miR-451, antisense miR-451 and empty plasmid adenovirus (containing green fluorescent protein; GFP), termed Ad-miR-451, Ad-asmiR-451 and Ad-GFP, respectively, were generated by Shanghai Genechem (Shanghai Genechem Co., Ltd., Shanghai, China) by cloning miR-451 or antisense miR-451 primary DNA, or the empty plasmid in the reverse orientation relative to the CMV promoter. The recombinant viruses were then amplified in HEK293 cells (Shanghai Genechem Co., Ltd.) and purified using the Adeno-X Purification kit (Microbix Biosystems, Inc., Mississauga, ON, Canada) in order to reach the titer of 1,011 pfu/ml.

The experimental protocol conformed to the Guideline for the Care and Use of Laboratory Animals (US National Institutes of Health publication, revised 2010) (20) and approved by the Institutional Animal Care and Use Committee of Wuhan University (Hubei, China). Sprague-Dawley rats (1–3 days old, n=96) were purchased from the Center of Experimental Animal (Wuhan University, Hubei, China). Rats were kept under identical housing conditions (temperature: 18–22°C, humidity: 50–60%) with a 12 h light-dark cycle and access to water and food ad libitum. Rats were sacrificed under etherization and the hearts were removed under sterile conditions. Neonatal rat ventricular myocytes were prepared by enzymatic dissociation as previously described (21). Briefly, the hearts were harvested, finely minced with scissors, and then dissociated with 0.125% (w/v) trypsin (HyClone Laboratories; GE Healthcare Life Sciences, Logan, UT, USA) and 0.08% collagenase I (HyClone Laboratories; GE Healthcare Life Sciences) for 40 min at 37°C. Cardiomyocytes were seeded at a density of 1×10⁶ cells/ml and cultured in Dulbecco's modified Eagle's medium nutrient mixture F-12 (DMEM/F12; HyClone Laboratories; GE Healthcare Life Sciences) supplemented with 15% fetal bovine serum (FBS; Shanghai Weike Biochemical Reagent Co., Ltd., Shanghai, China), 1% penicillin (100 U/ml; North China Pharmaceutical Huasheng Co., Ltd., Shijiazhuang, China) and 1% streptomycin (100 μg/ml; North China Pharmaceutical Huasheng Co., Ltd.) at 37°C, with 5% CO₂ for 4 days. Cells were then randomly allocated into five groups and treated as follows: i) Control group, DMEM/F12 with 15% FBS; ii) A/R group, DMEM/F12 for 4 h anoxia followed by 2 h reoxygenation with DMEM/F12 with 15% FBS; and iii) A/R + Ad-GFP; iv) A/R + Ad-miR-451; and v) A/R + Ad-asmiR-451 groups were subjected to Ad-GFP, Ad-miR-451 and Ad-asmiR-451 (final MOI=100, infection time=2 h), respectively, 24 h prior to A/R as described for group ii.

GFP expression was considered the marker of successful transfection of adenovirus, and the percentage of positive GFP fluorescence was considered the transfection ratio. Apoptosis was assessed by flow cytometric analysis of propidium iodide (PI) and Annexin V double staining (Multi Sciences (Lianke) Biotech Co., Ltd., Hangzhou, China). Briefly, cardiomyocytes were harvested following treatment, rinsed with phosphate-buffered saline (PBS) and suspended in 500 μl binding buffer (Multi Sciences (Lianke) Biotech Co., Ltd.). Cells were then incubated with 5 μl Annexin V and 5 μl PI, and the stained cells were analyzed using the BD LSRFORTESSA flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

