The mouse cardiomyocytes used in the present study were obtained from the Shanghai Cell Bank (Shanghai, China). The cardiomyocytes were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serumm(Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). All cells were maintained in a humidified atmosphere containing 5% CO₂ at 37°C. Cell transfection was performed using FuGENE^® HD transfection reagent (Roche Diagnostics, Indianapolis, IN, USA), according to the manufacturer's protocol. Briefly, cardiomyocytes were seeded into 6-well plates at a density of 2×10⁵ cells/well and cultured for 24 h to reach 70–80% confluence. Subsequently, 2 µg of plasmids encoding m-miR-423-5p and miR-Ctrl were diluted in 100 µl serum-free medium, and 5 µl FuGENE^® HD transfection reagent was added to the tubes containing the diluted DNA. The contents were mixed, and the transfection complex was incubated for 15 min at room temperature, followed by its addition to the 6-well plates. Medium alone was used as a blank control. When the cardiomyocytes reached 70–80% confluence in the 6-well plates, H₂O₂ was added to the wells at different concentrations (0, 0.1, 0.2, 0.4 and 0.8 mM). At 12, 24 and 48 h following transfection and/or H₂O₂ treatment, the cardiomyocytes were collected for further experiments.

The miR-423-5p and miR-423-5p-mimic expression plasmids were constructed, as previously (15). Briefy, each miRNA and plasmid oligonucleotide were annealed at 90°C for 3 min, cooled to 37°C and incubated for 1 h. The annealed double-stranded DNA oligonucleotides were ligated between the HindIII and SacI sites (Fermentas; Thermo Fisher Scientifc, Inc.) on the pMIR-REPORT vector (Ambion Life Technologies; Thermo Fisher Scientific, Inc.). The resulting recombinant plasmids were termed pMIR-miR-423-5p, pMIR-miR-423-5p-mimic and pMIR-miR-control (Ctrl). The three constructs were verified using DNA sequencing. The plasmids, pMIR-miR-423-5p, pMIR-miR-423-5p-mimic and pMIR-miR-control, were exacted and sent to the technology center of Invitrogen Life Technologies at Shanghai for DNA sequencing. The results were comprised with Jellyfish software (version 3.2) The plasmids were extracted using EndoFree Plasmid Giga kits (Qiagen GmbH, Hilden, Germany) from DH5α Escherichia coli transformants (Genewiz, Suzhou, China) and stored at −20°C until further use. The concentration was determined by measuring the A260/A280 ratio using a Thermo ND 2000 spectrophotometer (Thermo Fisher Scientific, Inc.).

A Cell Counting Kit-8 (CCK8) assay (Beyotime Institute of Biotechnology, Shanghai, China) was used to detect cell viability. The absorbance was determined at a wavelength of 450 nm using a plate reader. The same experiments were performed three times.

Total RNA was extracted from each experimental group using TRIzol^® reagent (Invitrogen Life Technologies, Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The RNA concentration was assessed spectrophotometrically at 260 nm (Thermo ND 2000; Thermo Fisher Scientific., Inc.). Reverse transcription was performed on the isolated total RNA using a Reverse Transcription kit (Takara Bio, Inc., Otsu, Japan), and qPCR was performed using a Real Time PCR kit (Takara Bio, Inc). Reverse transcription was performed using 1 µg total RNA in 2 µl water at 65°C for 5 min, 30°C for 10 min, 42°C for 10–30 min and 2°C for 3 min. The qPCR conditions were as follows: Denaturation at 94°C for 2 min, amplification for 30 cycles at 94°C for 0.5 min, annealing at 60°C for 0.5 min and extension at 72°C for 1 min, followed by a terminal elongation step at 72°C for 10 min. The RT-qPCR analysis was performed on a Bio-Rad CFX96 thermal cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA). U6 was amplified as an internal control and the Cq value of each PCR product was calculated, from which the fold change was analyzed (15). The m-miR-423-5p and m-U6 primers were supplied by Guangzhou RiboBio Co., Ltd. (Guangzhou, China). The sequences were not supplied due to the rules of the company.

