FEBS Letters 587 (2013) 1548–1555

journal homepage: www.FEBSLetters .org

MicroRNA profiling during rat ventricular maturation: A role for miR-29ain regulating cardiomyocyte cell cycle re-entry

0014-5793/$36.00 Crown Copyright � 2013 Published by Elsevier B.V. on behalf of Federation of European Biochemical society. All rights reserved.http://dx.doi.org/10.1016/j.febslet.2013.01.075

Abbreviations: miRNA, microRNAs; CCND2, cyclin D2; CDK2, cyclin-dependentkinase 2; P2, postnatal day 2; P4W, 4 weeks of age; GAPDH, glyceraldehyde-3phosphate dehydrogenase; UTR, untranslated regions⇑ Corresponding authors. Address: Department of Cardiac Surgery, Cardiovascu-

lar Institute & Fu Wai Hospital, 167 Beilishi Road, Beijing 100037, China. Fax: +86 1088396050.

E-mail addresses: shengshouhu@yahoo.com (S. Hu), zhengzhe@fuwai.com (Z.Zheng).

Xiaoqing Cao a,b,c, Jue Wang a,b,c,d, Zhenhua Wang a,b,c, Juan Du a,b,c, Xin Yuan a,b,c, Weicong Huang d,Jian Meng a,b,c, Haiyong Gu a,b,c, Yu Nie a,b,c, Bingyang Ji a,b,c, Shengshou Hu a,b,c,⇑, Zhe Zheng a,b,c,⇑a State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Center for Cardiovascular Disease, Chinese Academy of Medical Sciences, Peking Union MedicalCollege, Beijing 100037, Chinab Department of Surgery, Fuwai Hospital & Cardiovascular Institute, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, Chinac Key Laboratory of Cardiac Regenerative Medicine, Ministry of Health, National Center for Cardiovascular Diseases, Beijing 100037, Chinad Department of Thoracic and Cardiovascular Surgery, The First Affiliated Hospital of Wenzhou Medical College, Wenzhou 325000, Zhejiang Province, China

a r t i c l e i n f o

Article history:Received 10 November 2012Revised 19 January 2013Accepted 23 January 2013Available online 12 April 2013

Edited by Tamas Dalmay

Keywords:MicroRNACardiomyocyteProliferationMicroarrayMicroRNA-29a

a b s t r a c t

Recent studies demonstrated that the mammalian heart possesses some capacity to proliferate. Weobserved cardiomyocyte proliferation within 4 weeks of age (P4W) in rats. We found 95 microRNAsthat are differentially expressed in P4W cardiomyocytes. MicroRNA-29a was among the most highlyup-regulated microRNAs in P4W cardiomyocytes. Overexpression of microRNA-29a suppressed theproliferation of H9c2 cell line. MicroRNA-29a inhibition induced cardiomyocytes to proliferate,accelerated the G1/S and G2/M transition, and up-regulated the cell cycle gene expression. CyclinD2 (CCND2) was identified as a direct target of microRNA-29a. These findings indicate that microR-NA-29a is involved in cardiomyocyte proliferation during postnatal development.Crown Copyright � 2013 Published by Elsevier B.V. on behalf of Federation of European Biochemical

society. All rights reserved.

1. Introduction

It has long been thought that mammalian cardiomyocytes exitthe cell cycle soon after birth and become terminally differentiatedin the adult [1]. Recent studies indicated that postnatal mamma-lian cardiomyocytes are still capable of substantial plasticity [2].The capability of the cardiomyocyte to proliferate is lost by 7 daysof age in mouse [3] and a rapid switch from hyperplasia to hyper-trophy in rat cardiomyocyte occurred within 3–5 days after birth[4]. In human heart, the capability to undergo proliferation is lost3–6 months after birth [5]. Although many efforts have been madeto understand the postnatal development and maturation in the

heart [6], the potential mechanisms that regulate postnatal cardio-myocytes proliferation are still poorly understood.

MicroRNAs (miRNAs, miRs) are a class of endogenous evolu-tionarily conserved small (18–22 nucleotides) non-coding RNAs[6,7], which have emerged as pivotal regulators in cell prolifera-tion, differentiation and apoptosis [7,8]. Some differentiation asso-ciated miRNAs have been reported to coordinately restrictoncogene-induced proliferation [9]. miR-29 has displayed thecapacity for posttranscriptional regulation in myoblast [9] andrhabdomyosarcoma [10]. During cardiomyocyte postnatal devel-opment, miRNAs were reported to involve in cardiomyocyte prolif-eration [11,12].

