The rat myocardial H9c2 cell line was obtained from the Shanghai Institutes for Biological Sciences (Shanghai, China). Cells were grown in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (TBD Science, Tianjin, China), 100 units of penicillin/ml and 100 µg of streptomycin/ml in a humidified atmosphere at 37°C with 5% CO₂. H9c2 cells were treated with 400 µM PA (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for consecutive 6, 12 or 24 h to induce myocardial lipotoxic injury according to the previously published study (4).

Following the aforementioned treatment with PA for 24 h, cells were fixed in 4% paraformaldehyde for 30 min at room temperature. Following washing three times with phosphate-buffered saline, cells were incubated with 3 mg/ml Oil Red O working solution (Sigma-Aldrich; Merck KGaA) for 30 min at room temperature. Cell nuclei were then stained with hematoxylin for 2 min at room temperature, followed by imaging with a light microscope (Olympus Corporation, Tokyo, Japan).

GAS5-specific small interfering RNA (siRNA) (sense, 5′-TCTCACAGGCAGTTCTGTGG-3′, antisense, 5′-ATCCATCCAGTCACCTCTGG-3′), scrambled siRNA [serving as the negative control (NC)], (sense, 5′-UUCUCCGAACGUGUCACGUTT-3′, antisense, 5′-ACGUGACACGUUCGGAGAATT-3′) miR-26a mimics (5′-UUCAAGUAAUCCAGGAUAGGCU-3′), mimics NC (5′-UUCUCCGAACGUGUCACGUTT-3′), miR-26a inhibitor (5′-AGCCTATCCTGGATTACTTGAA-3′) and inhibitor NC (5′-CAGUACUUUUGUGUAGUACAA-3′) were designed and synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). Briefly, cells were transfected with the 5 µl siRNAs (20 µM) and 200 µl miRNA nucleotide sequences (100 nM) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. At 24 h post-transfection, cells were then washed and cultured in fresh medium, followed by treated with 400 µM PA for 24 h.

Total RNA was extracted from the cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. RNA concentration and A260/280 was measured using a NanoPhotometer P-Class (Implen GmbH, Munich, Germany). To detect mRNA expression levels, the PrimeScript RT reagent kit with gDNA Eraser (Takara Bio, Inc., Otsu, Japan) and SYBR Premix Ex Taq II (Takara Bio, Inc.) were used for RT and qPCR, respectively, according to the manufacturer's protocol. To detect miR-26a expression, Mir-X™ miRNA First Strand Synthesis kit and Mir-X™ miRNA RT-qPCR SYBR^® kit (Takara Bio, Inc., Otsu, Japan) were used for RT and qPCR, respectively, according to the manufacturer's protocol. PCR was performed at 42°C for 30 min; 95°C for 10 min, followed by 45 cycles of amplification at 95°C for 20 sec, 62°C for 30 sec, 72°C for 30 sec. The melt curve stage was performed at 95°C for 15 sec, 60°C for 1 min, 95°C for 15 sec. GAPDH and U6 were used as the internal controls in the detection of mRNA and miRNA expression levels. All oligonucleotide primers were designed by Shanghai Sangon Biotech Co., Ltd. (Table I). The relative expression was analyzed using 2^−ΔΔCq method (23).

Cells were seeded at 5×10³ cells/well in 96-well plates. Following transfection for 24 h, the cell viability was determined by CCK-8 assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) according to the manufacturer's protocol. The absorbance value was detected at 450 nm by an Epoch 2 Microplate Spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA).

Cells were seeded at 1×10⁵ cells/well in 24-well plates. Following transfection for 24 h, LDH released from the cardiomyocytes into the culture medium was measured using an LDH assay kit (cat. no. A020-1; Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's protocol. The absorbance value was detected at 450 nm by an Epoch 2 Microplate Spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA).

Cells were seeded at 1×10⁵ cells/well in 24-well plates. Following transfection for 24 h, the concentrations of tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β) in the culture medium were detected using commercial rat TNF-α or IL-1β ELISA kits (cat. no. EK3822/2 and EK301B2/2, respectively; Hangzhou MultiSciences Biotech Co., Ltd., Hangzhou, China) according to the manufacturer's protocol. To measure the activity of NF-κB, cells were harvested after transfection and PA treatment, and nuclear extract lysates were extracted from harvested cells using Nuclear Extraction kit (cat. no. ab113474; Abcam, Cambridge, UK), according to the manufacturer's protocol. The activity of NF-κB in nuclear extract lysates was detected using NF-κB p65 transcription factor assay kit (cat. no. ab133112; Abcam) according to the recommended experimental protocol.

starBase v2.0 (http://starbase.sysu.edu.cn/index.php) and LncBase Predicted v.2 tool (http://carolina.imis.athena-innovation.gr/diana_tools/web/index.php?r=lncbasev2/index-predicted) were employed to predict the binding cites between GAS5 and miRNAs.

Luciferase reporter plasmids (pmirGLO-GAS5-wt and pmirGLO-GAS5-mut) were obtained from Shanghai GenePharma Co., Ltd. Briefly, 2×10⁵ cells were plated into each well of a 24-well plates and transfected with pmirGLO-GAS5-wt or pmirGLO-GAS5-mut together with miR-26a mimics or mimics NC, as previously described (18). At 48 h post-transfection, luciferase activity was analyzed using the Dual-Glo Luciferase assay system (Promega Corporation, Madison, WI, USA) according to the manufacturer's protocol.