26S proteasome function was assayed, as described previously (16). Briefly, the cells were washed with phosphate-buffered saline, followed by washing with buffer I, containing 50 mM Tris (pH 7.4), 2 mM DTT, 5 mM MgCl₂ and 2 mM ATP), and pelleted by centrifugation at 1,500 g for 3 min at 4°C. Glass beads and homogenization buffer containing 50 mM Tris (pH 7.4), 1 mM DTT, 5 mM MgCl₂, 2 mM ATP and 250 mM sucrose, were added and vortexed for 1 min. The beads and cell debris were removed by centrifugation at 1,000 × g for 5 min and 10,000 × g for 20 min. Protein concentration was determined using a bicinchoninic acid assay protocol (Pierce Biotechnology, Inc., Rockford, IL, USA). The protein from each sample (100 µg) was diluted with buffer I to a final volume of 1,000 µl, and the fluorogenic proteasome substrate, SucLLVY-7-amido-4-methylcou-marin (chymotrypsin-like; Sigma-Aldrich) was added to a final concentration of 80 µM in 1% DMSO. To access 26S function, buffer I was replaced with ATP-free buffer containing 20 mM HEPES (pH 7.8), 0.5 mM EDTA and 0.03%SDS (16). Cleavage activity was monitored continuously by the detection of free 7-amido-4-methylcoumarin using a fluorescence plate reader (Gemini, Molecular Devices LLC, Sunnyvale, CA, USA) at 380/460 nm and 37°C. As controls for the drug experiments, 7-amido-4-methylcoumarin (2 µM) was incubated with the drugs in buffer I without cell extracts, and measurements of proteasome function were corrected when necessary.

Following transfection of the cardiomyocytes with m-miR-423-5p and miR-Ctrl (48 h), total protein was collected. The cells were lysed on ice for 30 min with radioimmunoprecipitation assay lysis buffer containing 50 mM Tris-HCl (pH 7.4), 1% NP-40; 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 µg/ml each of aprotinin, leupeptin and pepstatin, 1 mM Na₃VO₄ and 1 mM NaF. The proteins (20 µg) were separated by 10% SDS-polyacrylamide gel electrophoresis and electronically transferred onto a polyvinylidene difluoride membrane (EMD Millipore, Bedford, MA, USA). Following blocking with 5% non-fat milk in Tris-buffered saline containing 0.5% Tween-20 (TBST), the membranes were incubated with the recommended dilutions of primary antibodies against OGT (rabbit polyclonal; 1:800; cat. no. sc-32921; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 1 h at 37°C, phosphorylated (p) adenosine monophosphate (AMP)-activated protein kinase (p-AMPK; cat. no. 4188; 1:1,000), AMPK (cat. no. 2532; 1:1,000), p53 (cat. no. 2527; 1:1,200) and caspase 3 (cat. no. 9662; 1:600) for 1 h at 37°C (all purchased from Cell Signaling Technology, Inc., Danvers, MA, USA) and GAPDH (cat. no. sc-25778; 1:5,000; Santa Cruz Biotechnology, Inc.). Following washing with TBST, the membranes were incubated with peroxidase-conjugated secondary antibodies against goat anti-mouse immunoglobulin G (cat. no. ab97040; 1:5,000) or goat anti-rabbit (cat. no. ab191866; 1:5,000; each from Abcam, Cambridge, MA, USA) at 37°C for 1 h. The peroxidase-labeled bands were visualized using an enhanced chemiluminescence kit (Pierce Biotechnology, Inc.). The ratios of OGT and p-AMPK to GAPDH were calculated using densitometry, and values were normalized by dividing the ratios by the ratio in the blank control.

In order to detect apoptotic cells in the cardiomyocytes, a TUNEL assay was performed using a DeadEndTM Fluorometric TUNEL system (Promega Corporation, Madison, WI, USA), according to the manufacturer's protocol. Cell nuclei with dark green fuorescent staining were defined as TUNEL-positive nuclei, which were visualized using a fluorescence microscope (DTX500; Nikon Corporation, Tokyo, Japan). In order to quantify the TUNEL-positive cells, the number of green fluorescent-positive cells were counted in randomly selected fields (n=5) at magnification, ×200. The cell nuclei were then counterstained with 4′,6-diamidino-2-phenylindole (Beyotime Institute of Biotechnology).

Statistical comparisons of all results were analyzed using one-way analysis of variance. Statistical analyses were performed using SPSS version 19.0 (IBM SPSS, Armonk, NY, USA). Values are expressed as the mean ± standard error of the mean. P<0.05 was considered to indicate a statistically significant difference.

The effect of H₂O₂ treatment on cardiomyocyte viability was determined using a CCK8 assay. As shown in Fig. 1A, H₂O₂ treatment (0.4 mM) for 12 h significantly reduced cell viability. Increasing the duration of treatment with H₂O₂ to 24 and 48 h, markedly inhibited the viability of the cardiomyocytes (Fig. 1A). Furthermore, the effect of H₂O₂ concentration on cardiomyocyte viability was determined. As the results in Fig. 1B show, treatment with 0.1 mM H₂O₂ for 24 h had no significant effect on cardiomyocytes viability. However, treatment with 0.2, 0.4 and 0.8 mM H₂O₂ for 24 h significantly inhibited cardiomyocyte growth (Fig. 1B). These results indicated that the effect of H₂O₂ treatment on cell viability was time- and concentration-dependent.

