Received Date : 11-Dec-2014

Accepted Article

Revised Date

: 31-Jan-2015

Accepted Date : 20-Mar-2015 Article type

: Original Research Article

Cardioprotective Effect of MicroRNA-21 in Murine Myocardial Infarction

Running title: MicroRNA-21 and MI

Ji Zhang 1, #, Xiao-Lin Xu 2, #, Xiao-Tian Sun 2, 3, Chang-Fa Guo 3, Chun-Sheng Wang 3, Yi-Qing Wang 2, Guo-Long Gu 4 , Bing Sun 5, * Gong-Liang Guo 5, Ke Ma 5, Yuan-Yuan Huang 5 & Li-Qun Sun 5 1 Department of Cardiology, Shanghai Tenth People’s Hospital, Tenth people's Hospital of Tongji University, Shanghai 200072, P. R. China; 2 Department of Cardiothoracic Surgery, Huashan Hospital, Fudan University, Shanghai 200040, P. R. China; 3

Department of Cardiac Surgery, Zhongshan Hospital of Fudan University & Shanghai Institute of Cardiovascular Diseases, Shanghai 200032, P. R. China;


Department of cardiovascular diseases, Jiangyin Hospital of traditional Chinese medicine affiliated Nanjing University of Chinese Medicine, Jiangyin 214400, P. R. China; 5

Department of Cardiology, Tongji Hospital, Tongji University, Shanghai 200092, P. R. China.


Ji Zhang and Xiao-Lin Xu contributed equally to this paper.


Correspondence: Dr. Bing Sun, Department of Cardiology, Tongji Hospital, Tongji University, Village Road No. 389, Putuo District, Shanghai 200092, P. R. China; Email: [email protected] Tel. /Fax.: +86-021-56051080

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/1755-5922.12118 This article is protected by copyright. All rights reserved.


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Introduction: To investigate the cardioprotective effect of MicroRNA-21 (miR-21) in murine Myocardial infarction (MI). Methods: Forty C57BL/6 male mice were divided into sham group, MI group, LV-GFP group and miR-21 group. Mice the MI group, LV-GFP group and miR-21 group were subjected to MI by left anterior descending artery (LAD) ligation, while chest was opened/closed without ligation in sham group. In MI group, expression of miR-21 in the MI area and its surrounding areas was detected at 1st, 2nd and 4th week after experiment. Subsequently, lentivirus expressing miR-21, and lentivirus that did not express miR-21, were transfected into mice left ventricular cavity of miR-21 group and LV-GFP group, respectively. Cardiac function, MI size, miR-21 expression, collagen I level, fibronectin content, number of α-SMA-positive cells, number of apoptotic cells, apoptosis related factors were compared between the three groups. Results: Compared with sham group, miR-21 levels in MI group were significantly decreased in the 1st week and 2nd week, but was almost the same in the 4th week. Left ventricular fractional shortening (LVFS) and left ventricular ejection fraction (LVEF) in the miR-21 group improved compared to the LV-GFP group. In miR-21 group, myocardial infarct size reduced by 36.9% in comparison to LV-GFP group. Compared to sham group, miR-21 expression in the miR-21 group and LV-GFP group decreased significantly. In the miR-21 group, collagen I level, fibronectin content and number of α-SMA-positive cells of miR-21 decreased significantly compared to the LV-GFP group. The number of apoptotic cells in the MI areas of the miR-21 group was significantly less than the LV-GFP group. Compared with the LV-GFP group, Bcl-2 level and the ratio of Bcl-2 to Bax were significantly increased, and the levels of Bax and Caspase-3 decreased. Conclusions: Our results suggest miR-21 is an important regulatory molecule in the pathophysiology of MI.

