miR-139-5p inhibits isoproterenol-induced cardiac hypertrophy by targetting c-Jun

Hypertrophic cardiomyopathy (HCM) is a serious monogenic disease characterized by cardiac hypertrophy, fibrosis, sudden cardiac death, and heart failure. Previously, we identified that miR-139-5p was down-regulated in HCM patients. However, the regulatory effects of miR-139-5p remain unclear. Thus, we investigated the role of miR-139-5p in the regulation of cardiac hypertrophy. The expression of miR-139-5p in left ventricular tissues in HCM patients and mice subjected to transverse aortic constriction (TAC) was significantly down-regulated. Knockdown of miR-139-5p expression in neonatal rat cardiomyocytes (NRCMs) induced cardiomyocyte enlargement and increased atrial natriuretic polypeptide (ANP) expression. Overexpression of miR-139-5p antagonized isoproterenol (ISO)-induced cardiomyocyte enlargement and ANP/brain natriuretic peptide (BNP) up-regulation. More importantly, we found that c-Jun expression was inhibited by miR-139-5p in NRCMs. Knockdown of c-Jun expression significantly attenuated cardiac hypertrophy induced by miR-139-5p deprivation. Our data indicated that miR-139-5p was down-regulated in the hearts of HCM patients and that it inhibited cardiac hypertrophy by targetting c-Jun expression.

Hypertrophic cardiomyopathy (HCM) is a serious monogenic disease characterized by cardiac hypertrophy, fibrosis, sudden cardiac death, and heart failure. Previously, we identified that miR-139-5p was down-regulated in HCM patients. However, the regulatory effects of miR-139-5p remain unclear. Thus, we investigated the role of miR-139-5p in the regulation of cardiac hypertrophy. The expression of miR-139-5p in left ventricular tissues in HCM patients and mice subjected to transverse aortic constriction (TAC) was significantly down-regulated. Knockdown of miR-139-5p expression in neonatal rat cardiomyocytes (NRCMs) induced cardiomyocyte enlargement and increased atrial natriuretic polypeptide (ANP) expression. Overexpression of miR-139-5p antagonized isoproterenol (ISO)-induced cardiomyocyte enlargement and ANP/brain natriuretic peptide (BNP) up-regulation. More importantly, we found that c-Jun expression was inhibited by miR-139-5p in NRCMs. Knockdown of c-Jun expression significantly attenuated cardiac hypertrophy induced by miR-139-5p deprivation. Our data indicated that miR-139-5p was down-regulated in the hearts of HCM patients and that it inhibited cardiac hypertrophy by targetting c-Jun expression.
Hypertrophic cardiomyopathy (HCM) is a monogenic disease with an estimated prevalence of 0.2% in the population [1]. This disease is characterized by cardiac hypertrophy, fibrosis, sudden cardiac death, and heart failure [2,3]. Although it has been revealed that HCM is mostly caused by mutations in sarcomere genes [4][5][6], the regulatory mechanisms of cardiac hypertrophy in HCM are not fully understood.
MicroRNAs (miRs) are intracellular small, non-coding RNAs that mainly down-regulate specific target genes by binding to the 3 -UTR of corresponding mRNAs [7]. Accumulating studies have revealed that miRs play important roles in regulating cardiac remodeling in HCM, thereby providing potential therapeutic targets for this disease [8][9][10][11]. By using microarray analyses, we previously screened for miRs in left ventricular tissues that were differentially expressed between HCM patients and healthy donors [9]. We identified that miR-139-5p was one of the most down-regulated miRs in the hearts of HCM patients [9]. However, whether miR-139-5p participates in the regulation of cardiac hypertrophy remains unclear.
The β-adrenergic receptor (β-AR) plays an important role in the regulation of cardiac excitation-contraction; however, hyperactivation of β-AR leads to cardiac remodeling [12,13]. The stimulation of β-AR activates the downstream activating protein-1 (AP-1), which then transmits signaling to induce cardiac hypertrophy [14,15]. AP-1 is a transcription factor consisting of a group of immediate early genes, such as c-Jun and c-Fos. They form homo-or heterodimers to regulate the expression of their target genes. By using online bioinformative assays (www.targetscan.org), we predicted that c-Jun might be a target of miR-139-5p in cardiomyocytes, which might participate in the regulation of cardiac hypertrophy. Therefore, we investigated this aspect in the present study. We found that isoproterenol (ISO), a potent β-AR agonist, strongly induced the expression of c-Jun, whereas exogenous addition of miR-139-5p mimic antagonized its expression and attenuated ISO-induced cardiac hypertrophy.

