The H9C2 cardiomyocytes were purchased from the American Type Culture Collection. Cells were cultivated in 10% fetal bovine serum (FBS; Beyotime Institute of Biotechnology) and 1% penicillin/streptomycin DMEM (Beyotime Institute of Biotechnology) and incubated at 37°C in an atmosphere containing 5% CO₂. The cells were subjected to H₂O₂ (50, 100 and 200 µM; Merck KGaA) solution in DMEM without FBS treatment for 4 h.

H9C2 cells were seeded into 12-well plates with 1×10⁶ cells/well and cultured in serum-free DMEM at 37°C with 5% CO₂ continuously to ensure that cell confluence reached 80% before transfection. According to the manufacturer's protocol, miR-370 mimics (cat. no. miR10000722-1-5), negative control (NC) mimic (cat. no. miR1190315051351), small interfering RNA (siR-/siRNA)-Forkhead box O1 (FOXO1)-1 (5′-GGACAACAACAGTAAATTT-3′), siR-FOXO1-2 (5′-GCACCGACTTTATGAGCAA-3′) and NC siRNA (cat. no. siT0000003-1-5) (Each, 100 nM; all, Guangzhou RiboBio Co., Ltd.) were transfected into H9C2 cells for 48 h using Lipofectamine 2000™ reagent (Invitrogen; Thermo Fisher Scientific, Inc.).

Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc.) was used to assess cardiomyocyte viability. After H9C2 cells were treated with H₂O_2, 10 µl CCK-8 solution was added to each well and the cells were incubated at 37°C for 4 h. Finally, the optical density of each well was measured at a wavelength of 490 nm using a plate reader (Infinite^® 200 PRO NanoQuant; Tecan Group, Ltd.).

The concentrations of plasma superoxide dismutase (SOD; cat. no. ml077379), malondialdehyde (MDA; cat. no. ml077384) and lactate dehydrogenase (LDH, ml003416) were measured using commercial ELISA kits (Shanghai Enzyme-linked Biotechnology Co., Ltd.) according to the manufacturer's protocols. The experimental groups were as follows: Control group, H9C2 cells untreated with H₂O₂; H₂O₂ group, H9C2 cells treated with H₂O₂; NC mimic +H₂O₂ group, H9C2 cells treated with NC mimic after H₂O₂; miR-370 mimic+ H₂O₂ group, H9C2 cells treated with miR-370 mimic after H₂O₂; NC siRNA+ H₂O₂ group, H9C2 cells treated with NC siRNA after H₂O₂; and siR-FOXO1-1+ H₂O₂ group, H9C2 cells treated with siR-FOXO1-1 after H₂O₂.

Cell apoptosis was evaluated using an Annexin V-FITC Apoptosis Detection kit (BD Biosciences; Becton, Dickson and Company) in accordance with the manufacturer's protocol. The cells were treated as described above. All adhering and floating cells were harvested, washed twice with PBS, transferred into sterile centrifuge tube and then stained successively with propidium iodide (10 µl) and Annexin-V-FITC (10 µl; Nanjing KeyGen Biotech Co., Ltd.) for 15 min at 37°C. A flow cytometer (BD Biosciences; Becton, Dickson and Company) was used to assess apoptotic cells and data were analyzed using CellQuest software (version 3.1, BD Biosciences). Experiments were repeated three times independently.

Total RNA was extracted from cells using TRIzol reagent (Thermo Fisher Scientific, Inc.). Then, the total RNA was used to synthesize cDNA using a PimeScript RT master mix kit (Takara Biotechnology Co., Ltd.) following the manufacturer's protocol. The RT conditions were as follows: 10 min at 25°C, 30 min at 45°C and 5 min at 95°C. qPCR was performed with the following thermocycling condition: 95°C for an initial 10 min followed by 40 cycles of denaturation for 15 sec at 95°C, annealing at 60°C for 30 sec and extension at 72°C for 30 sec with the SYBR Premix Ex Taq (Applied Biosystems; Thermo Fisher Scientific, Inc.). Fold-changes of miR-370 and mRNA levels were calculated using the 2^−ΔΔCq method (22). U6 and GAPDH were used as the endogenous controls. The following primers were used: GAPDH forward, 5′-AGACAGCCGCATCTTCTTGT-3′ and reverse, 5′-TGATGGCAACAATGTCCACT-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′, and reverse, 5′-AACGCTTCACGAATTTGCGT-3′; miR-370 forward, 5′-TACTCAGGATCCTGTGCAAGGCGGGCTACT-3′ and reverse, 5′-TACTCAAAGCTTCCCTCCCTCACCCAAATC-3′; FOXO1 forward, 5′-AGGATCCGATGTCACCATGGCCG-3′ and reverse, 5′-AAAGGATCCACCATGGCCG-3′.

The total proteins from cells were isolated using RIPA lysis buffer (Beyotime Institute of Biotechnology) and the concentration of protein was measured with a bicinchoninic acid kit (Beyotime Institute of Biotechnology). Subsequently, an equal quantity of proteins (20 µg) was separated by 10% SDS-PAGE and then transferred onto polyvinylidene fluoride membranes (EMD Millipore). Then the membranes were blocked with 5% non-fat dry milk for 1 h at room temperature. After washing, the membranes were incubated with primary antibodies against FOXO1 (1:1,000; cat. no. 2880) and GAPDH (1:2,000; cat. no. 5174) from Cell Signaling Technology, Inc. at 4°C overnight and washed with 0.1% Tris-Buffered Saline with Tween 20, followed by incubation with horseradish peroxidase conjugated secondary antibodies (1:3,000; cat. no. ab214880; Abcam) for 1 h at room temperature. Blots were developed using an enhanced chemiluminescence film kit (Perkin Elmer, Inc.) and the images were analyzed using ImageJ software (version 1.8.0; National Institutes of Health). All experiments were repeated three times independently.

