MicroRNA-125b protects against myocardial ischemia/reperfusion injury via targeting p53-mediated apoptotic signaling and TRAF6

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MicroRNA-125b protects against myocardialischaemia/reperfusion injury via targetingp53-mediated apoptotic signalling and TRAF6Xiaohui Wang1, Tuanzhu Ha1, Jianghuan Zou2, Danyang Ren1, Li Liu3,Xia Zhang1, John Kalbfleisch4, Xiang Gao2, David Williams1, and Chuanfu Li1*

1Department of Surgery, James H. Quillen College of Medicine, East Tennessee State University, Campus Box 70575, Johnson City, TN 37614-0575, USA; 2Animal Model Research Center,Nanjing University, Nanjing 210093, China; 3Department of Geriatrics, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China; and 4Department of Biometry andMedical Computing, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, USA

Received 3 January 2013; revised 7 February 2014; accepted 13 February 2014

Time for primary review: 37 days

Aims The present study examined the role of microRNA-125b (miR-125b) in myocardial ischaemia/reperfusion (I/R) injury.We constructed lentivirus-expressing miR-125b (LmiR-125b) and developed transgenic mice with overexpression ofmiR-125b.

Methodsand results

LmiR-125b was transfected into mouse hearts through the right common carotid artery. Lentivirus vector (LmiR-Con)served as vector control. Untreated mice served as I/R control. Sham operation served as sham control. Seven days aftertransfection, the heartswere subjected to ischaemia (45 min) followed by reperfusion (4 h). Myocardial infarct sizewas ana-lysed by 2,3,5-triphenyltetrazolium chloride staining. In separate experiments, hearts were subjected to ischaemia (45 min)followed by reperfusion for up to 7 days.Cardiac functionwasmeasured byechocardiographybefore, aswell as 3 and 7 daysafter myocardial I/R. Increased expression of miR-125b significantly decreased I/R-induced myocardial infarct size by 60%and prevented I/R-induced decreases in ejection fraction (EF%) and fractional shortening (%FS). Transgenic mice withoverexpression of miR-125b also showed the protection against myocardial I/R injury. Increased expression of miR-125battenuated I/R-induced myocardial apoptosis and caspase-3/7 and -8 activities. Western blot showed that increasedexpression of miR-125b suppresses p53 and Bak1 expression in the myocardium. In addition, transfection of LmiR-125bdecreased the levels of TNF receptor-associated factor 6 (TRAF6) and prevented I/R-induced NF-kB activation.

Conclusion miR-125 protects the myocardium from I/R injury by preventing p53-mediated apoptotic signalling and suppressingTRAF6-mediated NF-kB activation.

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -Keywords MicroRNA-125b † Myocardial I/R † NF-kB † Apoptosis † p53

1. IntroductionIt has well been documented that activation of NF-kB mediated byToll-like receptor/interleukin-1 receptor (TLR/IL-1R) contributes tomyocardial ischaemia/reperfusion (I/R) injury.1 –4 Inhibition of NF-kB-binding activity has been shown to protect against myocardial I/Rinjury.2 –6 The protective effects involve inhibition of innate immuneand inflammatory responses and attenuated I/R-induced cardiacmyocyte apoptosis.2– 6 However, the mechanisms by which inhibitionof NF-kB-binding activity decreases innate immune and inflammatoryresponses and attenuates cardiac myocyte apoptosis during myocardialI/R are still unclear.

Recent studies have demonstrated that activation of NF-kB regulatesmicroRNA expression which, in turn, negatively regulates NF-kB-binding activity,7,8 thereby decreasing innate immune and inflammatoryresponses.9–11 MicroRNAs (miRs) are 21–23 nucleotide non-codingRNA molecules and have been identified as novel regulators of gene ex-pression at the post-transcriptional level by binding to target messengerRNAs.9–14 miRshavebeendemonstratedtoplayacritical role in thenega-tive regulation of innate immune and inflammatory responses by regula-tion of NF-kB-binding activity.9–11 Importantly, recent studies haveshown that NF-kBactivation regulates theexpressionofmiRs,7,8 includingmiR-146, miR-155, and miR-21, etc., while these miRs, in turn, down-regulate NF-kB-binding activity. miR-21 has been demonstrated to play

* Corresponding author. Tel: +1 423 439 6349; fax: +1 423 439 6259. Email: [email protected]

Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2014. For permissions please email: [email protected].

