogenesis and Disease