The embryonic rat cardiomyocyte-derived H9C2 cell line was purchased from the American Type Culture Collection and grown in Gibco Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc.), which contained Gibco 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (MilliporeSigma) in a humidified incubator under standard conditions (5% CO₂). After a 24-h incubation at 37°C, cells were washed three times with phosphate-buffered saline (PBS) and the growth media was replaced with glucose- and FBS-free DMEM. Cells were then cultured in a Thermo 3131 hypoxic incubator (<0.1% O₂, 5% CO₂ and 95% N₂; Thermo Fisher Scientific, Inc.) at 37°C for 24 h (Hypoxia-ischemic; HI). Following treatment, the cells then were reoxygenated in an incubator in a 5% CO₂ atmosphere for 2 h (Hypoxia-ischemic-reoxygenate; HI/R). H9C2 cells were used to HI/R conditions to induce a myocardial I/R injury model in vitro.

H9C2 cells were seeded into 24-well plates at a density of 1×10⁵ cells/well. The cells were cultured overnight before transfection at 37°C. Next, transfection with SNHG8 siRNA (100 nM), RASA1 siRNA (100 nM), negative control (NC; scrambled sequence) siRNA (Santa Cruz Biotechnology, Inc.), RASA1 plasmid, NC (empty plasmid) plasmid (OriGene Technologies, Inc.), miR-335 mimics, NC-mimics inhibitor or NC-inhibitor (Guangzhou RiboBio Co., Ltd.) into H9C2 cells was performed using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The transfection medium was replaced with complete medium 6 h after transfection, and the cells were incubated for the indicated times. All treatments were started 24 h after transfection. The siRNA sequences are presented in Table I and Fig. S1 demonstrates the transfection efficacy as determined by reverse transcription-quantitative PCR (RT-qPCR).

Cell viability was examined using a Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Inc.). The H9C2 cells transfected with SNHG8 siRNA, miR-335 mimics, inhibitor, RASA1 siRNA or NC were seeded into 96-well plates at a density of 3×10³ cells/well incubation at 37°C for 24 h. Then, 10 µl CCK-8 solution was added to the cells (100 µl/well) and incubated at 37°C for 3 h. An MRX II microplate reader (Dynex Technologies, Inc.) was used to detect the absorbance at 450 nm.

The cells were lysed by the cell lysis buffer (Cell Signaling Technology, Inc.) and samples centrifuged at 300 × g for 5 min at 4°C after the lysis treatment. The supernatant was collected and the protein concentrations measured with a BCA Protein Assay kit (Sigma-Aldrich; Merck KGaA). The proteins (20 µg/lane) were separated by 10% SDS-PAGE and then transferred to polyvinylidene difluoride membranes (EMD Millipore). The membranes were blocked with Tris-buffered saline (TBS) and 0.1% Tween 20 (TBS-T) containing 5% bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA) at room temperature and then incubated with anti-Bax (cat. no. ab182734), anti-Bcl-2 (cat. no. ab194583) or anti-cleaved caspase-3 (cat. no. ab2302; all from Abcam; 1:1,000 in TBST containing 5% BSA) primary antibodies overnight at 4°C. The membranes were washed with TBST three times and then incubated with the appropriate horseradish peroxidase-labeled secondary antibody (1:2,000, Cell Signaling Technology, Inc.; cat. no. 7076) for 2 h at room temperature. Finally, the ECL Plus detection system (EMD Millipore) was used to examine the protein bands and the expression levels of the target proteins were semi-quantified by detecting the optical density value of each band. GAPDH (Cell Signaling Technology, Inc.; cat. no. 5174; 1:1,000) served as an internal control.

The H9C2 cells were seeded in 96-well plates at a density of 3×10³ cells/well in culture media. The medium was replaced with serum-free medium to synchronize the cells. After 24 h, the serum-free medium was replaced with growth media for 48 h. Cell proliferation was assessed using a Click-iT EdU imaging kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions.

TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to extract total RNA from the H9C2 cells according to the manufacturer's instructions. Total RNA was then reverse transcribed to cDNA using a PrimeScript RT Reagent kit (Takara Biotechnology, Co., Ltd.; cat. no. RR047A). qPCR was then performed using a SYBR Green RT-PCR Kit (Takara Biotechnology, Co., Ltd.). The PCR conditions were as follows: 40 cycles of 95°C for 30 sec, 60°C for 34 sec and 72°C for 30 sec. U6 (Takara Biotechnology, Co., Ltd.; cat. no. 638315) and β-actin served as the internal controls. The U6 was used for miRNA, and mRNA and lncRNA was normalization by β-actin. The relative expression levels of lncRNA SNHG8, miR-335 and RASA1 expression were assessed using the comparative 2^−ΔΔCq method (25) and primer sequences are presented in Table II and Table SI.

The cDNA fragments of SNHG8 carrying the wild-type (WT) or mutated (MuT) binding sites of miR-335 and the 3′-untranslated region (UTR) of the amplified RASA1 fragment containing the predicated target sites for miR-335 were synthesized by Shanghai GenePharma Co., Ltd. H9C2 cells (1×10⁵ cells/well) were seeded into 24-well plates and co-transfected with 50 ng recombinant luciferase vectors, 10 ng pGL3 vectors and 50 nM miR-335 mimics, miR-335 inhibitor or NC-mimic, NC-inhibitor using Lipofectamine^® 2000 (Thermo Fisher Scientific, Inc.). After transfection for 48 h, the cells were lysed and luciferase activities were evaluated using the Dual-Luciferase Reporter Assay System (Promega Corporation) and a luminometer (Glomax20/20; Promega Corporation).