Keywords: myocardial infarction; MicroRNA-21; cell apoptosis; cardioprotective effect; cardiac function; transfection

Introduction Myocardial infarction (MI) leads to the formation of scar and remodeling of left ventricle, including cardiac dilatation and cardiomyocyte fibrosis, due to insufficient cardiomyocyte replacement, and is the leading cause of morbidity and mortality worldwide [1,2]. Myocardial fibrosis could further lead to mechanical stiffness and ventricular contractile dysfunction [3]. MI could be induced by plaque disruption due to coronary thrombosis and an imbalance between myocardial oxygen demand and supply [4]. Although the outcomes of ST-elevation MI (STEMI) have improved significantly in the last 2 decades with reperfusion therapy, improving patient survival, the incidence of left ventricular thrombus has increased from 2.5% to 15% in anterior MI within the same period [5]. According to the WHO, ischemic heart disease led to 7.25 million deaths worldwide (12.8%) in 2008 [6]. MI treatment with anti-platelet drugs, such as heparin, reduces adverse ischemic incidents, but has the disadvantage of increasing the frequency of hemorrhages [7]. Beta-blocker therapy, recommended for STEMI, combined with thrombolytic therapy and primary percutaneous coronary intervention, significantly reduces the mortality in MI [8]. Additionally, early reperfusion strategies, both pharmacological and interventional, also reduced the mortality due to MI [9]. Despite the progress in MI treatment, many patients still die early during MI and survivors are still at a high-risk of heart failure [10]. Thus additional treatment strategies are needed to prevent heart failure following MI. Cardiovascular gene therapy has been an attractive are for treatment for cardiovascular

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diseases, such as venous ulcer, peripheral artery disease, atherosclerosis, pulmonary hypertension, and Fabry disease [11]. Compared to pharmacological agents, microRNAs (miRs) have the ability to regulate multiple downstream mediators to alter various signaling cascades. A class of small RNAs, called micro RNAs (miRs), are 22 nucleotides in length and regulate post-transcriptional gene expression by binding to complementary sequences within mRNAs to alter the mRNA translation and mRNA stability [12]. There are approximately 1,000 miRNAs encoded by the human genome, with individual miRNAs targeting several mRNAs and individual mRNAs targeted by multiple miRNAs, participating in a complex regulatory network to ultimately alter cell behavior, important for normal cell functions [13]. MiRNAs play a major role in relation to cardiac functions, by regulating both normal cardiac development and functions, and as the underlying cause in pathological conditions [14].Several miRNAs regulate cardiac fibrosis by altering fibroblast proliferation, suppressing collagen expression, inhibiting connective tissue growth factor functions in cardiac interstitial cells [15]. The miR-21 gene, located on chromosome 17q23.2, displays a strong evolutionary conservation across vertebrates and is highly expressed in multiple cell types of the cardiovascular system, including endothelial cell, vascular smooth muscle cell, cardiomyocyte and cardiac fibroblast [16]. miR-21 plays a fundamental role in cardiac fibroblast and post-MI remodeling, influencing endothelium by activating the nitric oxide pathway and decreasing apoptosis [17]. MiR-21 might also lead to coronary atherosclerotic plaque instability by regulating MMP-9 expression levels via reversion-inducing cysteine-rich protein with Kazal motifs [18]. Thus, miR-21 has several regulatory roles in the cardiovascular system and could be a high value therapeutic target in MI. In this study, we established mouse MI model and transfected miR-21 into mice left ventricular cavity. MiR-21 expression levels, collagen I, fibronectin and α-SMA protein were detected to examine the cardioprotective mechanism of miR-21, with an aim of providing new therapeutic targets for the clinical treatment of MI.

Materials and Methods Animal Care C57BL/6 mice were purchased from the experimental animal center of the Third Hospital of Jilin University. All mice were kept under pathogen-free conditions at 23 ±1 °C, humidity 55 ± 5%, under a 12 h dark/light cycle and were allowed unlimited food and water for one week. All animal experiments were approved by the Animals Care and Use Committee of Jilin Province and all animal procedures complied with NIH guidance for the Care and Use of Laboratory Animals [19].

MI Model A total of 40 healthy, 12-week old C57BL/6 male mice weighing 25-30 g were randomized into the following groups: the sham group (n = 10), the MI group (n = 10), the blank virus group (Lentiviral vectors (LV)-GFP group, n = 10) and the miR-21 group (n = 10). The left anterior descending artery (LAD) was ligated to induce MI in the MI group, the LV-GFP group and the miR-21 group. Mice were quickly anesthetized with 2 % chloral hydrate intraperitoneal injection. A left thoracotomy was performed under a small animal ventilator (Harvard Apparatus, U.S.) with endotracheal intubation. The LAD was ligated with a silk suture, approximately midway between the left atrium and the apex of the heart. The judgment of successful MI model is based on the pale color of left ventricle anterior part and MI appearance on ECG oscilloscope. Mice

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chests were closed layer by layer with silk suture and mice were rewarmed in an incubator for 30 to 60 minutes. In the sham group, mice chests were opened /closed without ligation and other operations were identical.