Ethics statement
All protocols pertaining to human subjects were approved by the Ethics Committee of Fuwai Hospital (Beijing, China) in accordance with the Declaration of Helsinki. Written informed consent was obtained from all the participants. Left ventricular tissues were obtained as previously described [9]. All the protocols pertaining to animals were approved by the Ethics Committee for Animal Study of Fuwai Hospital (Beijing, China) (approval number: 0088-R/M-300/200-GZ(X)).

Transverse aortic constriction operation
Transverse aortic constriction (TAC) operation in mice was performed as previously described [16]. In brief, 6-8 weeks old male C57BL/6 mice were weighed and anesthetized with 2.5% avertin (0.018 ml/g) through intraperitoneal injection. The transverse aorta was ligated for TAC operation. Sham-operated mice served as controls. All mice were killed at 4 weeks post operation, and the hearts were harvested for miR-139-5p expression analysis.

Cell culture
Primary neonatal rat cardiomyocytes (NRCMs) were cultured as previously described [17]. In brief, ventricles from neonatal Wistar rats were isolated and cut into small pieces. The tissues were washed with PBS and digested with 0.06% collagenase II (Worthington Biochemical Corporation, Lakewood, NJ, U.S.A.) at 37 • C. The cell suspension was centrifuged and resuspended with Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS. For differential adhesion, the cells were maintained in an atmosphere with 5% CO 2 at 37 • C for 90 min. The supernatants were resuspended with complete DMEM containing 0.1 mM bromodeoxyuridine, seeded in corresponding plates and routinely cultured for 24 h. Then, the medium was replaced with DMEM containing 1% FBS for experiments.

Cell transfection and treatment
NRCMs were transiently transfected with miR-139-5p mimics or inhibitors (GenePharma, Suzhou, China) at a final concentration of 100 nM. For the knockdown of c-Jun expression, a predesigned specific siRNA was used (Silencer R Select, s127979, Thermo Fisher, Carlsbad, CA, U.S.A.). All the above small RNAs were transfected by using Lipofectamine R RNAiMAX Reagent (Thermo Fisher) according to the manufacturer's protocol. For inducing cardiac hypertrophy in vitro, NRCMs were treated with 100 nmol/l ISO for 24 h [18].

Determination of cell surface area
NRCMs (5 × 10 5 cells/ml) were seeded in 24-well plates and routinely cultured. The cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100 in PBS for 5 min. The cells were then washed with PBS and stained with Texas Red-Phalloidin (Thermo Fisher) for 30 min at room temperature. The nuclei were stained with 0.1 μg/ml DAPI for 5 min. The cells were visualized with a fluorescence microscope, and the cell surface area was measured by analyzing 30 NRCMs from at least five random fields with ImagePro Plus 6.0 Software (Media Cybernetics, Bethesda, MD, U.S.A.).

Western blotting
Total protein was extracted with RIPA lysis buffer, quantitated, and normalized. Approximately 20 μg protein from each sample was separated using SDS/PAGE and blotted on to a nitrocellulose membrane. The membrane was blocked with 5% BSA at room temperature for 1 h and then incubated with the following primary antibodies at

Statistical analysis
Statistical analyses were performed with IBM SPSS 19.0 Software (SPSS, Inc., Chicago, IL, U.S.A.). Two-tailed Student's t test or ANOVA was used to determine the differences. The data are expressed as the mean + − S.E.M., and P<0.05 was considered statistically significant.

miR-139-5p is down-regulated in hypertrophic hearts
To validate whether miR-139-5p is down-regulated in hypertrophic hearts, we detected its expression in left ventricular tissues from 16 HCM patients and 8 healthy donors. We found that the expression of miR-139-5p in left ventricular tissues of HCM patients was down-regulated to 57.6% of that in the healthy donors (P<0.05, power: 0.505) ( Figure 1A). We then performed TAC operation to induce cardiac hypertrophy in mice. Consistently, the expression of miR-139-5p in the hearts of TAC-operated mice was significantly down-regulated compared with that in sham-operated mice, indicating that miR-139-5p is down-regulated in hypertrophic hearts (P<0.01, power: 0.765) ( Figure 1B).

miR-139-5p is required for maintaining normal cell size in cardiomyocytes
To investigate the role of miR-139-5p in cardiac hypertrophy, we knocked down its expression in NRCMs by transfecting a specific miR inhibitor. The levels of miR-139-5p were down-regulated to 25.4% of that in control NRCMs at 48 h post transfection (Figure 2A). We then measured changes in cell surface area by staining NRCMs with Texas Red-conjugated Phalloidin. We found that the cell surface area was significantly increased in miR-139-5p-deprived NRCMs ( Figure 2B). ANP and BNP are two known biomarkers of cardiac hypertrophy. The knockdown of miR-139-5p induced a 2.06-fold increase in ANP expression (P<0.05); however, the expression of BNP was unchanged ( Figure 2C). These results indicated that miR-139-5p is required to maintain normal cell size in cardiomyocytes.