Total RNA was extracted from cells with TRIzol reagent according to the manufacturer's instructions (Roche Diagnostics, Indianapolis, IN, USA) and reverse transcribed to produce cDNA from total RNA with oligo-dT primers (Generay Biotech Co, Ltd., Shanghai, China). The cDNA synthesis reaction was performed using 2 μg of total RNA and a Transcriptor First Strand cDNA Synthesis kit (Roche Diagnostics). The assessment of RNA integrity was conducted with an optical density (OD) measurement via a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and an OD 260/280 ratio >2 was considered an acceptable indicator of subsequent cDNA synthesis. RT-qPCR reactions were performed in 20 μl volumes using the LightCycler 480 SYBR Green I Master Mix (Roche Diagnostics) and the LightCycler 480 Real-time PCR system (Roche Diagnostics) according to the manufacturer's instructions. The reaction conditions were as follows: 1 cycle of 95°C for 10 min, followed by 40 cycles of 95°C for 10 sec, 60°C for 10 sec, 72°C for 20 sec, 1 cycle for 95°C for 5 sec and 65°C for 1 min. The samples were quantified by normalizing the gene expression levels of HMGB1 to that of the standard housekeeping β-actin gene, and the gene expression levels of miR-451 to that of U6 gene, and were expressed as relative mRNA levels compared with the internal controls using the 2^−ΔΔCq normalization method (22).

Cell viability was determined using the cell counting kit (CCK)-8 assay (Dojindo Molecular Technologies, Inc., Tokyo, Japan) according to the manufacturer's instructions. Briefly, the cardiomyocytes were seeded and incubated for 4 days in 96-well plates at 1×10⁵ cells/well prior to group separation as mentioned. The absorbance (optical density, OD) of each well was measured at 490 nm with an iMark microplate absorbance reader (Bio-Rad Laboratories, Hercules, CA, USA). The percentage cell viability (CV) was calculated using the following formula: % CV = (mean OD of test wells)/(mean OD of control well) × 100.

The oxidative stress injury was measured by the indexes of oxygen free radical in myocytes. SOD activity was measured using the SOD assay kit (Nanjing JianCheng Bioengineering Institute, Nanjing, China; cat. no. A001-1) according to the manufacturer's instructions.

Equal quantities of proteins (15 μl) were separated on 10% SDS-polyacrylamide gels (run on 160 V for 70 min) and then electro-transferred to a polyvinylidene difluoride membrane. The membranes were blocked with 5% non-fat milk for 1 h at room temperature, and rinsed twice for 2 min in Tris-buffered saline and Tween 20. Membranes were incubated with mouse monoclonal anti-HMGB1 (1:1,000; Sigma-Aldrich, St. Louis, MO, USA; cat. no. H9537), and rabbit polyclonal anti-caspase-3 and anti-GAPDH (1:1,000 and 1:5,000; Cell Signaling Technology, Inc., Danvers, MA, USA; cat. nos. 9662 and 5174, respectively) primary antibodies as previously described (23), with an overnight incubation at 4°C. The membranes were then incubated with the corresponding polyclonal goat anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA; cat. nos. 115-035-003 and 111-035-003, respectively) for 1 h at room temperature. The proteins were visualized with the Pierce ECL kit (Thermo Fisher Scientific, Inc.; cat. no. NCI4106). The expression of proteins of interest were normalized to the GAPDH expression.

To confirm whether miR-451 was able to directly recognize the 3′-untranslated region of HMGB1 and inhibit its expression, a luciferase assay was performed using the Dual-Luciferase Reporter Assay System (Promega Corporation, Madison, WI, USA) according to the manufacturer's instructions. Briefly, construct in which a fragment of the 3′-UTR of HMGB1 mRNA (containing either putative miR-451 binding sequence or mutant putative miR-451 binding sequence) was cloned into a firefly luciferase reporter construct and transfected into HEK293 cells with an empty plasmid (pDNR-CMV) or a plasmid expressing miR-451 (pmiR-451). The luciferase activity was normalized to the Renilla luciferase signal in HEK293 cells. Results were determined as the firefly to Renilla luciferase activity ratio (Fluc/Rluc).

Data are expressed as the mean ± standard deviation. Statistical analysis was performed using one-way analysis of variance followed by the Student-Newman-Keuls test. SPSS software, version 17.0 (SPSS, Inc., Chicago, IL, USA) was used for analysis. P<0.05 was considered to indicate a statistically significant difference.

