A total of 30 blood samples were collected from 30 patients with AMI (18 males and 12 females, aged 27–57 years old) who received percutaneous coronary intervention at the coronary care unit of the Second Affiliated Hospital of Harbin Medical University (Harbin, China) between May 2016 and May 2017. For patients with AMI, the following exclusion criteria applied: Previous history of myocardial infarction; having received percutaneous coronary intervention treatment; having acute heart failure upon admission; having myocardial disease, infectious pericarditis or pericardial disease; having an infectious disease, severe diabetes mellitus, malignant tumor, liver/kidney disease, pulmonary fibrosis, bone metabolic disorder, systemic immune disease or complications caused by malignant tumors; having cardiac shock. A total of 30 blood samples from 30 healthy individuals (15 males and 15 females, aged 28–61 years) admitted for general physical examination at the Second Affiliated Hospital of Harbin Medical University (Harbin, China) between May 2016 and May 2017 were recruited as the control group. None of the healthy individuals had a history of heart disease, vascular disease or other major diseases. Blood samples (5 ml) were collected from patients 6 h following the onset of AMI and frozen at −80°C prior to the extraction of total RNA. The present study was approved by the Ethical Committee of The Second Affiliated Hospital of Harbin Medical University. All patients provided written informed consent.

The rat cardiomyocyte cell line H9c2 was obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were grown in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin mixed solution (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The cells were incubated to confluence at 37°C with 5% CO₂ and passaged every 2–3 days (7).

A cell model of myocardial hypoxia/ischemia injury was established by exposing H9c2 cardiomyocytes to hypoxia for 48 h. In brief, H9c2 cells were cultured under standard conditions to reach 80% confluence, and subsequently grown in a hypoxia chamber (Thermo Fisher Scientific, Inc.) containing a gas mixture of 1% O₂, 5% CO₂ and 94% N₂ in a humidified incubator (Thermo Fisher Scientific, Inc.) at 37°C for 48 h.

TargetScan 7.1 bioinformatics software (www.targetscan.org/vert_71) was employed to identify putative targets of miR-124-3p. It was revealed that nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) repressing factor (NKRF) may be a target of miR-124-3p. To investigate this prediction, the wild-type (NKRF-WT) and mutant (NKRF-MUT) 3′-UTR of NKRF was cloned into a pmiR-RB-Report™ dual luciferase reporter gene plasmid vector (Guangzhou RiboBio Co., Ltd., Guangzhou, China). To point-mutate the miR-124-3p binding domain in the 3′-UTR of NKRF, a QuikChange Site-Directed Mutagenesis kit (Stratagene; Agilent Technologies, Inc., Santa Clara, CA, USA) was used, according to the manufacturer's protocols. H9c2 cells were co-transfected with 100 ng NKRF-WT or NKRF-MUT plasmid and 50 nM miR-124-3p mimic (5′-UAAGGCACGCGGUGAAUGCC-3′; Shanghai GenePharma Co., Ltd., Shanghai, China) or mimic control (5′-UUCUCCATCGUGCCUCUAT-3′; Shanghai GenePharma Co., Ltd.) using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. Luciferase activity was determined 48 h following transfection at 37°C using the Dual-Luciferase Assay system (Promega Corporation, Madison, WI, USA), according to the manufacturer's protocols, and was normalized to Renilla luciferase activity. Experiments were repeated in triplicate.

H9c2 cells were seeded in 6-well plates (4×10⁵ cells/well). Subsequently, 100 nM negative control of miR-124-3p inhibitor (NC; 5′-CCGUACUUCGCUAGAUCA-3′; Shanghai GenePharma Co., Ltd.), 100 nM miR-124-3p inhibitor (5′-UAAGGCACGCGGUGAAUGCC-3′; Shanghai GenePharma Co., Ltd.), 1 µM control-small interfering RNA (siRNA; cat no. sc-36869; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), 1 µM NKRF-siRNA (cat no. sc-72275; Santa Cruz Biotechnology, Inc.) or 100 nM miR-124-3p inhibitor + 1 µM NKRF-siRNA was transfected into H9c2 cells using Lipofectamine 3000, according to the manufacturer's protocols. Cells were subjected to subsequent experiments 48 h following transfection at 37°C. Transfection efficiency was determined via reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis.