LV Construction and Transfection

LV was purchased from Jikai Biological Company (Shanghai, China) and experiments followed standard procedures. Briefly, (LV)-miR-21-GFP was generated by cloning a 312 bp PCR fragment containing mouse miR-21 precursor hairpin loops into LV vector. RT-PCR was employed to amplify the miR-21 coding sequence. The LV-vector system containing pGC-FU plasmids, pDNA encoding miR-21 and two others pHelper plasmids were co-transfected into 293T cell. GFP was fused to the C-terminal of miR-21 to generate a recombinant for tracking miR-21 expression. Finally, virus titers were determined by qRT-PCR to detect GFP expression or by counting labeled cells. Approximately, 1×109 TU/mL miR-21 lentiviral particles were injected within the mice myocardium. Similar methods were followed to assemble blank lentiviruses, LV-GFP, containing only GFP. In the miR-21 group, left ventricular cavity was injected with (LV)-miR-21-GFP lentiviruses, while in LV-GFP group, left ventricular cavity was injected with LV-GFP lentiviruses.

Echocardiography Transthoracic echocardiographic analyses were undertaken on lightly anesthetized mice 2 weeks after MI to detect changes in cardiac function in miR-21 group and LV-GFP group. Two-dimensional short-axis images at the level of papillary muscles were obtained with a Vevo770 high-resolution system (VisualSonics Inc., Toronto, Canada) and a 40-MHz RMV 704 scan probe. Left ventricular internal diameter at end-diastole (LVIDD), 1eft ventricular end systolic dimension (LVESD), left ventricular fractional shortening (LVFS) and 1eft ventricular ejection fraction (LVEF) were calculated with Vevo Analysis software (version 2.2.3).

Masson Trichrome Staining for Infarct Size Left ventricles were excised and fixed by 10% zinc formalin solution and were embedded in paraffin and sectioned (10 μM thick) using standard protocols. Masson’s trichrome staining was performed following manufacturer’s protocol (Sigma-Aldrich, St Louis, MO, USA). Infarct size was calculated at 2 mm intervals from apex to the base from the average of four coronal section samples using the following formula: infarct size = [coronal infarct perimeter (epicardial plus endocardial)/total coronal perimeter (epicardial plus endocardial)] × 100. Vascular structures within 0.5 mm of the infarct border were observed under the microscope and scored by micrographs.

Real-Time Quantitative PCR

To detect the endogenous expression of miR-21 in the MI group, in MI and its surrounding areas, at 1st, 2nd and 4th week after ligation, and also in the Sham group, LV-GFP group and the miR-21 group at 2nd week after ligation, the total RNA was isolated from myocardial tissue by Trizol method (Invitrogen, USA) according to manufacturer’s protocol. The purity of total RNA was measured by UV spectrophotometer and the integrity of RNA was verified by agarose gel electrophoresis. Total microRNA cDNAs and miR-21 cDNA

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was generated with TaqMan MicroRNA Reverse Transcript Kit (ABI Company, US.). The miR-21 expression was detected using TaqMan MicroRNA Assay Kit (ABI, US.) with miRNA-specific TaqMan probes and real-time PCR primers. U6 snRNA was used as internal control and miR-21 relative expressions in each group were calculated using 2 –ΔΔCt method.

Immunohistochemical Staining

Immunohistochemical (IHC) staining detected fibronectin and α-SMA positive cells in the MI and surrounding areas. Paraformaldehyde-fixed (4%) and paraffin-embedded frozen specimens were sectioned at 5-μm intervals and the endogenous hydrogen peroxide was eliminated. For α-SMA and fibronectin staining, sections were immune-labeled with the primary antibodies: anti-α-SMA (clone 1A4; 1:200; Sigma–Aldrich), anti-fibronectin (1:200; Abcam, Cambridge, UK). Then horseradish peroxidase (HRP)-labeled secondary antibody or fluorochrome-conjugated secondary antibodies was labeled. A Dako EnVision + System-HRP method was used to visualize positive staining. Images were taken by an inverted microscope (IX71; Olympus, Tokyo, Japan) and the positive area was quantified by Image-Pro plus (Media Cybernetics, Bethesda, MD, USA). For the negative control, primary antibodies were replaced by PBS.