The transfection ratio was determined by the GFP expression in the cells, and the ideal MOI was measured at 100 (transfection ratio, 87.6%). Gain- and loss-of-function approaches were utilized to regulate miR-451 expression in the cultured cardiomyocytes. As demonstrated in Fig. 1A, miR-451 expression was significantly increased in the Ad-miR-451 group compared with the control group (P<0.05). In the Ad-asmiR-451 group, miR-451 expression was decreased compared with the Ad-miR-451 group (Fig. 1A). Treatment with 4 h anoxia followed by 2 h reoxygenation decreased the expression of miR-451 in the A/R alone group compared with the control group (P<0.05; Fig. 1B). A/R treatment significantly increased miR-451 expression in the Ad-miR-451 group compared with the A/R alone group (P<0.05; Fig. 1B). miR-451 expression was markedly decreased in the Ad-asmiR-451 group compared with the Ad-miR-451 group (Fig. 1B). Furthermore, Ad-GFP alone had no significant effect on the expression of miR-451 in the A/R cardiomyocytes (P>0.05; Fig. 1).

As demonstrated in Fig. 2, the viability of cardiomyocytes was significantly decreased in the A/R alone group compared with the control group (P<0.05). Ad-miR-451 significantly attenuated cell viability during A/R compared with the A/R alone group (P<0.05; Fig. 2). In addition, Ad-asmiR-451 and Ad-GFP alone had no significant effect on the cell viability compared with the A/R alone group (P>0.05; Fig. 2).

SOD activity was significantly decreased in the A/R alone group compared with the control group (P<0.05; Fig. 3). Ad-miR-451 significantly inhibited the increase of SOD during A/R compared with the control group (P<0.05; Fig. 3), and significantly increased SOD activity compared with the A/R group (P<0.05; Fig. 3). Ad-asmiR-451 could slightly aggravate the increase of SOD during A/R compare with the Ad-miR-451 group (P>0.05; Fig. 3). Ad-GFP alone had no significant effect on SOD activity during A/R (P>0.05; Fig. 3).

The apoptosis rate of cardiomyocytes was significantly increased in the A/R alone group compared with control group (P<0.05; Fig. 4). Ad-miR-451 significantly suppressed the apoptosis of cardiomyocytes during A/R compared with the A/R alone group (P<0.05; Fig. 4). Ad-asmiR-451 and Ad-GFP alone had no significant effect on the apoptosis of cardiomyocytes during A/R (P>0.05; Fig. 4).

RT-qPCR revealed that HMGB1 mRNA expression was significantly decreased in the Ad-miR-451 group during A/R compared with the A/R alone group (P<0.05; Fig. 5). The mRNA expression of HMGB1 was not significantly changed in the Ad-asmiR-451 group compared with that in the A/R group (P>0.05; Fig. 5).

HMGB1 relative protein expression was significantly increased during A/R compared with the control group (P<0.05; Fig. 6A and B). In the Ad-miR-451 group, HMGB1 relative protein expression was significantly decreased compared with the A/R alone group (P<0.05; Fig. 6A and B). The HMGB1 relative protein expression was not significantly changed in the Ad-asmiR-451 group compared with the A/R alone group (P>0.05; Fig. 6A and B).

The cleaved caspase-3 relative expression was significantly increased during A/R compared with the control group (P<0.05; Fig. 6A and C), and significantly decreased in the Ad-miR-451 group compared with the A/R alone group (P<0.05; Fig. 6A and C). In the Ad-asmiR-451 no significant change was observed in the protein levels of cleaved caspase-3 compared with that in the A/R alone group (P>0.05; Fig. 6A and C).

The luciferase assay was performed to verify HMGB1 as one of the target genes of miR-451 in cardiomyocytes. A construct in which a fragment of the 3′-UTR of HMGB1 mRNA containing the putative or mutant-putative miR-451 binding sequence, was cloned into a firefly luciferase reporter and cotransfected into HEK293 cells with the plasmid expressing miR-451. The luciferase assay was conducted to further confirm that HMGB1 was a direct target of miR-451 in the HEK-293T cells. The results demonstrated that miR-451 transfection significantly inhibited the luciferase activity of wild type HMGB1 3′ UTR and not the mutant HMGB1 3′ UTR with the mutant binding site of miR-451 (Fig. 7). These results demonstrated that HMGB1 is a direct target of miR-451 in HEK-293T. The results suggested that miR-451 binds to HMGB1 mRNA directly and inhibits its expression.