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a protective role in myocardial I/R injury.15 We have reported thatincreased expression of miR-146a significantly decreases myocardialinfarct size and attenuates I/R-induced cardiac dysfunction via down-regulation of NF-kB activation by targeting Interleukin-1 receptorassociatedkinase1andTNFreceptor-associated factor6 (TRAF6).16 Col-lectively, the data suggest that the miRs that regulate TLR/IL-R1-mediatedNF-kB activation may be a new approach for the management andtreatment of myocardial I/R injury.

MicroRNA-125b (miR-125b) is a homologue of lin-4, which is the firstmiR discovered and an important regulator of developmental timing inCaenorhabditis elegans.17 Recent studies have shown that activation ofNF-kB decreases the expression of miR-125b.18,19 Tili et al. reportedthat the treatment of Raw 264.7 cell with lipopolysaccharide (LPS), aTLR4 ligand, suppresses the expression of miR-125b,18 while miR-125bsuppresses TNF-a expression by targeting the 3′-untranslated region ofTNF-a mRNA.18,19 miR-125b has been reported to play a role in down-regulation of apoptosis by repressing p53 and Bak-1.20,21 p53 is a tumoursuppressor protein that plays a critical role in regulating cell cycle andapoptosis in response to hypoxia and ischaemic stress.22,23 Inhibition ofp53-mediated apoptotic signalling significantly reduces I/R-induced myo-cardial injury.24 We have reported that increased expression of miR-125bin macrophages attenuates hypoxia/reoxygenation (H/R)-induced cellinjury.25 However, whether miR-125b serves a protective role inmyocardial I/R injury in vivo has not been investigated. miR-125b hasbeen shown to target TNF-a26 and inhibit p53-mediated apoptoticsignalling,27 therefore, it is possible that miR-125b serves a protectiverole in myocardial I/R injury.

In the present study, we examined the role of miR-125b in myocardialI/R injury. We observed that increased expression of miR-125b in themyocardium significantly decreases myocardial infarct size and preventsI/R-induced cardiac dysfunction. The mechanisms involve the inhibitionof I/R-induced activation of NF-kB and the prevention of I/R-activatedp53-mediated apoptotic signalling in the myocardium.

2. Methods

2.1 AnimalsMale wild-type (WT) C57BL/6J mice were obtained from Jackson Labora-tory. The experiments outlined in this manuscript conform to the Guidefor the Care and Use of Laboratory Animals published by the National Insti-tutes of Health (NIH Publication, 8th Edition, 2011). The animal care andexperimental protocols were approved by the ETSU Committee onAnimal Care.

2.2 qPCR assay of miRsmiRs were isolated using the mirVanaTM miR isolation kit (Ambion)16,25

(see Supplementary material online, Methods).

2.3 Construction of miR-125b intolentivirus-expressing systemmiR-125b, mature sequence mmu-miR-125b-5p (MIMAT0000136), wasconstructed into lentivirus expression vector using a lentivirus-expressingsystem (Invitrogen Corporation) as described previously16,25 (see Supple-mentary material online, Methods).

2.4 Transgenic miceTransgenic (Tg) mice with overexpression of miR-125b were developedwith C57BL/6J background (see Supplementary material online, Methods).

2.5 In vitro experimentsThe H9C2 rat cardiomyoblasts were obtained from the American TypeCulture Collection (Rockville, MD, USA) and were maintained in Dulbecco’sModified Eagle’s Medium (DMEM) supplemented 10% foetal bovine serumunder 5% CO2 at 378C.28 The cells were plated in six well plates at 1 ×105 cells/well. The cells were transfected with lentivirus-expressingmiR-125b (LmiR-125b) or lentivirus-expressing vector that served ascontrol (LmiR-con). The lentivirus-expressing vector contains a non-sensemiR sequence that allows formation of a pre-miRNA hairpin predicatednot to target any known vertebrate gene (Invitrogen Corporation). Stablytransfected cells were selected using a blasticidin-resistant marker. Thecells were subjected to hypoxia for 2 h followed by reoxygenation (H/R)25

for 24 h. The cells that were not subjected to H/R served as control (nor-moxia). There were three independent experiments in each group. Thecells were harvested at 24 h for isolation of cellular protein.

In separate experiments, adult cardiac myocytes were isolated from ninemale mice, as described previously.29 The cells were transfected withmiR-125b, miR-scrambled control (miR-con), or anti-miR-125b, respective-ly, carried by exosomes that were isolated from bone marrow stromal cells(BMSCs)30 (see Supplementary material online, Methods). The cardiac myo-cytes were subjected to hypoxia (2 h) followed by reoxygenation for 24 h.Cardiac myocytes were harvested for analysis of the effect of miR-125b onH/R-induced cardiac myocyte injury.