Total proteins were extracted with RIPA buffer (Beyotime Institute of Biotechnology, Shanghai, China). The nuclear and cytoplasmic proteins were separately extracted using Nuclear and Cytoplasmic Protein Extraction kit (Beyotime Institute of Biotechnology), according to the manufacturer's protocol, and the protein concentration was measured by Enhanced BCA Protein Assay kit (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. Next, 50 µg heat-denatured proteins were separated by 8% or 10% SDS-PAGE and then transferred to polyvinylidene difluoride membranes, followed by blocking with 1% bovine serum albumin solution at room temperature for 1 h. The membranes were then incubated at 4°C overnight with primary antibodies, including anti-HMGB1 (cat. no. ab128129; 1:1,000; Abcam), anti-NF-κB p65 (cat. no. ab193238; 1:1,000; Abcam), anti-GAPDH (cat. no. TA336768; 1:1,000; Zhongshan Jinqiao Biotechnology, Beijing, China) and anti-Lamin A (cat. no. ab26300; 1:1,000; Abcam), followed by incubation with horseradish peroxidase conjugated goat anti-rabbit or goat anti-mouse immunoglobulin G (cat. no. E030120-01; 1:4,000; EarthOx Life Sciences, Millbrae, CA, USA) at room temperature for 30 min. Detection of protein bands was performed using the enhanced chemiluminescence for western blotting kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. GAPDH or Lamin A was used as an internal control. Relative protein expression was determined by densitometry using Image J2× analysis software (National Institutes of Health, Bethesda, MD, USA).

All experiments were repeated three times. Data are presented as the mean ± standard error of the mean. Statistical analysis was performed with SPSS version 17.0 software (SPSS, Inc., Chicago, IL, USA). Differences between groups were initially evaluated using one-way analysis of variance, and multiple comparison analysis was further performed in the case that differences were statistically significant using Fisher's least significant difference test. P<0.05 was considered to indicate a statistically significant difference.

Oil Red O staining illustrated that lipids were accumulated in H9c2 cells following treatment with 400 µM PA (Fig. 1A), and the results of CCK-8 assay demonstrated that cell viability was markedly decreased by PA treatment (Fig. 1B), which suggested that PA induced lipotoxic injury in cardiomyocytes. In addition, the pro-inflammatory cytokine TNF-α and IL-1β mRNA and protein levels were significantly increased by PA treatment in H9c2 cells, as compared with the control group (Fig. 1C and D). This further suggests that PA exposure caused inflammation. Furthermore, the results of RT-qPCR revealed that the expression of GAS5 in H9c2 cells was significantly increased by 3.65- and 7.82-fold following treatment with 400 µM PA for 12 and 24 h, respectively (Fig. 1E). Therefore, these results demonstrated that GAS5 expression was induced by PA treatment in H9c2 myocardial cells.

The expression of GAS5 in H9c2 cells was reduced by nearly 70% following transfection with GAS5-specific siRNA (Fig. 2A), suggesting that GAS5 siRNA effectively inhibited the endogenous expression of GAS5 in cardiomyocytes. It was further observed that the cell viability was markedly increased (Fig. 2B) and the activity of LDH was significantly decreased by the downregulation of GAS5 in PA-treated cells (Fig. 2C). Taken together, these results demonstrated that the knockdown of GAS5 expression alleviated PA-induced myocardial injury.

To evaluate whether GAS5 regulated PA-induced inflammation, the present study detected the mRNA expression and concentration levels of the pro-inflammatory cytokines TNF-α and IL-1β. The results demonstrated that the mRNA expression levels of TNF-α and IL-1β in PA-treated H9c2 cells were notably reduced by the downregulation of GAS5 as compared with the non-transfected PA-treated group (Fig. 3A and B). In addition, the concentrations of TNF-α and IL-1β in PA-treated H9c2 cells were significantly decreased by the downregulation of GAS5 (Fig. 3C and D). Taken together, these results demonstrated that the downregulation of GAS5 inhibits PA-induced inflammation.

Bioinformatics analysis (starBase v2.0; http://starbase.sysu.edu.cn/index.php) revealed that the GAS5 transcript sequence contained a miR-26a binding region (Fig. 4A). Subsequently, the results of the luciferase activity assay demonstrated that luciferase activity in the pmirGLO-GAS5 group was reduced by 61% in the group transfected with miR-26a mimics compared with mimics NC (Fig. 4B). In the pmirGLO-GAS5-mut group, miR-26a mimics did not have a significant inhibitory effect on luciferase activity when compared with the mimics NC (Fig. 4B). These results demonstrated that GAS5 specifically binds with miR-26a.

Furthermore, the knockdown of GAS5 was demonstrated to markedly upregulate miR-26a expression, while treatment with miR-26a inhibitor significantly increased GAS5 expression (Fig. 4C and D). Therefore, these results further demonstrated that a reciprocal negative regulation exists between miR-26a and GAS5.

As HMGB1 is a known target of miR-26a (24), the present study investigated whether GAS5, acting as a ceRNA, regulated the expression of HMGB1, and examined whether the regulation of GAS5 on the expression of HMGB1 was mediated by miR-26a. The results demonstrated that the mRNA and protein expression levels of HMGB1 were repressed by transfection with the GAS5-specific siRNA alone in PA-treated cells. However, co-transfection with the GAS5-specific siRNA and miR-26a inhibitor prevented the inhibition of the mRNA and protein expression levels of HMGB1 (Fig. 5A and B). Similarly, transfection with the GAS5-specific siRNA alone decreased the protein expression of NF-κB in the nucleus, increased the cytoplasmic protein expression of NF-κB and inhibited the activity of NF-κB in cardiomyocytes. However, these effects of GAS5 knockdown on NF-κB were abolished by co-transfection with the miR-26a inhibitor (Fig. 5C and D). Taken together, these results demonstrated that the knockdown of GAS5 expression inhibited the HMGB1/NF-κB signaling pathway, and this effect was mediated by miR-26a.