The online bioinformatics tool, TargetScan (http://www.targetscan.org/vert_71/), was used to predict the possible binding targets of miR-370 and FOXO1. A fragment of the FOXO1-3′ untranslated region (UTR) containing the miR-370 predicted seed region (wild-type; WT) was amplified from the cDNA of cells and inserted into pGL-3 plasmids (Promega Corporation). The H9C2 cells were co-transfected with the WT 3′UTR of FOXO1 containing the miR-370 binding sites and mutant FOXO1 3′UTR with NC mimics or miR-370 mimics using Lipofectamine 2000™ (Thermo Fisher Scientific, Inc.). Luciferase activity was measured by the Dual Luciferase Reporter Assay system (Promega Corporation), normalizing to Renilla luciferase activity. All experiments were repeated three times independently.

All data were presented as the mean ± standard deviation. SPSS v20.0 software (IBM Corp.) was used to analyze the data. Additionally, a Student's t-test was used to determine differences between two groups, while one-way analysis of variance followed by the Dunnett's post hoc test were used for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

The H9C2 cells were treated with H₂O₂ at different concentrations (0, 50, 100 and 200 µmol/l) for 4 h. Then, the cell viability was detected using a CCK-8 assay. As shown in Fig. 1, when compared with the control (0 µmol/l H₂O₂) group, the activity of H9C2 cells treated with oxidative stress was concentration dependent and cell viability significantly decreased with the increasing concentration (P<0.01). A dose of 100 µmol/l (cell viability, 50%) was selected for subsequent experiments.

The H₂O₂-induced H9C2 cells were transfected with the miR-370 mimic or NC mimic vectors. Subsequently, RT-qPCR was performed to detect the expression levels of miR-370. As shown in Fig. 2A, the expression of miR-370 was significantly decreased in the H₂O₂-induced H9C2 cells when compared with the normal H9C2 cells (P<0.001); however, the expression of miR-370 was significantly increased in H9C2 cells transfected with miR-370 mimic when compared with the NC mimic group (P<0.001; Fig. 2A). The results indicated that miR-370 expression could be inhibited by H₂O₂ in H9C2 cells, which in turn suggested that miR-370 may be downregulated in myocardial cells with ischemia and hypoxia.

To verify miR-370 function in oxidative stress and apoptosis, an ELISA was used to detect SOD, MDA and LDH levels in the supernatant, and a flow cytometry assay was used to detect apoptosis. As exhibited in Fig. 2B, when compared with the control group, SOD expression significantly decreased (P<0.001) and MDA and LDH expression significantly increased in the H₂O₂ and NC mimic groups (P<0.01). Compared with the H₂O₂ and NC mimic groups, SOD expression increased, and MDA and LDH expression reduced in the miR-370 mimics group. Furthermore, the results of flow cytometry revealed that the rate of apoptosis was significantly increased in the H₂O₂ and NC mimic groups when compared with the control group (P<0.001; Fig. 2C and D). Compared with the H₂O₂ and NC mimic groups, the rate of apoptosis was decreased in the miR-370 mimics group. These findings suggested that the overexpression of miR-370 could markedly suppress the oxidative stress and apoptosis induced by H₂O₂.

The results of the online bioinformatics tool TargetScan suggested that FOXO1 was predicted to be a possible target gene of miR-370. A luciferase reporter assay was conducted to verify this prediction. As shown in Fig. 3, the 3′-UTR of the gene FOXO1 was shown to contain the binding sequences for miR-370, suggesting that FOXO1 may be a downstream target gene of miR-370. Luciferase activity was significantly reduced in the FOXO1 3′UTR WT group transfected with miR-370-3p mimics (P<0.001), whereas there was no variation in the mutant-type FOXO1 3′UTR, therefore also suggesting that FOXO1 may be a direct target gene of miR-370.

The mRNA and protein levels of FOXO1 were measured by RT-qPCR and western blotting assay. As shown in Fig. 4A and B, the expression of FOXO1 was markedly increased in the H₂O₂ and NC mimic groups when compared with the control group. Compared with the H₂O₂ and NC mimic groups, FOXO1 expression was decreased in the miR-370 mimic group. Then, the interference efficiency of FOXO1 siRNA plasmids was detected using RT-qPCR and western blot assays. As shown in Fig. 4C and D, the expression of FOXO1 was significantly reduced in the siR-FOXO-1 and siR-FOXO1-2 groups when compared with the NC siRNA group (P<0.001). The siR-FOXO1-1 plasmid with the best interference effects was selected for subsequent experiments.

ELISA was used to detect the levels of SOD, MDA and LDH in the supernatant, and a flow cytometry assay was used to detect apoptosis in H₂O₂-induced H9C2 cells. As shown in Fig. 5A, compared with the control group, the level of SOD significantly decreased (P<0.001) and the levels of MDA and LDH were significantly increased in the H₂O₂ and NC siRNA groups (P<0.001). However, compared with the H₂O₂ and NC siRNA groups, SOD expression significantly increased (P<0.01), and MDA and LDH expression significantly decreased in the siR-FOXO1-1 group (P<0.01). The results of flow cytometry revealed that the rate of apoptosis was significantly increased in the H₂O₂ and NC siRNA groups when compared with the control group (P<0.001; Fig. 5B and C). In addition, compared with the H₂O₂ and NC siRNA groups, the rate of apoptosis was significantly decreased in the siR-FOXO1-1 group (P<0.01). These results suggested that knockdown of FOXO1 could markedly suppress the oxidative stress and apoptosis of H9C2 cells induced by H₂O₂.