Hoechst 33258, bicinchoninic acid (BCA) protein assay kit, enhanced chemiluminescence (ECL) solution, and radioimmunoprecipitation assay (RIPA) buffer were supplied by Beyotime Institute of Biotechnology (Haimen, China). The cell counting kit-8 (CCK-8) was obtained from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). F12/Dulbecco's modified Eagle's medium (DMEM-F12) and fetal bovine serum (FBS) were purchased from HyClone (GE Healthcare Life Sciences, Logan, UT, USA). Primary antibodies for caspase-3 (cat. no. 9662) and GAPDH (cat. no. 5174) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA), and horseradish peroxidase (HRP)-conjugated secondary antibodies (cat. no. 00001-9) were obtained from ProteinTech Group, Inc. (Danvers, MA, USA). Annexin V/propidium iodide (PI) apoptosis kit was supplied by BD Pharmingen (BD Biosciences, San Jose, CA, USA). Lipofectamine RNAiMAX and Opti-MEM medium were supplied by Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The miRcute miRNA Isolation kit, miRcute miRNA First-strand cDNA Synthesis kit and miRcute miRNA quantitative polymerase chain reaction (qPCR) detection kit were obtained from Tiangen Biotech Co., Ltd. (Beijing, China).

The AC16 human adult ventricular cardiomyocyte cell line was purchased from American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM-F12 medium with 10% FBS and 1% penicillin-streptomycin at 37°C in a humidified incubator containing 5% CO₂. For HG treatment, AC16 cells grown to ~70% confluence were treated with HG (33 mM) for different durations (0, 24, 48 or 72 h). For miR-186-5p mimic treatment, cells were grown to 80–90% confluence and incubated at 37°C in fresh, serum-free and antibiotic-free medium for 3 h, and then transfected with miR-186-5p mimic (2 µg) or negative-control miRNA (2 µg) for 5 h prior to incubation at 37°C in HG for 24 h. For miR-186-5p inhibition treatment, cells were grown to 80–90% confluence and transfected with miR-186-5p inhibitor (2 µg) for 5 h and then incubated at 37°C with normal glucose (5.5 mM) for 24 h. The sequence information on miR-186-5p mimics and inhibitors are as follows: miR-186-5p mimics, 5′-CAAAGAAUUCUCCUUUUGGGCU-3′ and miR-186-5p inhibitor, 5′-AGCCCAAAAGGAGAAUUCUUUG-3′.

AC16 cells were seeded on 96-well plates at 5×10⁴ cells/well overnight at 37°C. Following treatment of AC16 cells with miR-186-5p mimic or inhibitor in the presence and absence of HG, respectively, for 24 h, and 10 µl MTS reagent was added to each well for 1 h at 37°C. The absorbance values were measured at 490 nm using a microplate spectrophotometer (Thermo Fisher Scientific, Inc.). Results were expressed as a percentage of control cells (transfected with negative-control miRNA). Each assay was independently performed in triplicate. Appreciation rate (%) = (mean OD value at time point/mean OD value at 0 Day-1) ×100. Suppression rate (%)=(1-mean OD value of experimental group/control group) ×100.

Nuclear morphology of AC16 cells was assessed via Hoechst 33258 staining. Briefly, AC16 cells were seeded into a 24-well plate at a density of 1×10⁵ cells/well overnight. Following treated as described above for 24 h, AC16 cells were washed with PBS three times and fixed with 4% formaldehyde for 10 min at 4°C. Cells were washed three times with PBS again and incubated with 10 µg/ml Hoechst 33258 at room temperature for 10 min in the dark. The morphological changes of AC16 cells were observed at ×200 magnification under a fluorescence microscope (Eclipse Ti; Nikon Corporation, Tokyo, Japan). Apoptosis rate was calculated as the number of apoptotic cells/total cells from the average of 5 random fields.

The apoptosis of AC16 cells was evaluated using an Annexin V-PI double staining assay kit according to the manufacturer's protocol. Briefly, AC16 cells were treated for 24 h as detailed above and the culture supernatant was collected. Subsequently, AC16 cells were washed and harvested with cold PBS. AC16 cells and supernatant were co-centrifuged at 1,000 × g at room temperature for 5 min. Following washing with PBS twice, cells were resuspended in 500 µl binding buffer from the Annexin V-PI kit and then co-incubated with 5 µl Annexin V-FITC reagent and 10 µl PI reagent for 10 min at 4°C in the dark. Cellular apoptosis was detected by flow cytometry (FC500; Beckman Coulter, Inc., Brea, CA, USA).

The level of miR-186-5p in AC16 cells, following treatment as described above, was detected with RT-qPCR. Total RNA was extracted from AC16 cells using an miRcute miRNA Isolation kit according to manufacturer's protocol. Single-strand cDNA was synthesized using an miRcute miRNA First-strand cDNA Synthesis kit according to manufacturer's protocol. qPCR was performed via a one step method with an miRcute miRNA qPCR Detection kit according to the manufacturer's protocol. U6 small nuclear RNA was used as an internal reference. Bulge-loop miRNA RT-qPCR Primer Sets (one RT primer and a pair of qPCR primers for each set) specific for miR-186-5p were designed by RiboBio (Guangzhou, China). Primer sequence of miR-186-5p was as follows: forward, 5′-TCAAAGAATTCTCCTTTTGGGCT-3′ and reverse 5′-CGCTTCACGAATTTGCGTGTCAT-3′. PCR was performed for 2 min at 94°C, followed by 40–45 cycles of 94°C for 20 sec and 60°C for 34 sec. The amplified products were measured using 1% agarose gel electrophoresis and densitometry of bands was analyzed using Image J software bundled with Java 1.8.0–112 (National Institutes of Health, Bethesda, MD, USA). Relative quantitative values were calculated using the 2^−ΔΔCq method (16).