2.6 miR microarrayCardiac myocytes were isolated from three adult mice29 and subjected tohypoxia (2 h) followed by reoxygenation (24 h) (see Supplementary mater-ial online). The cells were harvested and total RNA was isolated for miRmicroarray analysis (see Supplementary material online, Methods).

2.7 In vivo transfection of LmiR-125b intomouse heartsLentivirus-expressing miR-125 or miR-125b mimics were delivered into themyocardium of mice, as described previously16,31 (see Supplementarymaterial online, Methods).

2.8 Induction of myocardial I/R injuryMyocardial I/R injury was induced 7 days after transfection of LmiR-125b orLmi-con, as described previously2,3,28 (see Supplementary material online,Methods).

2.9 In situ apoptosis assayMyocardial apoptosis was examined, as described previously,2,3,28,32 usingthe in situ cell death detection kit (Roche, USA). Three slides from eachblock were evaluated for the percentage of apoptotic cells and four fieldson each slide were examined at the border areas using a defined rectangularfield area with 20× magnification. A total of 100 nuclei were accounted.Numbers of apoptotic cardiac myocytes are presented as the percentageof total cells counted.

2.10 Measurement of cell viability and LDDHactivityCell viability was assessed by measuring mitochondrial dehydrogenase activ-ity using the MTT assay kit (Sigma). Cell injury was assessed by measurementof lactate dehydrogenase (LDH) activity in culture medium using a commer-cial kit (Cytotoxicity Detection Kit, Sigma).

2.11 Western blotWestern blot was performed as described previously.2,3,28 The primaryantibodies (anti-Fas, anti-p-53, anti-Bax, Bak-1, and TRAF6) and peroxidase-conjugated secondary antibody were purchased from Cell Signaling Tech-nology, Inc. The signals were quantified using the G:Box gel imagingsystem by Syngene (Syngene, Fredrick, MD, USA).

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2.12 Electrophoretic mobility shift assay(EMSA)Nuclear proteins were isolated from heart samples as previouslydescribed.2,3,28 NF-kB-binding activity was measured using a LightShiftChemiluminescent EMSA kit (Thermo Fisher Scientific, Waltham, MA,USA).

2.13 Caspase activityCaspase 3/7 and -8 activities in heart tissues were measured as describedpreviously33 using a Caspase-Glo assay kit (Promega).

2.14 ELISA for cytokine assayThe levels of cytokines (TNF-a and IL-1b) were measured by ELISA usingOptEIA cytokine kits according to instructions provided by the manufacture(BD Biosciences).

2.15 Infiltration of neutrophils into themyocardiumNeutrophil accumulation in heart tissues was examined by staining with ananti-neutrophil marker antibody (NIMP-R14, Santa Cruz Biotechnology)as described previously34 (see Supplementary material online, Methods).

2.16 Statistical analysisThe data are expressed as mean+ SD. Comparisons of data betweengroups were made using one-way analysis of variance (ANOVA), andTukey’s procedure for multiple-range tests was performed. A P-value,0.05 was considered to be significant.

3. Results

3.1 H/R decreased miR-125b expression viaNF-kB activation in H9C2 cardiomyoblastsWe examined the effect of H/R on the expression of miR-125b in H9C2cardiomyoblasts. As shown in Figure 1A, hypoxia (2 h) followed by reox-ygenation (24 h) resulted in decreases in the levels of miR-125b by 33%compared with the normoxic control. To investigate whether miR-125bexpression is regulated by NF-kB activation, we measured NF-kB-binding activity following H/R. Figure 1B shows that H/R inducedincreases in NF-kB-binding activity by 27% compared with the normoxiccontrol. The treatment of the cells with an antioxidant, pyrrolidinedithiocarbamate (PDTC), which has been shown to inhibit NF-kB acti-vation,35 significantly increases the expression of miR-125b in normoxiccontrol and in H/R cells, when compared with the untreated normoxiaand H/R groups, respectively. Administration of PDTC also significantlydecreased NF-kB-binding activity following H/R.

LPS is a strong stimulator of NF-kB activation.36 Figure 1C shows thatLPS stimulation significantly induced decreases in miR-125b expressionin H9C2 cells. However, treatment of the cells with antioxidants, PDTCor N-acetyl cysteine (NAC), prevented LPS-mediated suppression ofmiR-125b expression. Collectively, the data suggest that miR-125b ex-pression during H/R is regulated by NF-kB activation.

We also analysed the effect of H/R on miR expression in adult cardiacmyocytes. miR array showed that 43 miRNAs were differentiallyexpressed in the cardiac myocytes after H/R at a false discovery rateof 0.05, when compared with non-H/R cells (normoxia) (see Supple-mentary material online, Tables S1 and S2).

