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Cardiac Hypertrophy is Positively Regulated by MicroRNA-24 in Rats

Background: MicroRNA-24 (miR-24) plays an important role in heart failure by reducing the efficiency of myocardial excitation-contraction coupling. Prolonged cardiac hypertrophy may lead to heart failure, but little is known about the role of miR-24 in cardiac hypertrophy. This study aimed to preliminarily investigate the function of miR-24 and its mechanisms in cardiac hypertrophy. Methods: Twelve Sprague-Dawley rats with a body weight of 50 ± 5 g were recruited and randomly divided into two groups: a transverse aortic constriction (TAC) group and a sham surgery group. Hypertrophy index was measured and calculated by echocardiography and hematoxylin and eosin staining. TargetScans algorithm-based prediction was used to search for the targets of miR-24, which was subsequently confirmed by a real-time polymerase chain reaction and luciferase assay. Immunofluorescence labeling was used to measure the cell surface area, and 3H-leucine incorporation was used to detect the synthesis of total protein in neonatal rat cardiac myocytes (NRCMs) with the overexpression of miR-24. In addition, flow cytometry was performed to observe the alteration in the cell cycle. Statistical analysis was carried out with GraphPad Prism v5.0 and SPSS 19.0. A two-sided P < 0.05 was considered as the threshold for significance. Results: The expression of miR-24 was abnormally increased in TAC rat cardiac tissue (t = −2.938, P < 0.05). TargetScans algorithm-based prediction demonstrated that CDKN1B (p27, Kip1), a cell cycle regulator, was a putative target of miR-24, and was confirmed by luciferase assay. The expression of p27 was decreased in TAC rat cardiac tissue (t = 2.896, P < 0.05). The overexpression of miR-24 in NRCMs led to the decreased expression of p27 (t = 4.400, P < 0.01), and decreased G0/G1 arrest in cell cycle and cardiomyocyte hypertrophy. Conclusion: MiR-24 promotes cardiac hypertrophy partly by affecting the cell cycle through down-regulation of p27 expression.

led to decreased efficiency of excitation-contraction coupling by down-regulating its target gene of Junctophilin-2. [12] Since target genes of miR-24 are different, we performed a miRNA functional enrichment analysis using TAM (tool for annotation of microRNA, a method for enrichment and depletion analysis of a miRNA category in a list of miRNAs). [13] The results showed that miR-24 was closely related with muscle development which is an important pathological basis of heart failure. In addition, the biological analysis showed that miR-24 was associated with cell cycle in cancer cells and cell proliferation. In cardiomyocytes, the activity of cell cycle controls cell proliferation, which plays an important role in cardiac hypertrophy. [14] These findings intrigued us to investigate the role of miR-24 in cardiac hypertrophy and its potential targets.
In the present study, we aimed to investigate the function of miR-24 in cardiac hypertrophy by establishing a transverse aortic constriction (TAC) rat model, and miR-24 overexpressing in neonatal rat cardiac myocytes (NRCMs). The underlying mechanisms of miR-24 in cardiac hypertrophy were also investigated by TargetScans algorithm-based prediction, luciferase assay, and flow cytometry.

Ethical approval
All the animal experiments were carried out according to the Guide for Care, and the use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85e23, revised 2011), and the study protocol was approved by the Institutional Animal Care and Use Committee of Peking University (No. LA2016145). Every effort was made to reduce the number of animals and minimize their suffering. A 4-week-old wild-type male Sprague-Dawley (SD) rats weighing 50 ± 5 g were obtained from the Medical Experimental Animal Center of Peking University, Beijing, China. The rats were divided into two groups: sham group (n = 6) and TAC group (n = 6). TAC and sham were induced as described previously. [15] Briefly, rats were anesthetized by intraperitoneal (i.p.) administration of 50 mg/kg pentobarbital (Merck, Whitehouse Station, NJ, USA). A longitudinal cut was made in the proximal portion of the sternum. The aorta under the upper left sternal border was ligated with a 27-gauge needle using 7-0 silk surgical thread. After ligation, the needle was promptly removed, and the thoracic cavity was closed. The sham procedure was identical except that the aorta was not ligated.

Echocardiography
Vevo2100 system (VisualSonics Inc., Toronto, Ontario, Canada) was used to evaluate cardiac structure and systolic function of rats. In brief, rats were anesthetized with 2.0% isoflurane (Baxter Healthcare Corp., CA, USA) until the heart rate was stabilized at about 400 beats/min. Parasternal long-axis images were acquired in B-mode with the maximum left ventricle length identified. The M-mode cursor was positioned perpendicularly to the maximum left ventricle dimension in end-diastole and systole, and M-mode images were obtained for measuring wall thickness and chamber dimensions.