Overexpression of miR-139-5p attenuates ISO-induced cardiac hypertrophy
Since miR-139-5p participates in the regulation of cell size in cardiomyocytes, we wanted to explore whether the exogenous administration of miR-139-5p is able to antagonize cardiac hypertrophy. ISO is a β-AR agonist that is widely used to induce cardiac hypertrophy. Treating NRCMs with 100 nmol/l ISO for 24 h induced a significant increase in cell size ( Figure 3A,B). Pre-transfection of NRCMs with the miR-139-5p mimic for 48 h attenuated ISO-induced cellular enlargement ( Figure 3A,B). Consistently, the expression of ANP and BNP was up-regulated by ISO treatment by 2.11-and 7.68-fold, respectively ( Figure 3C), whereas the transfection of NRCMs with the miR-139-5p mimic attenuated the ISO-induced increase in ANP and BNP expression ( Figure 3C). In addition, miR-139-5p did not affect cell surface area or ANP/BNP levels under basal conditions ( Figure 3A-C). These data indicated that miR-139-5p attenuates ISO-induced cardiac hypertrophy in vitro.

miR-139-5p down-regulates c-Jun expression in ISO-induced cardiac hypertrophy
miRs specifically bind to the 3 -UTR of its target mRNAs to regulate their expression. By using TargetScan (http: //www.targetscan.org/vert 71/), an online tool for predicting miR target genes, we identified c-Jun, IGF-1R, MyoCD, Wnt1, and β-catenin as candidate target genes of miR-139-5p. We transfected NRCMs with the miR-139-5p mimic for 48 h and found that the levels of c-Jun, IGF-1R, and β-catenin were all down-regulated, whereas the levels of My-oCD and Wnt1 were unaffected ( Figure 4A,B). Interestingly, miR-139-5p overexpression inhibited the ISO-induced up-regulation of c-Jun expression ( Figure 4A,B). In contrast, the knockdown of endogenous miR-139-5p increased the level of c-Jun ( Figure 4E,F). These results indicated that miR-139-5p inhibits the expression of c-Jun in cardiomyocytes and thus might play an important role in the regulation of cardiac hypertrophy.  The expression of IGF-1R was down-regulated by miR-139-5p in NRCMs, although ISO alone slightly down-regulated IGF-1R expression ( Figure 4A,B). The IGF-1R signaling pathway has been reported to regulate cardiac hypertrophy via PI3K/Akt [19]. As expected, miR-139-5p suppressed ISO-induced hyperphosphorylation of Akt in NRCMs, indicating that the IGF-1R/Akt pathway might also contribute to the antihypertrophic effects of miR-139-5p ( Figure 4C,D). Wnt1/β-catenin has been reported to be regulated by miR-139-5p [20]. We observed that miR-139-5p indeed down-regulated β-catenin expression under basal conditions, but it failed to inhibit Wnt1 or β-catenin expression in response to ISO stimulation ( Figure 4A,B).

Inhibition of c-Jun activation suppresses miR-139-5p knockdown-induced cardiac hypertrophy
As the expression of c-Jun was found to be regulated by miR-139-5p in NRCMs, we proposed that the up-regulation of c-Jun might be required in miR-139-5p deprivation-induced cardiac hypertrophy. To investigate this aspect, we knocked down c-Jun expression by transfecting c-Jun-specific siRNAs into miR-139-5p-deprived NRCMs. The knockdown of miR-139-5p expression in NRCMs significantly increase c-Jun levels, whereas the knockdown of c-Jun expression prevented this increase ( Figure 4E,F). As expected, the increase in cell surface area and the up-regulation of ANP expression induced by miR-139-5p deprivation were restored by c-Jun knockdown ( Figure 4G-I). These results indicated that c-Jun is indispensable in miR-139-5p deprivation-mediated cardiac hypertrophy in vitro.