Figure 1 H/R decreases miR-125b expression and increasedNF-kB-binding activity in H9C2 cells. H9C2 cells were treated withor without PDTC 15 min prior to hypoxia (2 h) followed by reoxy-genation (24 h). Cells were harvested. miRs were isolated fromharvested cells and miR-125b expression was examined by quantitativepolymerase chain reaction (qPCR). Nuclear proteins were isolated foranalysis of NF-kB-binding activity. H/R decreases the expression ofmiR-125b (A, P ¼ 0.001) and increases NF-kB-binding activity (B,P ¼ , 0.001). LPS treatment decreases the expression of miR-125b(C, P ¼ 0.006). H9C2 cells were treated with PDTC or NAC 15 minbefore LPS stimulation (24 h). The levels of miR-125b were measuredby qPCR. There were three independent experiments in each group.*P , 0.05 compared with indicated groups.

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3.2 Increased expression of miR-125battenuated H/R-induced cell injuryin H9C2 cardiomyoblastsTodeterminewhether miR-125bplays a role in the protection againstH/R-induced cell injury, we generated stably transfected H9C2 cells withLmiR-125b or LmiR-control (LmiR-Con). The stably transfected cellswere subjected to hypoxia (2 h) followed by reoxygenation (24 h).Untransfected H9C2 cells served as control. As shown in Figure 2A,transfection of H9C2 cells with LmiR-125b significantly increased thelevels of miR-125b in the cells. Figure 2B shows that H/R inducedincreases in LDH activity by 5.0-fold compared with non-H/R (nor-moxia) groups. However, H/R-induced LDH activity in H9C2 cellswas significantly attenuated by LmiR-125b transfection. LmiR-Contransfection did not affect H/R-induced LDH activity in H9C2 cells.Figure 2C shows that H/R markedly decreased cell viability (51%),compared with untreated normoxic cells. However, H/R-induced de-crease in cell viability was significantly attenuated by LmiR-125b trans-fection, when compared with untreated H/R cells. Transfection ofLmiR-con did not alter H/R-decreased cell viability.

Similarly, increased expression of miR-125b in isolated adult cardiacmyocytes significantly attenuated H/R-induced cell injury (Figure 2D–F). In addition, inhibition of miR-125b expression in adult cardiac myo-cytes increased the susceptible to H/R-induced cell injury (Figure 2D–F).

We also examined the effect of LmiR-125b transfection on H/R-induced caspase-3 and -8 activities in H9C2 cells. Figure 2G showsthat H/R induced caspase-3/7 activity by 79.8% and caspase-8 by44.2%, when compared with normoxic cells. However, increased ex-pression of miR-125b markedly attenuated H/R-increased caspase-3/7and -8 activities. There was no significant difference in caspase-3/7 and-8 activities between LmiR-Con H/R cells and untreated H/R cells. Thedata suggest that increased expression of miR-125b plays a protectiverole in H/R-induced cellular injury.

3.3 miR-125b suppresses p53 and Bak-1expression in H9C2 cardiomyoblastsTo understand the mechanisms by which overexpression of miR-125battenuated H/R-induced caspase-3/7 and -8 activities, we examined theeffectofmiR-125bonp53andBak-1expression inH9C2cardiomyoblastsin the presence and absence of H/R. Figure 3 shows that the levels of p53(A) and Bak-1 (B) were markedly lower in LmiR-125b-transfected nor-moxic cells than in normoxic control cells. H/R increased the levels ofp53 (64.7%) and Bak-1 (91.3%) compared with normoxic control cells.The levels of p53 and Bak-1 in LmiR-125b-transfected cells were alsoincreased following H/R stimulation. However, the levels of p53 andBak-1 in LmiR-125b H/R cells were significantly lower by 49.3 and42.5% compared with untransfected H/R cells and also were comparablewith the normoxic control group. LmiR-Con transfection did not alterH/R-induced increases in p53 and Bak-1 expression.

Increased expression of miR-125b in isolated adult cardiac myocytesinhibited H/R-increased expression of p53 and Bak1. In contrast,inhibition of miR-125b expression in cardiac myocytes increased ex-pression of p53 and Bak1 (Figure 3C and D).

H/R also induced increases in TNF-a production in the cardiomyo-blasts compared with normoxic control (Figure 3E). TNF-a interactswith TNF receptors (TNFRs), resulting in activation of extrinsic apop-totic signalling.37 However, increased expression of miR-125b preventsH/R-induced increases in TNF-a production (Figure 3E).