The expression of miR-124-3p and other genes was determined via RT-qPCR analysis. Total RNA was extracted from blood samples and cells using TRIzol^® reagent (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. cDNA was synthesized using the Moloney Murine Leukemia Virus RT kit (Takara Biotechnology Co., Ltd., Dalian, China), according to the manufacturer's protocols. Reaction conditions for RT were: 50°C for 5 min and 80°C for 2 min. qPCR was performed on the synthesized cDNAs with SYBR Green I (Applied Biosystems; Thermo Fisher Scientific, Inc.) using a CFX Connect Real-Time system (Bio-Rad Laboratories, Inc.) according to the manufacturers' protocols. The primer sequences for qPCR were as follows: GAPDH, forward 5′-CTTTGGTATCGTGGAAGGACTC-3′, reverse 5′-GTAGAGGCAGGGATGATGTTCT-3′; U6, forward 5′-GCTTCGGCAGCACATATACTAAAAT-3′, reverse 5′-CGCTTCACGAATTTGCGTGTCAT-3′; miR-124-3p, forward 5′-GCTAAGGCACGCGGTG-3′, reverse 5′-GTGCAGGGTCCGAGGT-3′; tumor necrosis factor-α (TNF-α), forward 5′-GAACTGGCAGAAGAGGCACT-3′, reverse 5′-GGTCTGGGCCATAGAACTGA-3′; interleukin (IL)-1β, forward 5′-TGTGAAATGCCACCTTTTGA-3′, reverse 5′-TGAGTGATACTGCCTGCCTG-3′; IL-6, forward 5′-CCGGAGAGGAGACTTCACAG-3′, reverse 5′-CAGAATTGCCATTGCACA-3′; and NKRF, forward 5′-TATTGATATTGGGGAGATGCC-3′ and reverse 5′-GGATCTTCCTGTCTTTCATCT-3′. Thermocycling was conducted as follows: 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 15 sec and annealing/elongation at 60°C for 30 sec. GAPDH (for mRNA) and U6 (for miR-124-3p) were used as internal controls. The 2^−ΔΔCq method (23) was used to determine the relative gene expression. Experiments were repeated in triplicate.

To measure the protein levels of NKRF, phosphorylated (p)-p65, B-cell lymphoma 2 (Bcl-2), and pro-caspases and cleaved caspases 3 and 9, western blot analysis was performed. Total protein was extracted from H9c2 cells using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China), according to the manufacturer's protocols. The concentration of protein was determined using a Bicinchoninic Acid Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.). Lysate samples (25 µg/lane) were separated via 12% SDS-PAGE and transferred onto polyvinylidene difluoride membranes prior to blocking with 5% skimmed milk at room temperature for 1.5 h. The membranes were incubated overnight at 4°C with primary antibodies against: NKRF (1:1,000; cat. no. ab168829; Abcam, Cambridge, MA, USA), p-p65 (1:1,000; cat no. 3033; Cell Signaling Technology, Inc., Danvers, MA, USA), p65 (1:1,000; cat no. 8242; Cell Signaling Technology, Inc.), Bcl-2 (1:1,000; cat no. 4223; Cell Signaling Technology, Inc.), cleaved caspase 3 (1:1,000; cat no. 9664; Cell Signaling Technology, Inc.), cleaved caspase 9 (1:1,000; cat no. 9505; Cell Signaling Technology, Inc.), pro-caspase 3 (1:1,000; cat. no. ab32150; Abcam), pro-caspase 9 (1:1,000; cat. no. ab135544; Abcam) and β-actin (1:1,000; cat. no. 4970; Cell Signaling Technology, Inc.). Membranes were subsequently incubated at room temperature for 4 h with the horseradish peroxidase-conjugated anti-rabbit immunoglobulin G antibodies (cat no. 7074; 1:2,000; Cell Signaling Technology, Inc.). Protein bands were visualized using a SuperSignal West Dura Extended Duration Substrate enhanced chemiluminescence detection system (Pierce; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. Protein expression was quantified using Gel-Pro Analyzer Version 6.3 densitometry software (Media Cybernetics, Inc., Rockville, MD, USA). Experiments were repeated in triplicate.