Neonatal rat cardiac myocytes culture
Cardiomyocytes isolated and cultured from newborn SD rats aged 1-2 days were obtained from the Medical Experimental Animal Center of Peking University. NRCMs were prepared as previously described. [16] In brief, central thoracotomy was performed in neonatal rats under anesthesia with 1.0% isoflurane. Hearts were quickly excised and immediately embedded in freezing Hank's Balanced Salt Solution (Thermo Fisher Scientific, MA, USA). Cardiomyocytes were dispersed by digestion with 0.25% trypsin (Gibco, NY, USA) and 0.05% collagenase II (Sigma, MO, USA) at 37°C. The isolated cardiomyocytes were then cultured with 10% fetal bovine serum (Hyclone, UT, USA) and antibiotics (50 U/ml penicillin and 50 mg/ml streptomycin) at 37°C in 5% CO 2 .

Quantitative reverse transcriptase-polymerase chain reaction analysis
Total RNA, containing miRNA, was extracted from cardiac tissues and NRCMs using miRNeasy Kits (QIAGEN, Germany), and genomic DNA was removed using a DNA-free kit (Invitrogen, CA, USA) following the manufacturer's instructions. First-strand cDNA was synthesized by miRNA-specific reverse-transcription primers (RiboBio Co., Ltd, China) (for miR-24) or oligodT15 (for p27) using SuperScript III Reverse Transcriptase (Invitrogen). One microgram of cDNA was included for real-time polymerase chain reaction (RT-PCR) amplification using the Brilliant II SYBR Green qPCR Master Mix (Stratagene, CA, USA), and the fluorescent signals were monitored with n Mx3000p Real-Time PCR System (Stratagene). The small nuclear RNA, U6, was used as control for miRNA samples, and glyceraldehyde 3-phosphate dehydrogenase was used as control for p27 mRNA quantification.

Measurement of cell surface area by immunofluorescence
NRCMs were seeded in 12-well plates at a concentration of 5 × 10 5 cells/ml. Over-expression of miR-24 was achieved following adenovirus transfection. After treatment for 24 h, the cells were fixed with 4% paraformaldehyde at 37°C for 15 min. After washing with phosphate-buffered saline (PBS), the cells were permeabilized with 2% Triton X-100 in PBS at 37°C for 30 min. After washing with PBS, the nuclei were stained with phalloidin (1:50-1:100; Sigma) at 37°C for 60 min. Immunofluorescence was determined using a fluorescence microscope (Leica, Germany). An average of 150 cardiomyocytes from 30 fields was chosen at random for measurement of cell size. Immunofluorescence images captured from cardiomyocytes stained with Texas red phalloidin were measured using the ImageJ Particle Analyzer algorithm.

Plasmid construction and luciferase assay
Human embryonic kidney HEK-293A cells were seeded at a concentration of 50,000 cells/well in 24-well plates. One construct contained the wild-type p27 3'UTR region with the miR-24 binding site intact (p27-WT). A matched control construct contained mutated bases in the miR-24 binding site (AAAGGUUGCAUA-cugagccA changed to AAAGGUUGCAUA-agtctaaA) (p27-MUT) (Shanghai Genechem Co., Ltd. China). HEK-293A cells were transfected with 500 ng of either p27-WT plasmid or p27-MUT, together with either mimic miR-24 (RiboBio Co., Ltd) or nontargeting negative control (scramble) at a concentration of 50 nM. Lipofectamine 2000 reagent (Invitrogen) was used for the transfection, and 50 ng Renilla luciferase vector was included in each well to control the transfection efficiency. Twenty-four hours after transfection, luciferase activity was measured using a Dual-Luciferase Reporter Assay System (Promega Corporation, WI, USA); and the firefly luciferase activity was normalized to Renilla luciferase activity.

Flow cytometry
NRCMs were harvested at the indicated time points, washed, fixed, and centrifuged (1500 ×g), and then labeled with propidium iodide (Invitrogen). Flow cytometric analysis was carried out on a FACScan Q4 (Becton Dickinson, CA, USA) cell sorter. Percentages of cells in G0-G1, S, and G2-M phases were calculated with Mod Fit software (Verity Software House, USA) for Windows.

Measurement of total protein content by 3 H leucine incorporation assay
NRCMs were seeded in 24-well plates at a concentration of 1 × 10 5 cells/ml. 3 H-leu (1 uci/ml) was added for 12 h before the cells were harvested. After washing with PBS, the cells were treated with precooled 10% trichloroacetic acid for 30 min on ice. The cell lysate was added and incubated at 37°C overnight so that the cells were fully cracked. Each sample was transferred to the flicker bottle according to the proportion of 0.5 ml cell lysate in the 6 ml scintillation liquid. The lid was covered, and the samples were shaken and mixed well, and counts per minute (CPM) and disintegrations per minute value were read on the stroboscope (Hidex 300SL, Finland).