Western Blotting Expressions of Collagen I, α-SMA in the MI border zone and expressions of Bcl-2, Bax and Caspase-3 in the MI area were detected by western blotting. Samples from the left ventricle were homogenized and lysed. Briefly, proteins were extracted by in presence of 10mM PMSF, 1M EDTA and 1M Tris•Cl and protein concentrations were determined by the BCA Protein Assay. Equal amounts of total protein were mixed with 5 × SDS sample buffer, boiled for 5 min to acquire denatured protein and loaded on 10% PAGE gels with 5% stacking gels for separation. The separated proteins were then transferred the onto nitrocellulose membranes by semi-dry transfer. Next, membranes were blocked in Tris Buffered Saline with Tween containing 5% nonfat milk for 1 h at room temperature to block the nonspecific immunoglobulin binding sites. The membranes were the rinsed TBS-T three times (15 min for each time) and incubated with antibodies for 10h to 12h at 4 °C with gentle shaking and with the appropriate diluted primary antibody. Membranes were then washed three times (15 min for each time) in TBS⁄T. Subsequently, membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature and then washed three times in TBS⁄T for three mes. Protein was visualized using an enhanced chemiluminescence (ECL) detection kit and exposed to X-ray films. GAPHD was used as an internal control for grayscale comparison.

Apoptosis Detection by TUNEL Assay Terminal deoxynucleotidyl transferase (TdT) dUTP Nick End Labeling (TUNEL) assay was used to detect apoptotic cells that undergo extensive DNA fragmentation during the late stages of apoptosis. The experiment was carried out using an In Situ Cell Death Detection kit (Roche, Basel, Switzerland) to detect the apoptosis level in MI tissues, according to manufacturer protocols, and the apoptosis rate was calculated as the number of apoptotic cells / total cells × 100%.

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Statistical Analyses

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Statistical software package SPSS17.0 was used analyze the data. Data were presented as mean ± standard deviation (SD). Differences between the two groups were compared using Student’s t test; ANOVA-LSD (T) pairwise method was applied to compare differences. P values less than 0.05 in all analyses were considered statistically significant.

Results Changes in Infarct Area and Heart Function by miR-21

The heart function detected by M-mode echocardiography is shown in Figure 1. Two weeks after MI, both LVFS and LVEF in the miR-21 group significantly improved compared with the LV-GFP group (P < 0.05), but was lower than the sham group (P < 0.05). However, no significant differences in LVIDD were detected between the miR-21 group and the LV-GFP group (P > 0.05). Figure 2 shows the comparison of infarct area between the miR-21 group and the LV-GFP group by Masson staining, demonstrating a clear thickening of scar tissues, a decrease of localized swelling, and shrinking of heart expansion in infarct area after transfection with miR-21 mimics. In addition, the Masson staining revealed that the infarct area in the miR-21 group reduced by 36.9% compared with the LV-GFP group. Expression of miR-21 in the MI Zone and MI Border Zone

Compared with the sham group, endogenous miR-21 levels in the MI group were significantly decreased in the 1st week and 2nd week after ligation (P < 0.05), but recovered to normal levels in the 4th week (P > 0.05) (Figure 3). Figure 4 shows that the miR-21 expression levels of the LV-GFP group and the miR-21 group in MI zone and MI border zone at 2nd week after MI were markedly lower than the sham group (all P < 0.05). However, there was no detectable difference in miR-21 expression levels between LV-GFP group and the miR-21 group in the MI zone and the MI border zone at 4 week after MI (all P > 0.05) (Figure 4).