The viability of cells was determined using an MTT assay. Briefly, H9c2 cells were seeded into 96-well culture plates and grown at 37°C for 24 h. Subsequently, 5 mg/ml MTT solution (Amresco, LLC, Solon, OH, USA) was added to each culture well, and cells were incubated for a further 4 h. Subsequently, 150 µl DMSO was used to dissolve the purple formazan and the absorbance was detected at 490 nm using a Synergy™ 2 Multi-Mode microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). Experiments were repeated in triplicate.

To determine the apoptosis of cells, an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (cat no. 70-AP101-100; Hangzhou MultiSciences Biotech, Co., Ltd., Hangzhou, China) was used. Briefly, H9c2 cells were washed with PBS three times and fixed in 70% ethanol at 4°C overnight, and stained with PI/Annexin V-FITC, according to the manufacturer's protocols. A flow cytometer was used (BD Biosciences, Franklin Lakes, NJ, USA) to analyze cell apoptosis, and the apoptosis rate was calculated as the total percentage of cells in the right-side quadrants (early + late apoptotic cells) using FlowJo software (version 7.6.1; FlowJo LLC, Ashland, OR, USA) was used for data analysis. Experiments were repeated in triplicate.

An ELISA was performed to determine the levels of TNF-α, IL-1β and IL-6 in cell supernatants. Briefly, H9c2 cells were transfected with NC, miR-124-3p inhibitor, or miR-124-3p inhibitor + NKRF-siRNA for 2 h and then subjected to hypoxia for 48 h. Cell supernatants were harvested via centrifugation (1,000 × g, 15 min, 4°C). The expression levels of IL-1β (cat no. PI305; Beyotime Institute of Biotechnology), TNF-α (cat no. PI518; Beyotime Institute of Biotechnology), and IL-6 (cat no. PI330; Beyotime Institute of Biotechnology) were detected using ELISA kits, according to the manufacturer's protocols. Experiments were repeated in triplicate.

Data are presented as the mean ± standard deviation of at least three experiments. All data were analyzed using SPSS version 17.0 software (SPSS, Inc., Chicago, IL, USA). Comparisons between groups were performed using Student's t-tests or one-way analyses of variance followed by a Tukey's test. P<0.05 was considered to indicate a statistically significant difference.

RT-qPCR analysis was performed to investigate the expression of miR-124-3p in AMI. As presented in Fig. 1A, the expression levels of miR-124-3p were significantly increased in the blood of patients with AMI compared with healthy controls. Additionally, it was revealed that the expression of miR-124-3p in the cardiomyocyte cell line H9c2 was significantly upregulated following exposure to hypoxic conditions for 48 h compared with the control (Fig. 1B).

TargetScan was employed to predict potential targets of miR-124-3p. Various target genes of miR-124-3p, including NKRF (Fig. 2A). Previous studies indicated that NKRF is a silencer protein that binds negative regulatory elements (NRE) to repress the transcription of various NF-κB-regulated genes (24). Therefore, NKRF serves important roles in inflammation responses and apoptosis; however, the involvement of this gene in AMI remains unclear. Thus, NKRF was selected for further investigation in the present study.

To determine whether miR-124-3p directly modulates the expression of NKRF via interactions with potential binding sites in the 3′-UTR, a luciferase reporter assay was performed. Transfection of H9c2 cells with miR-124-3p mimic upregulated the expression of miR-124-3p compared with the mimic control (Fig. 2B). Furthermore, co-transfection of H9c2 cells with miR-124-3p mimic and NKRF-WT plasmid significantly decreased the luciferase activity compared with co-transfection with miR-124-3p mimic and NKRF-MUT plasmid (Fig. 2C). The results indicated that miR-124-3p directly targeted NKRF mRNA.