3.4 In vivo increased expression of miR-125bdecreased infarct size and improved cardiacfunction following myocardial I/RTo examine whether increased expression of miR-125b will induceprotection against myocardial I/R injury, we transfected mouse heartswith LmiR-125b or LmiR-Con before the hearts were subjected to I/R.Figure 4A shows that the green fluorescent protein (GFP) that is carriedby LmiR-125b or LmiR-Con is mainly expressed in the myocardium.qPCR data showed that the levels of miR-125b in LmiR-125b-transfectedhearts were significantly increased by 8.2-fold compared with LmiR-Con-transfected hearts (Figure 4B). Figure 4C shows that I/R inducedsignificant injury as denoted by infarct size in untreated hearts. In contrast,infarct size was significantly reduced (60%) following I/R in LmiR-125b-transfected mice compared with the untreated I/R group.LmiR-Con transfection did not alter I/R-induced myocardial infarct size.

We also examined the effect of increased expression of miR-125b oncardiac function followingmyocardial I/R. As shown in Figure 4D, ejectionfraction (EF%) and fractional shortening (%FS) in untreated I/R heartswere significantly reduced by 39.6 and 44.6% on Day 3 and by 26.8and 32.4% on Day 7 after myocardial I/R compared with baseline.However, I/R-induced cardiac dysfunction was prevented byLmiR-125b transfection. EF% and %FS values in LmiR-125b-transfectedmicewere not significantly decreased at 3 and 7 days after myocardial I/Rcompared with LmiR-125b baseline. Transfection of miR-Con did notalter I/R-induced cardiac dysfunction.

3.5 I/R-induced myocardial apoptosis wasattenuated by LmiR-125b transfectionCardiac myocyte apoptosis contributes to myocardial I/R injury.38 Weexamined the effect of increased expression of miR-125b on I/R-induced myocardial apoptosis. Figure 5A shows that I/R markedlyinduced myocardial apoptosis compared with sham control. InLmiR-125b-transfected mice, I/R-induced myocardial apoptosis was sig-nificantly attenuated, when compared with the untreated I/R group(12.0+2.87 vs. 28.5+1.72%). LmiR-Con transfection did not affectI/R-induced myocardial apoptosis.

Figure 5B shows that I/R increased caspase-3/7 (31.4%) and caspase-8(40.5%) activities in the myocardium compared with sham control.However, increased expression of miR-125b prevented I/R-inducedcaspase-3/7 and -8 activities compared with the I/R group. There wasno significant difference in caspase-3/7 and -8 activities betweenLmiR-Con I/R mice and the untreated I/R group.

3.6 Transfection of LmiR-125b preventedthe increase in p53, Bak-1, Bax, and Fas levelsin the myocardium following I/RTo determine the mechanisms by which increased expression ofmiR-125b-attenuated I/R-induced myocardial apoptosis, we examinedthe levels of pro-apoptotic effectors including p53, Bak-1, Bax, and Fasin the myocardium. As shown in Figure 5C, I/R increased the levels ofp53 by 94%, Bak-1 by 72%, Bax by 90%, and Fas by 64.5%, respectively,compared with sham control. LmiR-Con transfection did not alter thelevels of pro-apoptotic effectors in the myocardium. However,increased expression of miR-125b prevented the increases in p53,Bak-1, Bax, and Fas levels in the myocardium following I/R. Both p53and Bak-1 levels in LmiR-125b-transfected hearts were markedlylower than in the untreated sham control.

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3.7 Increased expression of miR-125bdecreased TRAF6 expression and attenuatedneutrophil infiltration in the myocardiumActivation of TLR-mediated NF-kB signalling contributes to myocardialI/R injury by promoting the inflammatory responses.2 –4 We examined

the effect of LmiR-125b transfection on NF-kB activation during myo-cardial I/R. TRAF6 is an important effector in the TLR-mediatedNF-kB activation pathway.36 Figure 6A shows that the levels of TRAF6in LmiR-125b-transfected sham and I/R hearts were significantly lowerthan in untransfected sham and I/R groups. Transfection of LmiR-Con