MiR-21 Transfection Reducing Myocardial Fibrosis after MI Immunohistochemistry showed lower expressions of collagen I and fibronectin, and lower number of α-SMA positive cells in the miR-21 group compared to LV-GFP group (Figure 5). Western blotting showed that, compared with sham group, increased collagen I and fibronectin expressions and increased number of α-SMA positive cells were found in the miR-21 group and the LV-GFP group to different extents. A remarkably lower levels of collagen I and α-SMA protein expression was observed in the miR-21 group compared to the LV-GFP group (Figure 6). Apoptosis-related factors Bcl-2, Bax and Caspase-3 affected by miR-21 transfection Western-blot showed that, compared with the sham group, Bax and Caspase-3 levels increased but ratio of Bcl-2/Bax decreased in the LV-GFP group (all P < 0.05), suggesting apoptosis was caused by MI treatment. Compared with the LV-GFP group, Bcl-2 level and ratio of Bcl-2/Bax was significantly increased, while the levels of Bax and Caspase-3 were decreased in the miR-21 group (all P < 0.05) (Figure 7).

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Regulation of Apoptosis Levels after MI by miR-21

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TUNNEL staining was used to detect apoptosis in the MI zone and MI border zone after MI at one week. The result demonstrated markedly lower apoptotic cells (positive staining cells) in the miR-21 group compared to the LV-GFP group (Figure 8).

Discussion MI is a common presentation of coronary artery disease (CAD) and is characterized by acute myocardial necrosis induced by persistent and severe myocardial ischemia [20]. Thrombolytic therapy and percutaneous coronary intervention, for MI treatment, can open-up coronary infarction, save the dying myocardium and improve cardiac functions [21,22]. MiR-21 is highly expressed in all solid tumors, and promotes proliferation and migration of tumor cells [23]. MiR-21 is involved in cardiovascular diseases, and plays a regulatory role in early pathological process of MI, suggesting that miR-21 might be potential therapeutic target for MI [24]. One of the most important findings in our current study was that, after transfection of miR-21, the area of MI in the miR-21 group significantly decreased compared to LV-GFP group, implying that miR-21 played a protective role in MI. MiR-21 is highly expressed in almost all the main cell types of the cardiovascular system, including cardiac fibroblast, cardiomyocyte, endothelial cell and vascular smooth muscle cell (VSMC). In addition, miR-21 plays a vital role in the proliferation and apoptosis of VSMC and cardiac cells, and is important for cardiac fibroblast functions [17]. Masson staining in our study showed that after transfection of miR-21, scar tissue in the MI area was remarkably thick, accompanied by reduced local swelling and significant improvement in the degree of cardiac enlargement.

Another significant result in our study was that miR-21 expression levels of the LV-GFP group and the miR-21 group in the MI zone and the MI border zone at 2nd week after MI were lower than the sham group. The potential explanation might be that up-regulated expression of miR-21 in myocardial infarction zone caused a specific repression of downstream tensin homolog and effector phosphatases [25]. In addition, studies have indicated that miR-21 regulates cardiac angiogenesis, fibrosis and cardiomyocyte hypertrophy in MI [15,26]. Thum T et al. showed that down-regulation of miR-21 in heart, under hypertrophy-inducing conditions, reduces cardiomyocyte size, interstitial fibrosis and heart weight [27].

Our study also indicated that overexpression of miR-21 decreased collagen deposition in the peripheral areas of MI, reduced myocardial cell apoptosis and improved heart function, implying that miR-21 apoptosis of myocardial cells under hypoxic conditions. Overexpression of miR-21 reduced myocardial cell apoptosis induced by tumor necrosis factor α (TNF-α). Also, miR-21 regulates collagen synthesis of cardiac fibroblasts to regulate fibrosis in cardiac hypertrophy [28]. Notably, Smad 7 inhibition and overexpression of miR-21 can promote TGF-β functions on the differentiation and proliferation of fibroblasts, and further promote repair of heart tissue after MI, providing novel therapeutic areas for the effective prevention of cardiac rupture after MI [29]. TGF-β signaling pathway is the main pathway in the regulation of proliferation and differentiation of fibroblasts, and this pathway plays an important protective role in MI [30,31]. The overexpression of miR-21 can significantly activate the differentiation and proliferation of fibroblasts by regulating the expression of TGF-β receptor III (TGF-βRIII). Type I collagen and type III collagen are the main component of myocardial interstitial cells [32]. These two collagen tyoes constitute the spatial and structural network supporting the heart shape, and the ratio between them determined the heart strength and compliance. In pathological conditions, changes in collagen content and