In this study, we first characterized the proliferation pattern ofcardiomyocytes during the postnatal development. Then we inves-tigated the miRNA and mRNA expression profiles in isolatedcardiomyocytes from postnatal day two (P2) and P4W groups.We identified miR-29a as one of the most robustly up-regulatedmiRNAs in P4W cardiomyocytes and found that miR-29a couldsuppress cardiomyocytes proliferation. CCND2 was identified as amiR-29a target which may be involved in the process of cardiomy-ocyte cell cycle and proliferation regulation.

Fig. 1. Immunofluorescence staining of the expression of Ki-67 (green), H3P (green) and troponin T (red) in the heart of rats. (A) Sections from various postnatal stages werestained to label proliferating and mitotic cardiomyocytes (scale bars = 50 lm). Nuclei were stained by DAPI (blue). Quantification of the numbers of Ki-67 (B) and H3P (C)positive cardiomyocytes at sequential postnatal stages is shown. Values are presented as mean ± S.E.M., ⁄,#P-value < 0.05 vs. P1, n = 3.

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2. Materials and methods

2.1. Animals and cell lines

All animal procedures were conducted in accordance with hu-mane animal care standards approved by the Institutional AnimalCare Committee of Fuwai Hospital and Fuwai hospital Ethics Com-mittee (Beijing, China). Ventricular cardiomyocytes were isolatedfrom Sprague–Dawley rats (Animal Laboratory Center of PekingUniversity Health Science Center, China) as described previously[13,14]. H9c2 cells (CRL-1446) were from ATCC (American TypeCulture Collection, MD, USA).

2.2. Rat cardiomyocytes isolation and culture

Left ventricular cardiomyocytes were isolated with enzymaticdissociation of the whole heart from rats on a Langendorff appara-tus as described before [15]. Briefly, cardiomyocytes were isolatedby antegrade collagenase perfusion and purified by differentialcentrifugation as described [16,17]. After standard culture for48 h, cardiomyocyte purity was analyzed on the BD FACSCalibur™flow cytometer (Supplementary Fig. S1A) and immunofluorescence(Supplementary Fig. S1B) as previously described [18]. More than95% of cardiomyocytes were positive for the cardiomyocyte spe-cific marker.

2.3. miRNA microarray, mRNA microarray, and GO-network

Total RNA was extracted from cardiomyocytes using Trizol(Invitrogen, Carlsbad, CA, USA). miRNA and mRNA microarrayhybridizations were performed in triplicate with total RNA usingthe Affymetrix miRNA microarray service (miRNA 2.0) and Rat

Genome 230 2.0 Array (Affymetrix, CA, USA) in CapitalBio (Beijing,China) (Supplementary Materials and methods).

2.4. Cell culture and transfection

H9c2 and cardiomyocytes were cultured and a subcultivationratio of 1:3 was used for further culture before cells researchedthe 70% confluent state [19]. Transfection was performed usingthe siPORT NeoFX transfection agent with miRNA mimics (a small,synthetic, double-stranded RNAs used to mimic endogenous ma-ture miRNAs) or inhibitors (a small, synthetic, single-strandedRNAs used to inhibit mature miRNAs for functioning), accordingto the manufacturer’s instructions (Applied Biosystems, CA, USA)(Supplementary Materials and methods).

2.5. Cell proliferation assay

CellTiter 96� AQueous One Solution Cell Proliferation Assay(MTS, Promega, Madison, WI, USA) was conducted to evaluatethe effect of miRNAs on H9c2 proliferation, following the manufac-turer’s instructions. The absorption was determined at 490 nm on amicroplate reader (Model 680; BioRad, Tokyo, Japan).