Hematoxylin and eosin and Sirius Red staining
The left ventricular ischemic tissue from the rats was fixated by 4% for 24 h, then paraffin-embedded and sectioned at a 4-µm thickness through a rotary microtome. Hematoxylin and eosin (H and E) and Sirius Red staining was used to detect morphologic changes of cardiac myocytes and the degree of myocardial fibrosis as previously described. [17]

Statistical analysis
Statistical analysis was carried out with GraphPad Prism v5.0 (GraphPad Software Inc., San Diego, California, USA) and SPSS 19.0 (IBM Corp, Armonk, NY, USA). The Shapiro-Wilk was used to test the normal distribution in the continuous data, and the Levene's test was used to detect the equality of variances. Consistent with the measurement data, normal distribution was expressed as mean ± standard error (SE), and the independent samples Student's t-test was used to detect the differences between groups. If the data distribution was not normal, the Mann-Whitney test was employed for the nonparametric analysis. In all analyses, a two-sided P < 0.05 was considered as the threshold for significance.

Expression of microRNA-24 was increased in the left ventricular tissue of transverse aortic constriction rats
TAC was an established in vivo model of hemodynamic overload, [18] which induced significant and equivalent left ventricle pressure load in SD rats. Expression of the hypertrophic marker genes, atrial natriuretic factor (ANP) and brain natriuretic peptide (BNP) was increased following TAC injury (ANP: 0.93 ± 0.15 vs. 0.50 ± 0.07, P < 0.05; BNP: 0.04 ± 0.01 vs. 0.02 ± 0.00, t = −3.676, P < 0.01) [ Figure 1a and 1b]. TAC rats exhibited cardiac hypertrophic pathological changes by H and E staining compared with the sham group, and the myocardial cross-sectional area in the TAC group was significantly increased compared with the sham group (6307 ± 681 µm 2 vs. 3470 ± 260 µm 2 , t = −3.890, P < 0.01) [ Figure 1c]. The Sirius Red staining of the heart showed that the fibrosis area in TAC rats was also increased compared with the sham group (62.50% ± 0.01% vs. 13.10% ± 0.03%, t = −3.344, P < 0.01) [Supplementary Figure 1]. Echocardiography revealed that the left ventricular posterior wall thickness at diastole and left ventricular mass were both increased following TAC injury (2.61 ± 0.17 mm vs. 1.91 ± 0.10 mm, t = −3.564, P < 0.01; 1469.0 ± 140.6 mg vs. 969.1 ± 34.2 mg, t = −3.453, P < 0.01) [ Figure 1d]. In addition, the heart weight-to-body weight ratio and the heart weight-to-tibia length ratio were also elevated following TAC injury [Supplementary Table 1]. Quantitative RT-PCR (qRT-PCR) analysis was conducted to determine the expression of miR-24 in heart tissue of the TAC rat model. As shown in Figure 1e, increased expression level of miR-24 was observed in rat hypertrophic hearts induced by 6 weeks of TAC injury (2.61 ± 0.17 vs. 1.91 ± 0.10, t = −2.938, P < 0.05), suggesting that miR-24 was involved in cardiac hypertrophy.

Expression of p27 was repressed by microRNA-24 in hypertrophic cardiac tissue and Neonatal rat cardiac myocytes
To determine the mechanism by which miR-24 regulates cardiac hypertrophy, we used the TargetScans algorithm (http://www.TargetScans.org) to search for the targets of miR-24. As shown in Table 1, priorities were given to both human and rat targets, to higher expression in heart tissue, and to targets with clearly studied functions. The aggregate P CT value (the probability of conserved targeting) derived from the TargetScans software was also considered [Supplementary Figure 2]. Six potential targets of miR-24 were included in this study. qRT-PCR was performed to detect the expression of the screened targets following overexpression of miR-24 in NRCMs. The results showed that the expression of CDKN1B (p27, Kip1) was significantly decreased (0.69 ± 0.07 vs. 1.00 ± 0.03, t = 3.108, P < 0.01) [ Figure 2a]. Interestingly, in contrast to increased expression of miR-24 in hypertrophic cardiac tissue, the expression of p27 mRNA exhibited a 40.35% reduction (0.01 ± 0.00 vs. 0.02 ± 0.00, t = 2.896, P < 0.05) [ Figure 2b]. The protein level of p27 exhibited a 40.80% reduction in hypertrophic cardiac tissue (5.95 ± 0.44 vs. 10.05 ± 0.83, P < 0.01) [ Figure 2c].

