Introduction
Cardiovascular disease (CVD) is one of the primary diseases currently threatening human health. In particular, acute myocardial infarction (AMI) is a factor that primarily results in increasing morbidity of CVD patients (1). Therefore, early, timely and accurate diagnosis and evaluation of AMI are essential for actively administrating effective treatment (2). Furthermore, early diagnosis may result in timely cardiac reperfusion and reduce mortality (2). The early treatment of AMI is crucial in the favorable prognosis of patients. At present, creatine kinase isozyme and myoglobin are the predominant serum biomarkers used for the clinical diagnosis of AMI (3). Notably, the aforementioned markers have attained certain levels in sensitivity and specificity of the early diagnosis of AMI (3). However, researchers have continued their efforts in studying novel serum markers with increased sensitivity and specificity (4). Serum markers reflecting the prognosis for AMI patients are of primary research interest (4).
It has previously een demonstrated that non-coding microRNAs (miRNAs) are closely associated with the genesis, development and prognosis of all diseases (5). miRNA expression demonstrates tissue specificity and high stability in the blood (6). Therefore, myocardium specific miRNAs have been speculated to act as ideal biomarkers for the early diagnosis of AMI (5). Furthermore, miRNA is important in numerous pathophysiological processes. These include the genesis and development of Myocardial Infarction (MI), myocardial fibrosis following MI and myocardial remodeling (7). A previous study suggested that miRNA is crucial in the genesis and development of human diseases, including tumor initiation, cardiovascular disease, diabetes, immune system and renal diseases (7). Furthermore, the effects of miRNAs generally manifest as a complicated regulatory network formed by multiple miRNAs during regulation of disease genesis and development (7). Therefore, the study of miRNA is of great importance to the understanding of genesis and developmental mechanisms of various diseases.
Under normal physiological status, the myocardium is energy-supplemented primarily via fat oxidation. However, in the case of an insufficient coronary blood supply, the myocardium is under anoxic conditions (8) and fatty acid oxidation efficiency is low (8). Furthermore, glucose and glycogenolysis for energy supply only account for a small part of aerobic metabolism (9). The persistent ischemia and hypoxia results in irreversible mitochondrial injury and myocardial cell death (9). Cell death occurs in three ways: Necrosis, apoptosis and autophagy (10). Apoptosis and autophagy have previously been demonstrated to be involved in ischemia reperfusion injury (10).
The mechanistic target of rapamycin (mTOR) signaling pathway receives and integrates multiple signals (11). These signals include amino acids, glucose, oxidative stress and growth factors (11). Therefore, mTOR exhibits an important regulatory role in cell growth, proliferation, and protein synthesis (12). mTOR signaling has been verified to be one of the canonical autophagy regulatory pathways (13). It is the signaling pathway that senses the cell nutritional status (13) and primarily exerts an autophagy-associated inhibitory effect on cardiac cell apoptosis, whilst stimulating cell growth and proliferation (13).
The phosphoinositide 3-kinase (PI3K)/RAC-γ serine/threonine-protein kinase (Akt)/mTOR signaling pathway exhibits a critical regulatory role in autophagy. A previous study suggests that research has been successful in identifying inhibitory drugs targeting various signaling pathways in order to treat growth of tumors (14). mTOR is the downstream molecule of Akt in the PI3K/Akt regulatory pathway. It is involved in regulating protein synthesis, cell cycle and angiogenesis. PI3K/Akt/mTOR signaling pathway is a core pathway that promotes cell growth, movement, protein synthesis, survival, and hormone, growth factor and nutrient metabolism (15). The present study investigated the effects of miRNA-145 on AMI and the potential underlying mechanism.