2.6. RNA extraction and quantitative reverse transcription-PCR

Total RNA was extracted with Trizol reagent (Invitrogen, Carls-bad, CA, USA). QRT-PCR was performed using an ABI 7300 cycledetection system. MicroRNA Reverse Transcription kit and TaqManprobes (Applied Biosystems) were used for miRNA RT-PCR. Datawere analyzed using the 2�DDCT method [20,21] and the foldchanges of miRNA or mRNA expression were normalized to U6

Fig. 2. MicroRNA expression profiling in cardiomyocytes during postnatal cardiac development. (A) Heap-map depicts miRNAs that are either up-regulated or down-regulated between P2 and P4W cardiomyocytes. Y-Axis represents the normalized arbitary microarray hybridization signal. (Green): lower expression; (red): higherexpression. (B) Total numbers of miRNAs up-regulated (ratio P 2) or down-regulated (60.05) were illustrated in pie. (C) Temporal regulation of six miRNAs in ventriculartissues during postnatal development. Expression levels of each miRNA are normalized to U6 and represented as a fold change relative to P2 ventricular tissue. (D) Temporalregulation of six miRNAs in cardiomyocytes. Each sample was tested in triplicate. Expression levels of each miRNA are normalized to U6 and represented as a fold changerelative to P2 cardiomyocytes. ⁄,#P-value < 0.05 vs. P2, n = 3.

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snRNA or glyceraldehyde-3 phosphate dehydrogenase (GAPDH).Primers are listed in Supplementary Table S1.

2.7. Immunofluorescence staining and analysis

Hearts were isolated from rats and divided into different groups(days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 21, 28). Sections (5 lm thickness)were processed for immunofluorescence according to the standardprocedures [12] (Supplementary Materials and methods).

2.8. Cell cycle analysis

Neonatal cardiomyocytes were cultured for 48 h in 6-wellplates (0.5 � 106 cells per well), synchronized for 24 h and thentransfected with miR-29a mimics or inhibitors. The detailed proce-dures were performed as previously described [22] (Supplemen-tary Materials and methods).

2.9. Western blot analysis

20 lg of lysate was separated by 12.5% SDS–PAGE and trans-ferred to nitrocellulose. Antibodies to cyclin D2 (diluted 1:400 in

TBST) and b-actin (diluted 1:1000 in TBST) were purchased fromAbcam (Cambridge, UK). Secondary antibody was labeled withfar-red-fluorescencent Alexa Fluor 680 dye (Supplementary Mate-rials and methods).

2.10. Luciferase reporter assays

PsiCHECK™-2 based vectors (Promega) were used to constructthe reporters (PMIR-REPORT). The fragment of the 30untranslatedregion (30UTR) of CCND2 mRNA containing the putative (WT) ormutated (MU) miR-29a binding sequence was used to constructthe vectors. The sequences of miR-29a binding site and mutantsites were: WT (50-GAAGAAGAAGATTTT� � �TGGTGCTG-30) and mu-tated (MUT: 50-GAAGAAGAAGATTTT� � �CAACAAGG-30). The miR-29a binding sites were constructed using the XhoI and notI restric-tion sites of PMIR-REPORT. HEK 293T cells (1 � 105) were seededto 24-well and transfected with lipofectamine 2000 (Invitrogen),according to the manufacture’s instructions. After transfection for48 h, relative luciferase measurements were determined with thedual Luciferase Reporter Assay system (Promega, Madison, USA)for the firefly and renilla luciferase activity of triplicate wells inluminescence plate reader.

Fig. 3. miRNA–mRNA anti-correlated sets pathways. Representative network of differentially expressed miRNAs and mRNAs involved in cell cycle (A) and cell proliferation(B) during cardiomyocyte postnatal development. Box nodes represent upregulated miRNA (red), cycle nodes (blue) represent downregulated mRNAs and signal transductionpathway are also involved.

Fig. 4. Effects of six miRNAs on H9c2 proliferating. Overexpression of miR-29ainhibits H9c2 cell proliferation, while downregulation of miR-29a promotesproliferation. The absorbency were divided by the respective absorbance andpresented in percentage, relative to the negative control (n = 4). #P-value < 0.05 vs.pre-miRNA control, ⁄P-value < 0.05 vs. anti-miRNA control.

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2.11. Statistical analysis

All the experiments were repeated at least three times and thedata are shown as mean ± S.E.M. Significance was determined byusing the Student t-test for comparison of two groups and one-way ANOVA for multiple comparison in SPSS 13.0 analysis (SPSSInc, Chicago, IL, USA). P values less than 0.05 were considered sta-tistically significant.