Figure 2 Increased expression of miR-125b attenuates H/R-induced cell injury and cell death in H9C2 cardiomyoblasts and adult cardiac myocytes.Stably transfected H9C2 cells with LmiR-125b or LmiR-con were subjected to hypoxia (2 h) followed by reoxygenation (24 h). H9C2 cells that werenot transfected served as control. (A) Increased levels of miR-125b after transfection of cells with LmiR-125b. Overexpression of miR-125b decreasedLDH activity (B, P ¼ , 0.001), increased cell viability (C, P ¼ , 0.001), and attenuated caspase-3/7 and -8 activities (G, P ¼ 0.003) following H/R.There were three independent experiments in each group. *P , 0.05 compared with indicated groups. #P , 0.05 compared with the control H/Rgroup. Cardiac myocytes were isolated from adult male mice and transfected with miR-125b mimics, anti-miR-125b mimics, or miR-scrambled control(miR-con) that were carried by BMSC-derived exosomes, respectively. Untransfected cells served as control. Twenty-four hours after transfection, thecells were subjected to hypoxia (2 h) followed by reoxygenation (24 h). (D) Transfection of cardiac myocytes with BMSC-derived exosomes thatloaded with miR-125b mimics significantly increased the levels of miR-125b in cardiac myocytes (N ¼ 3). Increased expression of miR-125b attenuatedH/R-increased LDH activity (E) and H/R-decreased cell viability (F ). Inhibition of miR-125b expression by transfection of anti-miR-125b mimics increasedthe susceptibility to H/R-induced cell injury (E) and H/R-decreased cell viability (F ). A new cardiac myocyte isolation was performed for each independentexperiment (n ¼ 3, performed in triplicate). *P , 0.05 compared with indicated groups. #P , 0.05 compared with the control H/R group.

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Figure 3 Increased expression of miR-125b decreased p53 and Bak-1 expression in H9C2 cells and adult cardiac myocytes. LmiR-125b or LmiR-constably transfected H9C2 cells were subjected to hypoxia (2 h) followed by reoxygenation (24 h). Untransfected H9C2 cells served as control. Overexpres-sionofmiR-125bsuppresses theexpressionofp53 (A,P ¼ , 0.001) andBak-1 (B,P ¼ , 0.001)expression. (E,P ¼ 0.003)Thesupernatantswereharvestedfor the analysis ofTNF-a. *P , 0.05comparedwith indicated groups. #P , 0.05comparedwithnormoxic groups.Cardiac myocyteswere isolated fromadultmale mice and transfected with miR-125b mimics, anti-miR-125b mimics, or miR-scrambled controls (miR-con) that were carried by BMSC-derived exo-somes, respectively. Untransfected cells served as control. Twenty-four hours after transfection, the cells were subjected to hypoxia (2 h) followed by reox-ygenation (24 h). Increased expression of miR-125b inhibited expression of p53 (C) and Bak1 (D) in the presence and absence of H/R. Inhibition of miR-125bexpression by transfection of anti-miR-125b mimics increased the expression of p53 (C) and Bak1 (D) in the presence and absence of H/R. Cardiac myocyteswere isolated from four hearts. *P , 0.05 compared with indicated groups. #P , 0.05 compared with non-transfected control groups.

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Figure 4 LmiR-125b transfection protects the myocardium from I/R injury. Mouse hearts were transfected with either LmiR-125b or LmiR-con, respect-ively. Seven days after transfection, hearts were harvested and sectioned. (A) GFP expression was viewed using a fluorescent microscope (green) and GFPexpression was confirmed by staining with anti-GFP antibody (red). (B) Increased expression of miR-125b in the myocardium 7 days after LmiR-125b trans-fection. (C) Increased expression of miR-125b by transfection of LmiR-125b for 7 days reduced myocardial infarct size (P ¼ , 0.001). The infarct area(white) and the area at risk (red + white) from each section were measured using an image analyzer. Ratios of risk area vs. left ventricle area (RA/LV)and infarct area vs. risk area (IA/RA) were calculated and are presented in the graphs. Photographs of representative heart sections are shown above.(D) Increased expression of miR-125b by transfection of LmiR-125b for 7 days attenuated I/R-induced cardiac dysfunction (P ¼ , 0.001). Cardiac functionwas examined by echocardiography before (baseline), 3 and 7 days after I/R. There were 6–8 mice in each group. *P , 0.05 compared with indicatedgroups.

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Figure 5 Transfection of LmiR-125b attenuates I/R-induced myocardial apoptosis. Mice were transfected with LmiR-125b or LmiR-control for 7 daysbefore the hearts were subjected to myocardial ischaemia (45 min) followed by reperfusion (4 h). (A) Myocardial apoptosis were examined by the TUNELassay in the heart sections. DAPI stains nucleus (blue colour) and TUNEL-positive cells show green fluorescence. The bar graph shows the per cent apop-totic cells (P ¼ , 0.001). (B) Increased expression of miR-125b attenuated I/R-induced caspase-3/7 and -8 activities in the myocardium (P ¼ 0.002).(C) Increased expression of miR-125b prevents I/R-increased p53, Bak-1, Bax, and FasL levels in the myocardium (P ¼ , 0.001). There were five micein each group. *P , 0.05 compared with indicated groups. #P , 0.05 compared with the untreated I/R group.