Materials and methods
Ethics and AMI model
Adult male Sprague-Dawley (180–220 g; n=12) rats were maintained in cages at 21±2°C, under a 12 h light-dark cycle, with 55±5% constant humidity, and had free access to food and water. All rats were randomized into two groups: Control (n=10) and AMI model groups (n=10). All animal experimentation was performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals of Cangzhou Central Hospital, Hebei Medical University (Cangzhou, China), and approved by the Ethics Committee of Cangzhou Central Hospital. Rats were anesthetized with pentobarbital sodium (35 mg/kg, i.p.) and exposed following the skin incision in the fourth intercostal space of a left thoracotomy. A snare occlude was used to ligate the left anterior descending coronary artery with 6-0 silk suture. Cardiac ischemia was verified by visual observation and continuous electrocardiogram monitoring. The coronary artery was reperfused by releasing the knot following 1 h of occlusion. A total of 3 h following induction of AMI, cardiac tissue was harvested.
Haematoxylin and eosin (H&E) staining
Cardiac tissue was harvested under pentobarbital sodium (35 mg/kg, i.p.) and washed with PBS. Cardiac tissue was fixed with 4% paraformaldehyde for 24 h and embedded into paraffin. Samples were cut into 4.0 µm of sections and sections were stained with H&E for 5 min at room temperature and observed under a LSM 780 NLO confocal microscope (Carl Zeiss AG, Oberkochen, Germany).
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and gene expression microarrays
Total RNA was extracted from tissue samples or cells using TRIzol® kit (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and total RNA (200 ng) was then reverse transcribed to cDNA using an RT kit (Takara Biotechnology Co., Ltd., Dalian, China). RT-qPCR was performed using SYBR Premix Ex TaqII (Takara Biotechnology Co., Ltd.) and amplification occurred under the following conditions: Pre-denaturation for 10 min at 94°C; 40 cycles of 30 sec at 94°C, 30 sec at 55°C and 30 min at 72°C, followed by an extension step for 10 min at 72°C. Primers sequences sued were as follows: miRNA-145 forward, 5′-GGT CCA GTT TTC CCA GG-3′ and reverse 5′-CAG TGC GTG TCG TGG AGT-3′; U6 forward, 5′-AGA AGG CTG GGG CTC ATT TG-3′ and reverse 5′-AGG GGC CAT CCA CAG TCT TC-3′. Data using RT-qPCR was quantified using the 2−∆∆Cq method (16).
A total of 500 ng total RNA was used to execute the gene expression microarrays, and amplified using the Ovation PicoSL WTA System V2 kit (Nugen Technologies. Inc., CA, USA). cDNA samples were Cy3-labeled using the SureTag DNA labeling kit (Agilent Technologies, Inc., Santa Clara, CA, USA). The scanning was conducted using a SureScan Microarray Scanner and Feature Extraction software, version 10.7.3.1 (Agilent Technologies, Inc.).
Cell culture and cell transfection
The H9c2 cell line was cultured and maintained in Dulbecco's minimum essential medium (DMEM, Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) at 37°C, in an environment containing 5% CO2. 100 ng of miRNA-145 mimics (5′-GUC CAG UUU UCC CAG GAA UCC CU-3′), 100 ng of miRNA-145 inhibitors (5′-AGG GAU UCC UGG GAA AAC UGG AC-3′) and 100 ng of negative control (5′-CAG UAC UUU UGU GUA GUA CAA-3′) were transfected into H9c2 cells using Lipofectamine 2000 (Thermo Fisher Scientific, Inc) at 37°C according to the manufacturer's protocol. After transfection for 4 h, old medium was removed and new DMEM was added into H9c2 cells for 20, 44 or 68 h. Then, H9c2 cells were subjected to a hypoxia/reoxygenation protocol for 2 h.
Luciferase reporter gene assay
Bioinformatics software on http://www.targetscan.org was adopted to predict the targeted correlation between miRNA-145 and Akt3. AKT3-3′UTR-WT plasmid and miR-145 mimics were constructed by Shanghai GenePharma, Co., Ltd (Shanghai, China) and transfected using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.). The expression of reporter gene was presented using luminometer reading (TD20/20; Turner Designs, Sunnyvale, CA, USA) by the activity ratio of firefly luciferase and renilla luciferase.