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component results in remodelling of cardiac structure and function [33]. Bax exhibits pro-apoptotic effect whereas Bcl-2 has an anti-apoptotic effect, the ratio of Bcl-2 to Bax is an effective predictor for anti-apoptotic functions [34]. Caspase-3, a well-known executioner of apoptosis, activates many cell death proteins, such as poly ADP-ribose polymerase [35]. Limitations of our study are acknowledged. Importantly, the sample size was not large enough. Second, our study investigated the role of miR-21 in a few pathways, however miR-21 has diverse functions and did not further verify contribution of other miR-21 pathways to the observed effects in our study. Thus, whether miR-21 is involved in cardiac remodeling through other molecular pathways remains to be determined. Consequently, more studies with much larger sample sizes and more comprehensive information are needed to further confirm our findings.

Conclusions In conclusion, our study provides evidence, using mouse model of MI, that overexpression of miR-21 decreases collagen deposition in the area surrounding MI by reducing the expressions of collagen I and fibronectin, and by decreased number of α-SMA positive cells, reducing myocardial cell apoptosis by decreasing Bax and Caspase-3 levels and increasing Bcl-2/Bax ratio, further reducing the MI area and improving heart function. In addition, the findings in our study identified novel targets and pathways for MI therapy, and potentially anti-fibrosis therapy for other cardiac diseases.

Acknowledgments We would like to thank the director in the Department of Cardiology, the Third Hospital of Jilin University for his supporting. We also appreciate the hard work of our researchers and the valuable advice from reviewers.

Competing Interests All authors in our study have no conflict of interest.

Funding This work is supported by NSFC (NO. 81300150 and NO. 81370790) and Foundation of Shanghai Municipal Science and Technology Commission (NO. 124119a7000).

Author Contributions Gong-Liang Guo and Ke Ma were involved in the conception and design of the study. Gong-Liang Guo and Yuan-Yuan Huang were responsible for the analysis of data. Yuan-Yuan Huang and Li-Qun Sun drafted the manuscript, and all authors have revised the article for important intellectual content. Li-Qun Sun is the corresponding author. All authors have read and approved the final manuscript.

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Legends Figure 1 Change of heart functions in the miR-21 group, the LV-GFP group and the sham group two weeks after MI detected by M-mode echocardiography. miR-21, microRNA-21; MI, myocardial infarction; LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; LVIDD, left ventricular internal diameter at end-diastole. Figure 2 Decreased infarct size after MI in the miR-21 group compared with the LV-GFP group detected by Masson staining. miR-21, microRNA-21; MI, myocardial infarction; LV-GFP, lentiviruses-GFP; RV, right ventricular; LV, left ventricular.

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Figure 3 Expressions of miR-21 levels in the MI group in the 1st week, 2nd week and 4th week after MI treatment. miR-21, microRNA-21; MI, myocardial infarction. Figure 4 Expressions of miR-21 levels in the sham group, the LV-GFP group and the miR-21 group after suffering MI. miR-21, microRNA-21; MI, myocardial infarction; LV-GFP, lentiviruses-GFP.

Figure 5 Fibrosis progress by MiR-21 regulation two weeks after MI in the sham group, the LV-GFP group and the miR-21 group. miR-21, microRNA-21; MI, myocardial infarction; LV-GFP, lentiviruses-GFP. Figure 6 Expression of COL-1 and a-SMA in the MI border zone regulated by miR-21 in the sham group, the miR-21 group and the LV-GFP group. miR-21, microRNA-21; MI, myocardial infarction; LV-GFP, lentiviruses-GFP; COL-1, collagen I. Figure 7 Western blot detecting Bcl-2, Bax and Caspase-3 protein levels. Compared with Sham group, aP < 0.01; compared with the LV-GFP group, bP < 0.05, cP < 0.01. Figure 8 Regulation of apoptosis levels after MI in the sham group, the miR-21 group and the LV-GFP group (Tunnel staining positive cells were yellow-green, DAPI was blue, white arrows indicated Tunnel-positive cells). miR-21, microRNA-21; MI, myocardial infarction; LV-GFP, lentiviruses-GFP.

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Cardioprotective Effect of MicroRNA-21 in Murine Myocardial Infarction.

To investigate the cardioprotective effect of MicroRNA-21 (miR-21) in murine myocardial infarction (MI)...
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