Overexpression of microRNA-24-induced cardiomyocyte hypertrophy
To determine the effects of up-regulated miR-24 overexpression on NRCMs, we observed the changes of NRCMs after transfection with miR-24-AEVs [ Figure 2e].

Overexpression of microRNA-24 led to the altered cell cycle in neonatal rat cardiac myocytes
As a target gene of miR-24, p27 is known as a cyclin-dependent kinase inhibitor, which is involved in regulating the cell cycle through inducing G0/G1 arrest. In the adenovirus-mediated overexpression, up-regulated miR-24 led to the lower expression of p27. To determine whether the up-regulated expression of miR-24 caused any changes in the cell cycle, flow cytometry was used to detect alterations in the cell cycle [ Figure 4a-c]. It was shown that the G0/G1 phase population decreased from 87.97% to 81.21% (miR-24 vs. GFP: t = −4.515, P < 0.01) [ Figure 4d], whereas the S phase population increased from 12.35% to 6.29% (miR-24 vs. GFP: t = −3.453, P < 0.01) [ Figure 4e]. The percentage of G2/M phase cells was not significantly altered (miR-24 vs. GFP, t = −0.334, P = 0.745) [ Figure 4f]. These results indicated that up-regulated miR-24 resulted in cardiac hypertrophy by affecting the G0/G1 arrest through down-regulation of p27 expression.

dIscussIon
In the present study, we found that miR-24 expression increased in hypertrophic cardiac tissue and the up-regulated miR-24 would lead to cardiomyocyte hypertrophy. As a target gene of miR-24, p27 expression was decreased both in hypertrophic cardiac tissue and in NRCMs with miR-24 upregulation. The up-regulated miR-24 resulted in increased cell surface, total protein expression and cell cycle changes  in NRCMs. Our results suggested that miR-24 was a key mediator promoting cardiac hypertrophy, and one of the potential mechanisms was promoting cells in G0/G1 phase into S phase by repressing p27 expression. miR-24 is extensively expressed in various tissues, [19] and it plays an important role in various physiological or pathological conditions, such as heart failure, oral cancers, prostate cancer, hyperglycemia, and Alzheimer's disease. [20][21][22][23][24] In the present study, miR-24 was found to participate in the process of cardiac hypertrophy in rats, and the up-regulated expression of miR-24 in NRCMs led to the higher expression of ANP and BNP, the higher expression of the total protein, the higher increase of cell surface area and CPM in 3 H-Leu incorporation. These results showed that abnormally increased miR-24 promoted cardiac hypertrophy, which is not a concomitant phenomenon of cardiac hypertrophy. In general, cardiac myocytes rapidly proliferate during fetal life but withdraw from the cell cycle and lose the ability of proliferation soon after birth in mammals. [25,26] Although to what extent adult cardiac myocytes proliferate is controversial, it appears that the cell cycle changes are involved in cardiac hypertrophy in a predominant form of an increase in cell size (hypertrophy), but not in number. [27] As a kind of cyclin-dependent kinase (CDK) inhibitors (CKIs), p27 extensively suppresses the activities of various cyclins and CDKs, which drive forward the cell cycle, and consequently, cells in G0/G1 phase cannot enter into the S phase of cell cycle. [28] This is important in inhibiting the proliferation of tumor cells. [29,30] However, p27-knockout mice were reported to develop age-dependent cardiac hypertrophy and had exaggerated hypertrophic responses to aortic banding. [31] Recent evidence suggested that miRNAs controlled the levels of multiple cell cycle regulators, thus controlling cell proliferation, and miR-24 was reported to promote cell proliferation by targeting p27 in primary keratinocyte and different cancer types . [22,32] In our study, miR-24 up-regulation in NRCMs induced the down-regulation of p27, leading to cardiomyocyte hypertrophy. Analysis of the cell cycle showed that the percentage of cells in G0/G1 phase decreased, the percentage of cells in S phase increased, and the percentage of cells in G2/M phase remained unchanged. This suggested that the cell cycle changes in NRCMs resulted in cardiomyocyte hypertrophy. As the luciferase report assay showed that p27 was targeted by miR-24, we suggested that miR-24 could induce cardiac hypertrophy through repressing p27 expression.
In conclusion, the present study showed that miR-24 was a key mediator of cardiac hypertrophy by repressing p27 expression, which led to S phase arrest. As many other miRNAs and CKIs are involved in cell cycle changes, [33] more in-depth studies are needed.

Financial support and sponsorship
This work was supported by grant from National Natural Science Foundation of China (No. 91339105, and No. 81625001).

Conflicts of interest
There are no conflicts of interest.