The H9c2 cell line (American Type Culture Collection, Manassas, VA, USA) was grown at 37°C in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and 10% penicillin/streptomycin in an atmosphere containing 5% CO₂. The phenotype of myocardial cells was initially observed under a light microscope (magnification, ×40). The negative small interfering (si) RNA scramble control (siSCR), CUEDC2 siRNA (siCUE) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) primers were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). At ~80% confluence, cells were divided into the following groups: i) untreated cells (control); ii) cells transfected with siSCR; iii) cells treated with 1 µM DOX (Sigma-Aldrich; Merck KGaA) at 37°C for 24 h (DOX); iv) cells transfected with siSCR and treated with 1 µM DOX at 37°C for 24 h (DOX + siSCR); and v) cells transfected with siCUE and treated with 1 µM DOX at 37°C for 24 h (DOX + siCUE). All experiments were carried out independently at least three times.

According to the manufacturer's protocol, cells (1×10⁵) were seeded into 6-well plates and maintained in serum-free medium overnight. Briefly, Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was mixed with serum-free DMEM. After 5 min, the resulting regent was divided into three centrifuge tubes, containing: i) 10 µl Lipofectamine^® 2000 alone, ii) 10 µM siScr (cat. no. sc-37007; Santa Cruz Biotechnology, Inc.) or iii) 10 µM siCUE (cat. no. sc-90791; Santa Cruz Biotechnology, Inc.), according to the experimental design. The mixtures were incubated at room temperature for 20 min. Subsequently, the liposome mixtures were added to the cells. After a 6 h incubation at 37°C, the transfection medium was removed and supplemented with normal culture medium or DOX for 1 h. The cells were collected a total of 24 h post-transfection and then subjected to subsequent assays.

The CCK-8 method was used to determine cell viability. Briefly, cells (1×10⁵) were seeded in 96-well plates and treated with DOX (0.1, 0.5, 1 and 5 µM) for 24, 36 and 48 h time intervals. The collected cells were then treated with CCK-8 solution (10 µl; cat. no. C0038; Beyotime Institute of Biotechnology, Haimen, China) for 4 h at 37°C. The absorbance was measured at a wavelength of 450 nm using a microplate reader (Promega Corporation, Madison, WI, USA).

Cells were stained with the oxidant-sensitive fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA; cat. no. 35845; Sigma-Aldrich; Merck KGaA) for 15 min and were washed with PBS. 2′,7′-Dichlorofluorescein (DCF) was generated from DCFH-DA in the presence of peroxide. The fluorescence of DCF was detected using Accuri C6 flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA) with an excitation wavelength of 485 nm. Data analysis was performed using Accuri CFlow Plus software (version 1.0; BD Biosciences).

The JC-1 probe can emit green fluorescence at low membrane potentials and red fluorescence at increased potentials. MitoProbe™ JC-1 Assay kit (cat. no. M34152; Invitrogen; Thermo Fisher Scientific, Inc.) was used to determine MMP, according to the manufacturer's protocol. Briefly, the cells were stained with JC-1 for 20 min at 37°C and were then loaded onto an Accuri C6 flow cytometer (BD Biosciences). The ratio of red/green fluorescence was calculated and a decrease in the ratio indicated loss of MMP. Data analysis was performed with Accuri CFlow Plus software (version 1.0; BD Biosciences).

The Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit (cat. no. V13241; Invitrogen; Thermo Fisher Scientific, Inc.) was used to detect apoptosis. Annexin V-FITC⁺/PI⁻ indicated early apoptotic cells, whereas late apoptotic cells were represented as Annexin V-FITC⁺/PI⁺. According to the manufacturer's protocol, following treatment, cells in a 24-well plate (1×10⁵ cells/well) were stained with Annexin V-FITC and PI for 10 min at room temperature in the dark, after which fluorescence intensity was analyzed using Accuri C6 flow cytometry with Accuri CFlow Plus software version 1.0 (BD Biosciences).