Western blotting
Following cell transfection (n=3) for 48 h, cells were washed with PBS, and total protein was extracted using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Nanjing, China), and then quantified using a bicinchoninic acid assay (Beyotime Institute of Biotechnology). A total of 50 µg total protein was separated by 8–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride membrane. The membrane was blocked with 5% milk in Tris buffered saline Tween-20 (TBST) and incubated with primary antibodies against B cell lymphoma 2 associated apoptosis regulator (Bax, cat. no. sc-6236; 1:500; Santa Cruz Biotechnology; Inc., Dallas, TX, USA), p-Akt (cat. no. sc-7985-R; 1:500; Santa Cruz Biotechnology; Inc.), p-mTOR (cat. no. sc-101738; 1:500; Santa Cruz Biotechnology; Inc.), microtubule associated protein 1 light chain 3 (LC3, cat. no. sc-292354; 1:500; Santa Cruz Biotechnology; Inc.) and GAPDH (cat. no. sc-25778; 1:500; Santa Cruz Biotechnology; Inc.) at 4°C overnight. Following this, the membrane was washed with TBST and incubated with goat anti-rabbit IgG-horse radish peroxidase (cat. no. sc-2004; 1:5,000; Santa Cruz Biotechnology; Inc.) for 1 h at 37°C. Protein bands were exposed by BeyoECL Moon (Beyotime Institute of Biotechnology) and analyzed using Bio-Rad Laboratories Quantity One software, version 3.0 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Immunofluorescence
Following cell transfection, (n=3) for 48 h, cells were washed with PBS for 15 min and fixed with 4% paraformaldehyde for 15 min at room temperature. Cells were incubated with 0.1% Triton X-100 for 15 min at room temperature and blocked with 5% bovine serum albumin (Beyotime Institute of Biotechnology) in PBS for 1 h at room temperature and incubated with the primary antibody against LC3 (cat. no. sc-292354; 1:100; Santa Cruz Biotechnology; Inc.), p-Akt (cat. no. sc-7985-R; 1:100; Santa Cruz Biotechnology; Inc.) at 4°C overnight. Following washing with PBS, cells were incubated in a mixture of fluorescent secondary antibody (Alexa 488 anti-mouse immunoglobulin G; 1:100, cat. no. sc-516248; Santa Cruz Biotechnology; Inc.) for 1 h at room temperature, incubated with DAPI assay for 30 min and analyzed using a LSM 780 NLO confocal microscope (Carl Zeiss AG, Oberkochen, Germany).
MTT assay
Following cell transfection (n=3) for 24, 48 or 72 h, cells were stained with 20 µl MTT (5 g/l, G3582; Promega Corporation, Madison, WI, USA) for 4 h at 37°C, in an incubator in an environment containing 5% CO2. A total of 150 µl dimethyl sulfoxide was added to cells and shaken for 10 min. Cell proliferation was measured using a microplate reader (SpectraMax M5; Molecular Devices, LLC, Sunnyvale, CA, USA) at a wavelength of 490 nm.
Flow cytometry
Following cell transfection (n=3) for 48 h at 37°C, cells (1×106 cell/ml) were collected at 1,000 × g for 10 min at 4°C and washed with PBS, fixed with 4% paraformaldehyde for 15 min at room temperature and resuspended with 150 µl binding buffer. A total of 10 µl Annexin V-FITC and 5 µl propidium iodide (BB-4101-2; BestBio Science., Shanghai, China) staining solution were added to the cells for 15 min, in the dark. Flow cytometry was used to detect cell apoptosis and analyzed using Flowjo 7.6.1 (FlowJo, LLC,).
Statistical analysis
Data are presented as the mean ± standard deviation (n=3) using SPSS software, version 17.0 (SPSS Inc., Chicago, IL, USA), and were analyzed by one-way analysis of variance (ANOVA) or two-way ANOVA followed by Tukey's post-hoc test. P<0.05 was considered to indicate a statistically significant difference.
Results
Expression of miRNA-145 in AMI in vivo model
Firstly, the present study measured the alteration of miRNAs in the AMI in viv model using the microarray gene chip method. As presented in Fig. 1A, H&E staining of heart tissue indicated that there was myocardial damage in the AMI model, compared with normal group. miRNA-145 expression was downregulated in the AMI rat model, compared with control group (Fig. 1B). In addition, miRNA-145 expression was analyzed using RT-qPCR. Fig. 1C demonstrated that miRNA-145 expression was downregulated in the AMI rat model, compared with control group. It was therefore hypothesized that miRNA-145 may be a regulator for AMI.