In the subsequent experiment, the expression of miR-423-5p in cardiomyocytes following H₂O₂ treatment was examined using RT-q-PCR analysis. As shown in Fig. 1C, H₂O₂ treatment (0.4 mM) for 12 h significantly induced the expression of miR-423-5p in the cardiomyocytes. The expression of miR-423-5p in the cardiomyocytes was higher following H₂O₂ treatment for 24 and 48 h, compared with that at 12 h. Treatment with 0.1 mM H₂O₂ for 24 h had minimal effect on the expression of miR-423-5p in the cardiomyocytes, compared with 0.2, 0.4 and 0.8 mM H₂O₂ treatment, which significantly induced the expression of miR-423-5p in the cardiomyocytes (Fig. 1D). Taken together, these results indicated that H₂O₂ treatment significantly induced the expression of miR-423-5p in cardiomyocytes, in a time- and concentration-dependent manner.

To examine the effect of miR-423-5p on mediation of H₂O₂-induced apoptosis in cardiomyocytes, plasmids encoding miR-423-5p and miR-423-5p-mimic, which neutralized the activity of miR-423-5p, were constructed. RT-qPCR analysis demonstrated that transfection with the miR-423-5p plasmid significantly induced the expression of miR-423-5p in the cardiomyocytes (Fig. 2A). To examine the effect of the miR-423-5p-mimic on the expression of miR-423-5p, the cardiomyocytes were exposed to H₂O₂ (0.4 mM) following miR-423-5p-mimic transfection. After 48 h, the cells were collected for RT-qPCR analysis. The results demonstrated the ability of the miR-423-5p-mimic to inhibit the expression of miR-423-5p (Fig. 2B). Cardiomyocyte viability was determined in the following treatment groups: Blank, H₂O₂ (0.4 mM) alone, H₂O₂ combined with miR-Ctrl transfection, H₂O₂ combined with miR-423-5p-mimic transfection, and miR-423-5p transfection without H₂O₂ treatment. As shown in Fig. 2C, H₂O₂ treatment or transfection with miR-423-5p significantly decreased cardiomyocyte viability (Fig. 2C). No significant difference in cardiomyocyte viability was found between the blank control group and the H₂O₂ combined with miR-423-5p-mimic transfection group (Fig. 2C). These results indicated that cardiomyocyte viability was restored following inhibition of miR-423-5p activity. A TUNEL assay was used to detect apoptotic cardiomyocytes. It was found that H₂O₂ treatment or the overexpression of miR-423-5p significantly induced cardiomyocyte apoptosis, compared with the miR-423-5p-mimic, which markedly inhibited H₂O₂-induced apoptosis (Fig. 2D and E). These results demonstrated that miR-423-5p mediated H₂O₂-induced apoptosis in the cardiomyocytes.

In our previous study, it was demonstrated that OGT is the direct target of miR-423-5p. Thus, in the present study, the expression of OGT was examined. As shown in Fig. 3A and B, the expression of OGT was significantly inhibited following exposure to H₂O₂ for 12, 24 and 48 h. Further investigation demonstrated that H₂O₂ markedly decreased the expression of OGT in a concentration-dependent manner (Fig. 3C and D). To examine the role of miR-423-5p on mediation of the H₂O₂-regulated expression of OGT, the cardiomyocytes were treated in the following groups: Blank control, H₂O₂ (0.4 mM) treatment, H₂O₂ combined with miR-Ctrl transfection, H₂O₂ combined with miR-423-5p-mimic transfection, and miR-423-5p transfection without H₂O₂ treatment. The results indicated that treatment with H₂O₂ or transfection with miR-423-5p significantly inhibited the expression of OGT. Of note, the expression of OGT in cardiomyocytes was restored following inhibition of miR-423-5p activity by the miR-423-5p-mimic. No significant difference were observed between blank control group and the H₂O₂ combined with miR-423-5p-mimic transfection group. Collectively, miR-423-5p mediated the H₂O₂-induced downregulation of OGT.

The activities of AMPK and 26S proteasome activity, as the downstream targets of OGT, were also determined in the present study. The phosphorylation of AMPK was inhibited following H₂O₂ treatment or the overexpression miR-423-5p through a reduction in OGT (Fig. 4A). Whereas, no significant change in the expression of p-AMPK was observed between the blank control group and the H₂O₂ combined with miR-423-5p-mimic transfection group. According to the change in the expression of p-AMPK, 26S proteasome activity was also increased following H₂O₂ treatment or the overexpression miR-423-5p (Fig. 4C). Transfection with the miR-423-5p-mimic also restored 26S proteasome activity to a normal level (Fig. 4C). These results demonstrated that miR-423-5p mediated the H₂O₂-regulated expression of p-AMPK and activity of 26S proteasome, which are downstream targets of OGT.