To investigate the effects of miR-124-3p in AMI, H9c2 cells were transfected with NC, miR-124-3p inhibitor, control-siRNA, NKRF-siRNA or miR-124-3p inhibitor + NKRF-siRNA, and the transfection efficiency was determined 48 h following transfection. Transfection with miR-124-3p inhibitor significantly downregulated the expression of miR-124-3p compared with the control (Fig. 3A), whereas NKRF-siRNA significantly downregulated the expression of NKRF mRNA (Fig. 3B) and protein (Fig. 3C and D) in H9c2 cells compared with control-siRNA. It was further demonstrated that miR-124-3p inhibitor significantly increased the expression levels of NKRF at the mRNA (Fig. 3E) and protein (Fig. 3F and G) levels; these effects were eliminated by transfection with NKRF-siRNA.

An MTT assay was performed to determine the effects of miR-124-3p on the viability of H9c2 cells following hypoxia. It was revealed that miR-124-3p inhibitor significantly attenuated the hypoxia-induced decrease in the viability of H9c2 cells, whereas co-transfection with inhibitor and NKRF-siRNA eliminated the effects of the inhibitor (Fig. 4).

The effects of miR-124-3p on the apoptosis of H9c2 cells were determined via flow cytometry. It was demonstrated that hypoxia significantly promoted H9c2 cell apoptosis, whereas transfection with miR-124-3p inhibitor attenuated the effects of hypoxia (Fig. 5A and B). Furthermore, co-transfection with NKRF-siRNA significantly eliminated the effects of miR-124-3p inhibitor on the apoptosis of H9c2 cells.

To further investigate the antiapoptotic effects of miR-124-3p inhibitor, the expression of apoptosis-associated proteins was determined, including Bcl-2, cleaved caspase 3, cleaved caspase 9, procaspase 3 and procaspase 9. It was observed that the protein expression levels of Bcl-2, procaspase 3 and procaspase 9 were markedly decreased in H9c2 cells following hypoxia, and those of cleaved caspase 3 and cleaved caspase 9 were significantly increased in H9c2 cells treated with hypoxia, compared with the control; transfection with miR-124-3p inhibitor reversed the hypoxia-induced effects (Fig. 5C). Furthermore, the effects of miR-124-3p inhibitor on the expression of Bcl-2, cleaved caspase 3, cleaved caspase 9, procaspase 3 and procaspase 9 in H9c2 cells were eliminated by the silencing of NKRF.

As inflammation serves important roles in the development of AMI, the effects of miR-124-3p on inflammatory responses during AMI were investigated. As presented in Fig. 6, the protein and mRNA expression levels of TNF-α, IL-1β and IL-6 were significantly increased following hypoxia; however, transfection with miR-124-3p inhibitor significantly inhibited the hypoxia-induced upregulation of inflammatory cytokines, effects which were eliminated following co-transfection with NKRF-siRNA. The results indicated that miR-124-3p inhibitor suppressed hypoxia-induced inflammatory responses in cardiomyocytes.

To investigate the mechanisms underlying the effects of miR-124-3p inhibitor on the cardiomyocyte cell line H9c2, the activity of the NF-κB pathway was analyzed. As presented in Fig. 7, hypoxia significantly downregulated the expression of NKRF (Fig. 7A-C) and promoted the phosphorylation of NF-κB p65 in H9c2 cells (Fig. 7D-F); transfection with miR-124-3p inhibitor significantly attenuated the effects of hypoxia on NKRF expression and p65 phosphorylation. Additionally, it was demonstrated that NKRF-siRNA significantly eliminated the effects of miR-124-3p inhibitor on NKRF and p-p65 levels in H9c2 cells. The expression of p65 protein in H9c2 cells was not notably different across the various groups.