3. Results

3.1. Age-dependent expression of proliferation cardiomyocytes duringpostnatal development in rats

The temporal sequence of cardiomyocytes proliferation wascharacterized. The number of Ki-67 positive cardiomyocytes was

decreased at day 10 (69 ± 9 vs. 114 ± 6 at day 1) (Fig. 1A and B). To-tal mitotic indices determined by H3P were almost threefold lowerthan Ki-67 staining. The number of H3P positive cardiomyocyteswas significantly decreased at day 7 (almost threefold lower,10 ± 3 vs. 32 ± 3 at day 1, Fig. 1A and C). These data suggest thatthe proliferation and mitotic potentials of cardiomyocytes hasgradually disappeared after birth.

3.2. Comparative analysis of miRNA and mRNA profile

The pattern of altered miRNA expression between P2 and P4Wwas significantly distinct (Fig. 2A). 95 miRNAs were differentiallyregulated in P4W group (at least twofold) (SupplementaryTable S2). 7.7% (30/389) of miRNAs displayed increasing expres-sion and 16.7% (65/389) decreasing expression (Fig. 2B). Multiplelarge miRNA families were up-regulated, including the miRNA-29, miRNA-30 and miRNA-133 families. mRNA expression inP4W was also clustered together and separated from P2 (Supple-mentary Fig. S2A and B).

3.3. Correlation between differentially expressed miRNAs and mRNAsprofiling

Among the transcripts that were significantly repressed in P4Wcardiomyocytes, gene ontology cluster analysis revealed a strikingenrichment for genes involved in cell proliferation (SupplementaryFig. S2B). Through the biostatistitical and pathway analysis, wefound two important pathway networks, which were involved incell cycle (Fig. 3A) and cell proliferation (Fig. 3B). The most highlyinterconnected modules included several miRNAs: miR-1, miR-29,miR-30, miR-34a, miR-139, miR-185. Besides, those miRNAs werealso found to be increased in P4W cardiomyocytes (at least two-fold) (Supplementary Table 2). These findings help us to focus oncardiomyocytes proliferation and choose the above six miRNAsfor further investigation. Those miRNAs were validated in heart tis-sues harvested at multiple time points after birth (Fig. 2C). One ofthe most highly upregulated miRNAs between P2 and P4W was

Fig. 5. MiR-29a inhibition induces cell cycle re-entry in neonatal cardiomyocytes. (A) Representative cardiomyocytes stained with Ki-67 (green) and a-actin (red) aftermiRNA transfection in low (scale bars: 50 lm, up) and high (scale bars: 25 lm, down) magnification. Nuclei are stained with DAPI. (B) Quantitative analysis showed moreKi-67 positive cardiomyocytes in the miR-29a inhibition group. (C) qRT-PCR data of CCND2 and CDK2 expression after miRNAs transfection. Expression levels are normalizedto GAPDH. Data are expressed as mean ± S.E.M., P-value < 0.05 for ⁄⁄, ⁄ and #, compared with respective controls. Pc, Pre-control; P29a, Pre-miR29a; Ac, Anta-control; A29a,Anta-miR-29a. Arrow indicates proliferating cardiomyocyte.

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miR-29a. miR-29a expression was also found to be upregulated incardiomyocytes during the same period (Fig. 2D).

3.4. miR-29a inhibits H9c2 cell proliferation

H9c2 cell lines, a rat myoblast cell line derived from embryonichearts, were used in miRNAs functional screening to determine thefunction of miRNA in cardiomyocytes proliferation. FAM™ dye la-belled miRNA and qRT-PCR assays were used to determine thetransfection efficiency. After miRNA mimic and inhibitor transfec-tion, the expression levels of miRNAs increased (>10-fold) and de-creased (<10%), respectively (Supplementary Fig. 3A and B). Theoverexpression of miR-1, miR-34a, miR-30e, miR-139 and miR-185 in H9c2 could not increase cell proliferation 24 h after thetransfection compared with H9c2 transfected with the controls.However, overexpression of miR-29a could significantly suppressthe proliferation of H9c2, while inhibition of miR-29a would pro-mote the proliferation of H9c2 (Fig. 4).