Superoxide dismutase (SOD; cat. no. S0101), catalase (CAT; cat. no. S0051) and malondialdehyde (MDA; cat no. S0131) were spectrophotometrically determined in collected cells following treatment using commercial assay kits, according to the manufacturer's protocols (Beyotime Institute of Biotechnology). SOD and CAT activity was presented as units per mg of protein, and MDA content was presented as nmol/mg.

Total RNA was isolated from the cells using TRIzol^® regent (Invitrogen; Thermo Fisher Scientific, Inc). cDNA was synthesized from 2 µg RNA using oligo-dT primers (New England BioLabs, Inc., Ipswich, MA, USA) and M-MLV reverse transcriptase (Promega Corporation), according to manufacturer's protocol. qPCR was conducted on the Mastercycler^® ep realplex system (Eppendorf, Hamburg, Germany) using SYBR Green PCR master mix (Takara Bio, Inc., Otsu, Japan), according to the manufacturer's protocol. The thermocycling conditions used for qPCR were as follows: 95°C for 5 min; followed by 36 cycles of 95°C for 30 sec and 60°C for 30 sec; followed by 72°C for 7 min. The relative expression levels of target genes were normalized to the expression of the housekeeping gene GAPDH. The following primer sequences were used for qPCR: Bcl-2-associated X protein (Bax), forward 5′-TGGCCTCCTTTCCTACTTCG-3′, reverse 5′-AAAATGCCTTTCCCCGTTCC-3′; Bcl-2, forward 5′-CACACACACACATTCAGGCA-3′, reverse 5′-GGCAATTCCTGGTTCGGTTT-3′; caspase-3, forward 5′-CTCGCGTTAACAGGAAGGTG-3′, reverse 5′-GGCAGTGGTAGCGTACAAAG-3′; cytochrome c, forward 5′-AGAGTGAGTTCCAGGACAGC-3′, reverse 5′-ACTAACGAGGCCCCTTTGAA-3′; and GAPDH, forward 5′-GGGTCCCAGCTTAGGTTCAT-3′ and reverse 5′-CATTCTCGGCCTTGACTGTG-3′. Quantification of RT-qPCR products was performed using the 2^−ΔΔCq method (15).

Total proteins were extracted using radioimmunoprecipitation assay (Beijing Solarbio Science & Technology, Co., Ltd., Beijing, China) and were boiled. A BCA protein quantification kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to determine the protein concentration. Subsequently, protein samples from each group (20 µg) were separated by 10% SDS-PAGE and transferred onto PVDF membrane. Following blocking with fat-free milk for 2 h at room temperature, the membrane was incubated with primary antibodies overnight at 4°C. Prior to incubation with secondary antibodies at room temperature for 1 h, the membrane was washed with Tris-buffered saline containing 0.1% Tween. Protein bands were visualized using BeyoECL Plus reagent (Beyotime Institute of Biotechnology). The following antibodies were used for western blotting: Anti-phosphorylated (p)-FOXO3a (cat. no. ab53287; 1:1,000), anti-Bcl-2 (cat. no. ab59348; 1:700) and anti-Bax (cat. no. ab53154; 1:1,000) from Abcam (Cambridge, UK); anti-CUEDC2 (cat. no. 12294; 1:1,000), anti-FOXO3a (cat. no. 2497; 1:1,000), anti-cytochrome c (cat. no. 11940; 1:1,000), anti-cleaved caspase-3 (cat. no. 9664; 1:1,000), anti-p-AKT (Thr308; cat. no. 13038; 1:1,000), anti-AKT (cat. no. 4691; 1:1,000), anti-Bim (cat. no. 2819; 1:1,000) and anti-GAPDH (cat. no. 5174; 1:1,000) from Cell Signaling Technology, Inc. (Danvers, MA, USA). Horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (cat. no. sc-2004; 1:5,000) was supplied by Santa Cruz Biotechnology, Inc. Denistometric analysis was performed using Quantity One software (version 4.6.2; Bio-Rad Laboratories, Inc.).