AC16 cells were treated as detailed above and lysed in RIPA buffer containing 1 mM phenylmethane sulfonyl fluoride (Beyotime Institute of Biotechnology). Cellular protein was centrifuged at 12,000 × g for 10 min at 4°C and quantified using a BCA protein assay kit according to the manufacturer's protocol. Equal amounts of proteins (50 µl) were separated on 12% SDS-PAGE and electrotransferred onto a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). The membrane was then blocked with 5% no-fat milk in 0.05% Tween 20/TBS (TBST) for 2 h at room temperature. Following washing three times with TBST, the membrane was incubated with primary antibodies against cleaved caspase-3 (1:2,000) and GAPDH (1:2,000) overnight at 4°C. The membrane was subsequently incubated with a HRP-conjugated secondary antibody (1:5,000) for 2 h at room temperature and developed using an ECL solution. GAPDH was used as the internal loading control. The band densities were determined with Quantity One 4.6 software (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Each assay was independently performed in triplicate.

Data are presented as the mean ± standard error of the mean. The difference between groups was determined by one-way analysis of variance followed by Fisher's least significance difference test. P<0.05 was considered to indicate a statistically significant difference.

RT-qPCR was performed to measure the change of miR-186-5p level in HG-treated AC16 cardiomyocytes. As presented in Fig. 1, HG treatment for 12, 24 and 48 h significantly decreased the level of miR-186-5p in AC16 cells compared with control cells. The greatest decrease in miR-186-5p was observed following 24 h treatment. Therefore, 24 h was identified as the optimal treatment time for subsequent experiments.

To investigate whether miR-186-5p is associated with HG-induced myocardial cytotoxicity, we detected the effect of miR-186-6p mimic, which is able to simulate the high level of mature miR-186-6p in cells, or miR-186-5p inhibitor on the viability of AC16 cells. As presented in Fig. 2, pre-transfection of AC16 cells with miR-186-5p mimic significantly reversed the downregulation of cell viability induced by HG (Fig. 2A). In addition, HG induced a significant decrease in the appreciation rate of AC16 cells, compared with controls (Fig. 2B) and a significant increase in the suppression rate of AC16 cells (Fig. 2C), which were both significantly ameliorated by miR-186-5p mimic, which suggests that the AC16 cells transfected with miR-186-5p mimic had the ability to protect against long-term HG-induced injury. Furthermore, it was demonstrated that miR-186-5p inhibitor significantly downregulated the viability and appreciation rate (Fig. 2A and B) and significantly upregulated the suppression rate of AC16 cells (Fig. 2C) compared with controls, similar to treatment with HG, which suggests that miR-186-5p may have an important role in maintaining cell survival. These results showed that HG was able to induce cytotoxicity through downregulating the level of miR-186-5p in AC16 cells.

To determine whether an association exists between HG-induced cardiomyocyte apoptosis and miR-186-5p, the effect of miR-186-5p mimic and miR-186-5p inhibitor was observed on apoptosis in AC16 cells via Hoechst 33258 staining. As shown in Fig. 3, in the control group, AC16 cells exhibited regular-shaped nuclei and low intensity blue uniform fluorescence, whereas the numbers of AC16 cells with fragmented or condensed nuclei and bright blue fluorescence, which were characteristics of apoptotic cells. were markedly increased in HG-treated cells (Fig. 3A). However, compared with HG group, the number of AC16 cells with bright blue fluorescence was markedly decreased in the miR-186-5p mimic group. Furthermore, the rate of apoptosis was significantly upregulated by HG, in comparison with control cells; however, this was significantly ameliorated in miR-186-5p mimic cells (Fig. 3B). In addition, transfection with miR-186-5p inhibitor led to fragmented or condensed nuclei in AC16 cells. These results suggest that HG downregulated the level of miR-186-5p in AC16 cells, and therefore induced apoptosis.

Caspase-3 is considered as a key effector protease in the cellular apoptotic response, which is activated in the process of apoptosis followed by substrate binding, resulting in cell apoptosis through amplifying the cascade reaction (17). Therefore, the effect of miR-186-5p on cleaved caspase-3 level was measured in AC16 cells. As presented in Fig. 4, AC16 cells treated with HG or transfected with miR-186-5p inhibitor exhibited a significantly increased expression of cleaved caspase-3 protein compared with that of the control group. However, compared with the HG group, transfection with miR-186-5p mimic significantly decreased the expression of cleaved caspase-3 protein in AC16 cells. This suggests that HG induced an upregulation of cleaved caspase-3 protein expression via reducing the miR-186-5p level.

Finally, Annexin V/PI staining and flow cytometry were used to further test the effect of miR-186-5p on apoptosis in AC16 cells. As presented in Fig. 5, AC16 cells treated with HG or transfected with miR-186-6p inhibitor significantly increased the percentage of apoptotic cells in comparison with the control group, whereas transfection with miR-186-5p mimic significantly ameliorated the HG-induced increase in the apoptotic ratio. This result suggested that HG induced apoptosis through downregulating the miR-186-5p level in AC16 cells.