Discussion
Pathological cardiac remodeling in HCM is associated with the deregulated expression of many intracellular miRs [8][9][10][11]. In the present study, we reported that miR-139-5p was down-regulated in heart tissues of HCM patients. The knockdown of miR-139-5p expression in NRCMs induced cardiac hypertrophy, whereas the exogenous overexpression of miR-139-5p antagonized ISO-induced cardiac hypertrophy. These findings indicated that miR-139-5p acts an anti-hypertrophic miR in the heart.
HCM is a monogenic myocardial disease, which is characterized by cardiac hypertrophy, sarcomere disarrangement, cardiac fibrosis, heart failure, and cardiac sudden death [2]. By using microarray analysis, we found that miR-139-5p was significantly down-regulated in the left ventricular tissues of HCM patients. Moreover, the expression of miR-139-5p was down-regulated in the hearts of TAC-operated mice, and knockdown of miR-139-5p in NRCMs induced cardiomyocyte enlargement. These data indicated that miR-139-5p is indispensable in normal cardiomyocytes.
The β-AR signaling pathway, which is classically activated by catecholamines, is one of the well-known pathways in the induction of cardiac hypertrophy [12,13]. The activation of β-AR regulates cardiac excitation-contraction and leads to the increase in heart rate and cardiomyocyte contractility [21][22][23]. The hyperactivation of β-AR results in cardiac hypertrophy, fibrosis, senescence, inflammation, and cardiomyocyte apoptosis and necrosis [24][25][26][27][28][29]. The treatment of NRCMs with ISO caused cardiomyocyte enlargement and increased the expression of ANP and BNP. We observed that miR-139-5p overexpression attenuated ISO-induced cardiac hypertrophy, which indicated that miR-139-5p plays an antihypertrophic role in the heart. It has been reported that the stimulation of β-AR can induce the activation of AP-1, which is associated with the activation of the CaMKII, ERK, and JNK pathways [14,15]. AP-1 acts as a transcription factor for a group of immediate early genes, including c-Jun, c-Fos, JunB, and JunD. These proteins form homo-or heterodimers and bind to the promoters of various genes to regulate their expression, which subsequently induces cardiac hypertrophy [14,[30][31][32]. In the present study, we observed that c-Jun expression was strongly induced by ISO-treatment, whereas miR-139-5p inhibited this increase. Consistently, the knockdown of c-Jun expression significantly attenuated miR-139-5p deprivation-induced cardiac hypertrophy. The miR-139-5p/c-Jun axis has been reported to form a feedback loop in human gastric cancer cells [33]. Our data indicated that the down-regulation of c-Jun is indispensable for the anti-hypertrophic effects of miR-139-5p in cardiomyocytes.
The activation of β-AR induced the phosphorylation of Akt/GSK3β in NRCMs, which is associated with the up-regulation of ANP expression [34]. In colitis-associated tumorigenesis, miR-139-5p inhibits the cross-talk between the PI3K/Akt and Wnt pathways by targetting IGF-1R [35]. Our results indicated that overexpression of miR-139-5p in NRCMs antagonizes ISO-induced activation of Akt. miR-139-5p also inhibits the expression of IGF-1R in cells both under basal and ISO-stimulated conditions, indicating that the down-regulation of IGF-1R and inhibition of Akt might be involved in preventing cardiac hypertrophy. Mi et al. [20] reported that Wnt1 and β-catenin were directly targetted by miR-139-5p in C2C12 cells. In our study, we observed that miR-139-5p indeed down-regulated β-catenin expression under basal conditions; however, it failed to antagonize the ISO-induced up-regulation of β-catenin in NRCMs. The expression of Wnt1 was not altered by miR-139-5p overexpression in NRCMs. These data indicated that the Wnt1/β-catenin pathway might not be associated with the antihypertrophic effects of miR-139-5p. In addition, we found that the predicted MyoCD was not a target of miR-139-5p in NRCMs, as its expression was not altered under basal or ISO-stimulated conditions. Taken together, our study highlights that miR-139-5p is a novel antihypertrophic miR in cardiomyocytes. miR-139-5p attenuates cardiac hypertrophy possibly through the down-regulation of c-Jun expression in vitro. Our data indicated that the exogenous delivery of the miR-139-5p mimic to the heart might be a potential therapeutic strategy for pathological cardiac hypertrophy. These findings provide evidences that the miR-139-5p/c-Jun axis is a novel target for the prevention and treatment for cardiac remodeling. The future study will focus on the mechanism of miR-139-5p expression and its association with c-Jun.