3.5. Effects of miR-29a on cardiomyocytes proliferation

To further investigate whether miR-29a could regulate the pro-liferation of cardiomyocytes, we transfected the neonatal cardio-myocytes with miR-29a and its inhibitor; then we assessed theproliferation by Ki-67 staining. We found that miR-29a overex-pression (P29a) could decrease cardiomyocytes proliferation 48 hafter transfection compared with cells transfected with a controlmimic (Pc) (Fig. 5A). Compared with a control inhibitor (Ac),miR-29a inhibitor (A29a) significantly promoted the proliferationof cardiomyocyte (Fig. 5A). Quantification of Ki-67 positive cardio-

myocytes revealed a threefold increase (14.9 ± 0.3% vs. 4.9 ± 0.3%)after miR-29a inhibitor transfection than the control inhibitor(Fig. 5B). Cell cycle regulators have been implicated in cardiomyo-cytes proliferation. Herein, we detected the expression of two cellcycle-specific proteins, CCND2 and CDK2 in miR-29a transfectedcardiomyocytes. CCND2 and CDK2 gene expression were down-regulated after miR-29a transfection, but up-regulated when trea-ted with miR-29a inhibitors (Fig. 5C and D).

To demonstrate whether miR-29a also increased cardiomyo-cytes mitosis, we stained for histone H3 phosphorylated on serine10 (H3P), a G2/mitosis marker, in miR-29a transfected neonatalcardiomyocytes. We found that miR-29a inhibition resulted in athreefold increase in H3P-positive cardiomyocytes (4.2 ± 0.6% vs.1.5 ± 0.3%) (Fig. 6A and B). To further investigate the antiprolifera-tion effect of miR-29a on cardiomyocyte, we examined the effect ofmiR-29a on cell cycle progression. miR-29a inhibition reduced theamount of cardiomyocytes in G0/G1 phase, but increased the frac-tion of cells resided in S and G2/M phases, in comparison to controlinhibitor (Fig. 6C and D). Taken together, these results suggestedthat miR-29a inhibition could induce cardiomyocytes proliferation.miR-29a alters the proliferation of cardiomyocytes potentiallythrough the modulation of cell-cycle progression.

3.6. miR-29a directly inhibits the expression of CCND2 via its 30UTR

We used the TargetScan (http://www.targetscan.org) to predictthe candidate target genes for miR-29a and found that CCND2 isamong the top predicted targets, with a conserved site for miR-29a (Fig. 7A). Compared with the negative control, the luciferaseactivity for CCND2-WT-transfected cells decreased about 55% in

Fig. 6. MiR-29a inhibition induces mitosis and cell cycle progression in cardiomyocytes. (A) Representative images of cardiomyocyte stained for H3P (green) and a-actin (red)after miRNAs transfection. (B) Quantitative analysis showed more H3P positive cardiomyocytes in the miR-29a inhibition group. (C) Representative images of cell cycleanalysis after miRNAs transfection. (D) Flow cytometry analysis of cardiomyocytes stained with propidium iodide (PI) (n = 3). Data are presented as mean ± S.E.M., P-value < 0.05 for ⁄, #, and $, compared with respective controls. Arrow indicates mitosis cardiomyocyte.

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the miR-29a mimic transfection (Fig. 7B). The luciferase activitieswere inhibited by the overexpression of CCND2 at the presenceof the wild type 30UTR but not by the mutations (Fig. 7B). To fur-ther determine the expression of CCND2 in cardiomyocytes, wecollected cardiac ventricles from P2 and P4W rats and calculatedthe number of CCND2 positive cardiomyocytes. Immunofluores-cence staining suggested that the number of CCND2 positivecardiomyocytes was obviously downregulated in cardiomyocytesin P4W rat (Fig. 7C and D). Furthermore, we determined the CCND2protein expression in cardiomyocytes 48 h after miR-29a transfec-tion by western blot. miR-29a overexpression could significantlysuppressed CCND2 expression, while miR-29a inhibition up-regu-lated CCND2 expression (Fig. 7E and F). These data suggested thatCCND2 is downregulated during the postnatal development andmiR-29a targets CCND2 by binding to its 30UTR and negatively reg-ulated cardiomyocytes proliferation.