Statistical analyses were performed using GraphPad Prism (version 6.0; GraphPad Software, Inc., La Jolla, CA, USA). The difference between groups was analyzed using one-way analysis of variance followed by Dunnett's or Turkey's post hoc tests when appropriate. Data are presented as the means ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

The phenotype of myocardial cells are presented in Fig. 1A and B. Subsequently, cytotoxicity of DOX (0.1, 0.5, 1 and 5 µM) was determined in H9c2 cells. The results of CCK-8 assay demonstrated that viability of H9c2 cells was significantly decreased following incubation with 0.5 µM DOX for 24 h. Cytotoxicity of DOX increased in a dose-dependent manner (Fig. 1C). According to the results presented in Fig. 1C and a previous study (16), 1 µM DOX was selected to establish the cardiotoxicity model in the present study.

CUEDC2 is extensively expressed in the heart, and it has been reported that this protein is involved in modulation of the oxidative capacity of cardiomyocytes (17). Therefore, the potential function of CUEDC2 in cardiomyocytes was investigated in subsequent experiments. Following transfection with siRNA, the mRNA and protein expression levels of CUEDC2 were downregulated, thus indicating that knockdown of CUEDC2 was effective (Fig. 1D-F). Formation of ROS has previously been implicated in DOX-induced cardiotoxicity (17). Flow cytometric analysis demonstrated that the levels of ROS were elevated in the DOX group, whereas they were decreased in the DOX + siCUE group (Fig. 2A and B). Intracellular redox homeostasis is dependent on the generation and removal of ROS. Production of MDA and the activity of antioxidative enzymes, including SOD and CAT, can be used to monitor this redox state (18). The results of the present study revealed that DOX markedly increased the content of MDA, whereas depletion of CUEDC2 resulted in a significant decrease in MDA levels. In addition, DOX-reduced SOD activity was rescued by the depletion of CUEDC2, whereas the alterations in CAT activity were not significantly different in the DOX + siCUE group compared with in the DOX group (Fig. 2C).

Mitochondria are the primary target organelles of DOX-induced cardiotoxicity (19,20). MMP is necessary for the production of ATP, which serves crucial roles in living cells (21). The results of flow cytometry revealed that DOX induced MMP loss; however, MMP was recovered by the knockdown of CUEDC2 (Fig. 3A and B)

Mitochondrial dysfunction is closely associated with apoptosis (22); therefore, the levels of apoptosis were determined in the present study. The results of flow cytometry demonstrated that the rate of apoptosis was lower in the DOX + siCUE group compared with in the DOX group (Fig. 4A and B). The expression levels of apoptosis-associated genes were also detected in the present study. The mRNA expression levels of Bax, caspase-3 and cytochrome c were decreased in the DOX + siCUE group compared with in the DOX group (Fig. 5). Conversely, Bcl-2 expression was increased following depletion of CUEDC2. In addition, the protein expression levels of Bax, cleaved-caspase-3 and cytochrome c were lower in the DOX + siCUE group compared with in the DOX group, whereas Bcl-2 expression was elevated in the DOX + siCUE group compared with in the DOX group.

FOXO3a, as a member of the FOXO family of transcription factors, serves a role in the oxidative stress response. It has previously been reported that AKT signaling regulates the activity FOXO3a, which in turn modulates the expression of Bim (23,24). Levels of phosphorylated AKT and phosphorylated FOXO3a were significantly enhanced in the DOX + siCUE group compared with in the DOX group (Fig. 6). Conversely, the elevated protein expression of Bim following treatment with DOX was decreased following depletion of CUEDC2. These findings suggested that siCUE activated phosphorylated AKT, which subsequently phosphorylated FOXO3a and decreased Bim transcription.