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did not affect myocardial TRAF6 levels in the presence and absenceof I/R. I/R significantly induced NF-kB-binding activity comparedwith the sham control (Figure 6B). In contrast, increased expression ofmiR-125b prevented I/R-induced NF-kB-binding activity. There wasno significant difference in NF-kB-binding activity between the untrans-fected I/R group and LmiR-Con-transfected I/R mice. In addition, wehave observed that increased expression of miR-125b significantly atte-nuated I/R-induced neutrophil infiltration into the myocardium (seeSupplementary material online, Figure S1).

3.8 Transgenic mice with overexpressionof miR-125b protect against myocardialI/R injuryTo confirm our observation, we developed Tg mice with overexpres-sion of miR-125b. Figure 7A shows increased expression of miR-125bin the myocardium of Tg mice. Tg and WT mice were subjected to I/R.Figure 7B shows that I/R markedly induced myocardial infarct size inWT mice. However, infarct size in Tg mice was significantly reducedby 50% compared with WT I/R mice. In contrast, inhibition of

miR-125b by transfection of anti-miR-125b into the myocardium ofWT mice resulted in susceptible to I/R-induced injury. Infarct size wasmarkedly greater than in the untreated I/R group (Figure 7B). Tg micealso showed the prevention of cardiac dysfunction following I/R. Asshown in Figure 7C, both EF% and %FS values were significantly decreasedby 42.6 and 48.8% in WT mice after myocardial I/R. However, I/R-inducedcardiac dysfunction was prevented in Tg I/R mice.

4. DiscussionThe present study demonstrates that increased expression of miR-125bin the myocardium significantly decreased myocardial infarct size andprevented I/R-induced cardiac dysfunction. To the best of our knowl-edge, this is the first report that miR-125b exerts a protective role inmyocardial I/R injury. Inhibition of miR-125b resulted in significant sus-ceptibility to I/R-induced myocardial injury. The mechanisms by whichmiR-125b protects against myocardial I/R injury involve the preventionof I/R-induced NF-kB activation and p53-mediated apoptotic signalling.Our data suggest that modulation of miR-125b may be a useful strategyfor the induction of cardioprotection.

We have previously reported that scavenger receptor type A (SR-A)deficiency attenuates myocardial I/R injury.25 Interestingly, we haveobserved that the levels of miR-125b in SR-A deficient mice are signifi-cantly greater than in WT mice.25 Transfection of macrophages withmiR-125b mimics attenuated H/R-induced cell injury,25 indicating thatmiR-125b may play a protective role in myocardial I/R. Indeed, we dem-onstrate in the present study that increased expression of miR-125battenuates myocardial I/R injury and prevents I/R-induced cardiacdysfunction. In contrast, inhibition of miR-125b expression resulted inmore injury of the myocardium following I/R. miR-125b is highly con-served among mammals, vertebrates, and nematodes.8 miR-125b isexpressed in several organs, including brain, heart, lung, spleen, andskeletal muscle.39 Recent studies have reported that miR-125b expres-sion is regulated by NF-kBactivation,40 while miR-125bacts as a negativeregulator of the NF-kB pathway by reducing the levels of tumour necro-sis factor18,41 and by enhancing the stability of the NF-kB inhibitorNKIRAS2 (KBRAS2).41 We have observed that hypoxia followed byreoxygenation decreased the expression of miR-125b and increasedNF-kB-binding activity in H9C2 cells. Inhibition of NF-kB-binding activ-ity by anti-oxidant, PDTC, which has been reported to inhibit NF-kBactivation,35 prevents H/R-induced decreases in miR-125b expression.The data indicate that H/R-induced decreases in the expression ofmiR-125b are mediated, in part, by NF-kB activation.