4. Discussion

Before cardiomyocytes cease dividing and become terminallydifferentiated, they still have the ability to proliferate [23]. Here,we show that cardiomyocytes proliferative and mitosis activity re-mained constant during the first several days. The transition israther abrupt in postnatal day 10 and day 7 for proliferation andmitosis. This proliferation pattern is not consistent with the earlierobservations in isolated cardiomyocytes, the proliferation capacity

of which was lost within the first 3–4 days [4]. This discrepancymight be due to the possible effects of various factors on the pro-liferation of cardiomyocytes in vitro.

Our combined miRNA/mRNA expression profiles discriminatedseveral important miRNAs involved in the postnatal development.A handful of miRNAs involved in cell cycle and cell proliferationregulation were upregulated in P4W cardiomyocytes. miR-29awas one of the most highly up-regulated miRNAs in P4W. The pre-cise time of miR-29a up-regulation was around postnatal day 10, atime point that coincides with the onset of Ki-67 cardiomyocytesdownregulation.

Mature miR-290s are highly conserved among different species[24]. It has been reported that miR-29a was involved in manyphysiological and pathological processes. In the hematopoiesis sys-tem, miR-29a was found to promote progenitor proliferationthrough expediting G1 to S/G2 transitions [25]. A recent studydetermined miR-29a as an important tumor suppressor in acutemyeloid leukemia by regulating the expression of Ski [26]. Marziet al. [9] found a subset of differentiation associated miRNAs(miR-1, miR-34, miR-22, miR-365, miR-29, miR-145, and Let-7)acted coordinately to regulate cell cycle associated gene expressionin cancer research. Besides, one excellent study [10] showed thatmiR-29 can regulate the expression of CCND2 and other cell cycleregulator to decrease cell proliferation. We found that miR-29awas temporally up-regulated and miR-29a inhibition resulted inneonatal cardiomyocytes entering the cell cycle. These anti-prolif-erative effects of miR-29a on cardiomyocytes appeared to be med-

Fig. 7. MiR-29a directly targets CCND2. (A) Putative binding sites for rat miR-29a in the 30UTR of CCND2 and its seed sequence mutant (red). (B) Rat CCND2 gene 30UTRluciferase activity was inbibited by miR-29a, but had no effect on CCND2 30UTR mutant. (C) Sections from postnatal hearts at P2 and P4W stained with cyclin D2 (CCND2;green) and cardiac troponin T (red) to label cardiomyocytes (scale bars = 50 lm). (D) Quantification of the number of CCND2 cardiomyocytes per field in P2 and P4W hearts isshown. Data are presented as cells/field for six fields per heart for n = 3 samples per group, ⁄P < 0.05 vs. P4W group. (E) Immunoblots for CCND2 in cardiomyocytes transfectedwith miR-29a mimics or inhibitors. (F) CCND2 protein expression is down-regulated by overexpression of miR-29a but is up-regulated by miR-29a inhibitors. ⁄P < 0.05 vs. Pcgroup. &P < 0.05 vs. Ac group. #P < 0.05, compared with the normalized luciferase activity of control cells.

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iated through its negative effects on the cell cycle. It might beimportant for us to consider new strategies to control cardiomyo-cyte proliferation and open new approaches to cardiomyocyte cellcycle regulation.

Limitations still exist in the present study. First, our resultsshowed that inhibition of miR-29a could promote proliferation inneonatal cardiomyocyte. How miR-29a inhibition responds in theneonatal 10 days cardiomyocytes is still unknown, as the prolifer-ation capacity in rat heart is lost around the first 10 days. In thepresent study, we reported the proliferative effects of miR-29ainhibition on cardiomyocytes appeared to be mediated throughthe cell cycle progression. However, these proliferation effects re-ported could also be mediated through other cell cycle arrestcheckpoint. Further efforts should be made to explore other targetsof miR-29a to fully understand the role of miR-29a in cardiomyo-cyte proliferation.

In summary, our study identified a previously unrecognizedrole of miR-29a in determining cardiomyocyte proliferation andindicated that miR-29a inhibition could provide potential thera-peutic usage for cardiac cell cycle regulation.

Acknowledgements

The study was supported by the Natural Science Foundation ofChina (Grant number 81170130) and the National Basic ResearchDevelopment Program in China (Program 973; 2010CB529505).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.febslet.2013.01.075.

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