Downregulation of miRNA-145 increases cardiac cell apoptosis in an in vitro model of AMI
To test the function of miRNA-145 in an in vitr model of AMI, the present study downregulated miRNA-145 expression levels using anti-miRNA-145 inhibitor. As presented in Fig. 2A, anti-miRNA-145 mimics decreased miRNA-145 expression, compared with control group. Downregulation of miRNA-145 inhibited cell proliferation and increased apoptosis rate, compared with control group (Fig. 2B–D). Downregulation of miRNA-145 additionally promoted caspase-3 and -9 activities, and induced Bax protein expression, compared with control group (Fig. 2E–H).
Downregulation of miRNA-145 suppresses autophagy in an in vitro model of AMI
To validate the mechanism of miRNA-145 on apoptosis in AMI, the present study measured the alterations of autophagy. Downregulation of miRNA-145 suppressed LC3 and ATG5 protein expression compared with control group (Fig. 3A–C). Immunofluorescent staining demonstrated that downregulation of miRNA-145 suppressed LC3 protein expression compared with control group (Fig. 3D).
Downregulation of miRNA-145 suppresses p-Akt3 and p-mTOR protein expression in an in vitro model of AMI
Bioinformatics software (http://www.targetscan.org) was used to analyze the targeted association between miRNA-145 and Akt3. As presented in Fig. 4A, Akt3 was predicted to be the target gene of miRNA-145. The results of the western blotting demonstrated that downregulation of miRNA-145 suppressed p-Akt3 and p-mTOR protein expression, compared with control group (Fig. 4B–D). The immunofluorescent staining results presented in Fig. 5 revealed that downregulation of miRNA-145 suppressed p-Akt3 protein expression, compared with control group.
Overexpression of miRNA-145 decreases cardiac cell apoptosis in an in vitro model of AMI
To demonstrate the function of miRNA-145 in cardiac cell apoptosis, the present study used miRNA-145 mimics to increase miRNA-145 expression. As presented in Fig. 6A, miRNA-145 mimics increased miRNA-145 expression, compared with control group. Overexpression of miRNA-145 promoted cell proliferation, and decreased cardiac cell apoptosis, compared with control group (Fig. 6B–D). The overexpression of miRNA-145 reduced Bax protein expression and inhibited caspase-3/9 activities compared with control group (Fig. 6E–H).
Overexpression of miRNA-145 induces p-Akt3 and p-mTOR protein expression and promotes autophagy in an in vitro model of AMI
The overexpression of miRNA-145 promoted LC3 and AGT 5 protein expression, compared with control group (Fig 7A–C). Immunofluorescent staining indicated that the overexpression of miRNA-145 promoted LC3 protein expression compared with control group (Fig. 7D). In addition, it was demonstrated that p-Akt3 and p-mTOR protein expression levels were increased, compared with control group (Fig. 7E–G).
Inhibition of Akt3 suppresses the effects of miRNA-145 upregulation on cell apoptosis in an in vitro model of AMI
The present study next explored the function of Akt3 in the effects of miRNA-145 upregulation on cell apoptosis. As presented in Fig. 8A–C, administration of 1 nM Akt3 inhibitor GSK2110183 following miRNA-145 upregulation, suppressed p-Akt3 and p-mTOR protein expression, compared with upregulated miRNA-145 group. Furthermore, the inhibition of Akt3 following miRNA-145 upregulation suppressed LC3 and AGT5 protein expression, compared with upregulated miRNA-145 group (Fig. 8D–G). The effects of miRNA-145 upregulation on cell proliferation, cardiac cell apoptosis and Bax protein expression, in addition to caspase-3/9 activities, were reversed by Akt3 inhibitor, compared with upregulated miRNA-145 group (Fig. 9).