However,our in vivodata showthat increasedexpressionofmiR-125b inthe myocardium significantly prevents I/R-induced myocardialNF-kB-binding activity. Importantly, we have observed that the expressionof TRAF6 was suppressed by transfection of LmiR-125b. TRAF6 plays acrucial role in the induction of inflammatory responses via activation ofIkB kinases, leading to NF-kB nuclear translocation and activation.36,42

NF-kB activation regulates inflammatory cytokine expression.36,42 Wehave observed that transfection of H9C2 cells with LmiR-125b preventsH/R-induced increases in TNF-a production. Our observation is consist-ent with previous reports, showing that TNF-a mRNA is the target formiR-125b.18,19 Androulidaki et al.19 and Tili et al.18 reported that LPS, aTLR4 ligand, suppresses macrophage expression of miR-125b, whilemiR-125b negatively regulates TNF-a expression.18,19 Collectively, ourdata support the concept that NF-kB activation regulates miR-125b ex-pression during H/R, while increased expression of miR-125b negativelyregulates NF-kB activation. Therefore, inhibition of NF-kB activation by

Figure 6 LmiR-125b transfection decreases TRAF6 expression andprevents I/R-induced NF-kB binding activity. Mice were transfectedwithLmiR-125borLmiR-control for 7daysbefore theheartswere sub-jected to myocardial ischaemia (45 min) followed by reperfusion (4 h).Increased expression of miR-125b suppresses TRAF6 expression (A,P ¼ , 0.001) and NF-kB-binding activity (B, P ¼ , 0.001). Therewere five mice in each group. *P , 0.05 compared with indicatedgroups. #P , 0.05 compared with the control sham or control I/Rgroup.

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targeting TRAF6 in the myocardium could be an important mechanism formiR-125b protection against myocardial I/R injury. In addition, increasedexpression of miR-125b also down-regulated systemic inflammatoryresponses following myocardial I/R. We also observed that transfectionof LmiR-125b significantly attenuated I/R-induced infiltration of neutro-phils into the myocardium.

Myocardial apoptosis contributes to myocardial I/R injury.38 We haveobserved that increasedexpression of miR-125b significantly attenuatedI/R-induced myocardial apoptosis. The mechanisms by which miR-125battenuated I/R-induced myocardial apoptosis involve suppression ofp53-mediated apoptotic signalling in the myocardium following myocar-dial I/R. p53 is a tumour suppressor protein that regulates and interactswith the apoptotic protein Bax. Bax acts as an antagonist against anti-apoptotic Bcl2, resulting in increases in mitochondrial membrane

permeability and the release of cytochrome c.22,23 In addition, apoptoticlipid products serve as chemokines that promote infiltration of inflam-matory cells into the myocardium during myocardial I/R.22,23 Therefore,p53 is a critical pro-apoptotic effector for myocardial apoptosis duringmyocardial I/R injury.24 Inhibition of p53 expression is an important ap-proach for attenuation of myocardial I/R injury.24 Our in vitro datashowed that increased expression of miR-125b in H9C2 cells andadult cardiac myocytes suppressed the expression of p53 and Bak-1 inboth non-H/R and H/R cells, suggesting that miR-125b targets bothp53 and Bak-1 in cardiomyoblasts. In vivo data demonstrated that trans-fection of the myocardium with LmiR-125b prevents I/R-inducedincreases in the expression of p53 and Bak-1 in the myocardium. In add-ition, increased expression of miR-125b also prevents I/R-inducedincreases in Fas levels and caspase-3/7 and -8 activities in the

Figure 7 Reduced myocardial infarct size and attenuated cardiac dysfunction in the transgenic mice with overexpression of miR-125b. (A) Increasedlevels of miR-125b in the myocardium of Tg mice. (B) Tg mice show decreases in myocardial infarct size. Tg and WT mice that were treated with andwithout anti-miR-125b or anti-miR-scrambled control were subjected to ischaemia (45 min) followed by reperfusion (24 h). The hearts were harvestedand infarct size was analysed. Ratios of risk area vs. left ventricle area (RA/LV) and infarct area vs. risk area (IA/RA) were calculated and are presented in thegraphs. (C) Tg mice show the prevention of I/R-induced cardiac dysfunction (P ¼ , 0.001). Cardiac function was examined by echocardiography before(baseline) and 24 h after I/R. There were five mice in each group. *P , 0.05 compared with indicated groups.

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myocardium. The data indicate that anti-apoptotic properties ofmiR-125b include inhibition of both extrinsic and intrinsic apoptotic sig-nalling pathways during myocardial I/R.

In summary, we demonstrated in the present study that miR-125bplays a significant role in the protection against myocardial I/R injury.The mechanisms involve the inhibition of NF-kB activation as well asTNF-a production and the prevention of p53-mediated apoptotic sig-nalling following myocardial I/R. Our data suggest that miR-125b is atarget for the induction of protection against myocardial I/R injury.

Supplementary materialSupplementary material is available at Cardiovascular Research online.

Conflict of interest: none declared.

FundingThis work was supported by NIH (HL071837 to C.L., GM083016 to C.L. andD.L.W., GM53522 to D.L.W.).

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