Lysosomal inhibitors inhibit the effects of miRNA-145 upregulation on cell apoptosis in an in vitro model of AMI
Exposure to a total of 5 µM lysosomal inhibitor chloroquine diphosphate following miRNA-145 upregulation increased Bax protein expression levels compared with miRNA-145 upregulated group (Fig. 10A–C). In addition, chloroquine diphosphate reduced cell growth, increased cardiac cell apoptosis, and promoted caspase-3 and caspase-9 activities, compared with miRNA-145 upregulated group (Fig. 10D–H).
Discussion
AMI is the primary cause of coronary heart disease-associated mortalities (1). Early thrombolytic therapy or percutaneous coronary intervention has been adopted to recover blood perfusion in the ischemic site. This method effectively rescues the injured myocardium (1) and improves myocardial ischemia and necrosis (2). However, adverse effects include partial myocardial cell apoptosis and loss of heart function. These effects are termed ischemia reperfusion injury (2). At present, the potential cellular mechanism leading to ischemia reperfusion injury remains to be fully elucidated (3). The present study observed that miRNA-145 expression was downregulated in the AMI rat model, compared with control group.
miRNA is a highly conserved small non-coding RNA molecule (17) that regulates mRNA gene expression through complementary base pairing with mRNA (17). miRNA is intracellular RNA (17) and may regulate the post-transcription expression levels of multiple mRNAs. Therefore, it exhibits the potential to act as a useful therapeutic target. Numerous studies have been conducted researching the pathogenesis of CVD (18). Furthermore, miRNA alterations have been demonstrated to be involved in angiogenesis, myocardial hypertrophy, heart failure and myocardial fibrosis (18). The present study demonstrated that the downregulation of miRNA-145 increased cardiac cell apoptosis in an in vitr model of AMI. In accordance with the findings, Zhang et al (19) additionally suggests that miRNA-145 levels decrease in AMI.
Autophagy is commonly seen in acute and chronic myocardial ischemia and heart failure. It has previously been demonstrated that autophagy is markedly upregulated in ischemia reperfusion myocardial cells (20). Cell autophagy is the process by which lysosomes in the eukaryote degrade the damaged substances in the cell (21). In this process, various damaged proteins or organelles are consumed by the autopha-gosome with a bilayer structure (21). They are sent to the lysosome (animal) or vacuole (yeast and plant) for degradation, or are recycled (21). The results of the present study revealed that downregulation of miRNA-145 suppressed autophagy in an in vitr model of AMI. Higashi et al (22) suggests that miRNA-145 repairs infarcted myocardium by accelerating cardiomyocyte autophagy (21).
PI3K activates the serine/threonine Akt. Akt leads to the phosphorylation of serine/threonine mTOR via numerous regulators (23). mTOR is a serine/threonine protein kinase (23), highly conserved from fungus to mammal. The primary effect of mTOR is to regulate the cell cycle, cell growth and proliferation (24). Therefore, mTOR in mammals maintains a constant state of activation. This results in a dynamic balance between cell growth and metabolism. Research has verified that mTOR has a role as an active switch in regulatory cell autophagy. It senses the alterations of multiple intracellular and extracellular signals. In addition to this, it activates or inhibits the rate of autophagy (24). Subsequently, it enhances cell adaptability to environmental stress (24). The present study observed that the downregulation of miRNA-145 suppressed p-Akt3 and p-mTOR protein expression. Furthermore, the inhibition of Akt3 decreased the effects of miRNA-145 upregulation on cell apoptosis. The lysosomal inhibitor chloroquine diphosphate inhibited the effects of miRNA-145 upregulation on autophagy to adjust cell apoptosis, in an in vitr model of AMI (Fig. 11). Zhou et al (25) reported that an increase in miRNA-145 inhibits the proliferation and invasion of invasive pituitary adenoma cells through AKT3/mTOR signaling pathway in viv and in vitro (25).
In conclusion, the results of the present study demonstrated that miRNA-145 inhibited myocardial infarction-induced apoptosis by induction of autophagy via the Akt3/mTOR signaling pathway, in viv and in vitro. This finding increases the understanding of miRNA in the field of molecular biology research, and will potentially be useful for treating myocardial infarction patients in the future. However, further analysis and larger samples are necessary in order to validate the results.