The expression of LDH was determined by Cytotoxicity LDH Assay Kit-WST (Roche Diagnostics). Briefly, 50 µl cell suspension (2.5×10⁴ cells) was added to each well of a 96-well plate after transfection with or without SNHG8 siRNA, RASA1 siRNA or miR-335 inhibitor and incubated at 37°C for 1 h. Lysis buffer (10 µl) was added to each well and incubated at 37°C for 30 min. Then, 100 µl working solution was added to each well. The plate was protected from light and incubated at room temperature for 30 min. Finally, 50 µl stop solution was added to each well and the absorbance was measured at 490 nm using a microplate reader.

The number of apoptotic cells was determined by flow cytometry using the Annexin V-FITC Apoptosis Detection Kit (Dojindo Molecular Technologies, Inc.). Different treatment groups of H9C2 cells were treated with a trypsin-EDTA (0.25%) solution and centrifuged at 300 × g for 3 min at 4°C. Annexin V Binding Solution, diluted 10-fold, was added to make a final cell concentration of 1×10⁶ cells/ml. The cell suspension (100 µl) was transferred to a new tube, 5 µl Annexin V-FITC conjugate was added and then 5 µl PI solution was added to the cell suspension. The samples were protected from light and incubated for 15 min at room temperature. Finally, 10-fold diluted Annexin V binding solution (400 µl) was added and the percentage of early + late apoptotic cells was quantified by flow cytometry with a FACSCalibur system equipped with the CellQuest software (version 5.1; BD Biosciences).

Bioinformatics analysis was performed using StarBase v 3.0 (http://starbase.sysu.edu.cn/index.php) and miRTarBase (http://mirtarbase.cuhk.edu.cn/php/index.php) to determine the association between miRNA and lncRNA (26,27).

Data are expressed as the mean ± SD, and statistical analysis was performed using GraphPad Prism 8 software (GraphPad Software, Inc.). Statistical comparisons were conducted using unpaired Student's t-test or one-way analysis of variance with Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To determine the protective effect of lncRNA on HI/R myocardial injury, the expression levels of lncRNA were detected in the control group and HI/R myocardial cells. The results revealed that lncRNA SNHG8 was the most markedly upregulated lncRNA (Fig. 1A). It was also found that treatment with SNHG8 siRNA significantly increased HI/R-induced cell viability and proliferation, as analyzed by the CCK-8 assay and EdU analysis. LDH analysis demonstrated that treatment with SNHG8 siRNA reduced HI/R-induced cell damage (Fig. 1B-D). Subsequently, apoptotic rates and the change of apoptotic-related protein expressions following SNHG8 siRNA treatment in the HI/R myocardial cells was examined. As presented in Fig. 1E and F, HI/R-induction increased apoptosis and the protein expression levels of Bax and cleaved caspase-3 and reduced the expression of Bcl-2 protein, whereas HI/R treated cells transfected with SNHG8 siRNA exhibited decreased cell apoptosis and Bcl-2 and cleaved caspase-3 expression and increased the expression of Bcl-2 protein. These data indicated that inhibition of SNHG8 may protect against HI/R-induced myocardial cell injury.

It has previously been reported that lncRNA mediates HI/R injury through the regulation of miRNAs, such as lncRNA MALAT1 which can regulate miRNA-20b after HI/R injury (9,28). Bioinformatics analysis was performed to screen potential miRNAs that have complementary base pairing with SNHG8. We firstly determined the expression of miRNA after under HI/R condition, showing that miR-335 was (Fig. S2A). StarBase analysis identified miR-335 as a potential target of SNHG8 (Fig. 2A). Consistent with our predication, the dual-luciferase reporter assay verified that miR-335 mimics could decrease the luciferase activity of pGL3-SNHG8-wild-type (WT), whereas no significant difference on pGL3-SNHG8-mutant (Mut) was observed (Fig. 2B). The schematic diagram of the relationship between SNHG8 and miR-335 was shown in Fig. 2C. Furthermore, silencing of SNHG8 resulted in upregulation of miR-335 expression (Fig. 2D).

To further determine the role of miR-335 on HI/R myocardial injury, H9C2 cells were co-transfected with a miR-335 inhibitor and SNHG8 siRNA. As shown in Fig. 3A, treatment with the miR-335 inhibitor reduced cell viability compared with HI/R, whereas inhibition of SNHG8 increased cell viability after co-transfected with the miR-335 inhibitor on HI/R-induced H9C2 myocardial cell injury. Furthermore, SNHG8 siRNA combined with the miR-335 inhibitor was able to enhance cell proliferation and reduce LDH release compared with the miR-335 inhibitor alone group (Fig. 3B and C, respectively). SNHG8 siRNA reduced the miR-335 inhibitor-induced increase in the number of apoptotic cells under the HI/R condition in H9C2 cells (Fig. 3D). Western blot analysis of apoptosis-related proteins demonstrated that, Bax was decreased and Bcl-2 was increased following co-transfection with SNHG8 siRNA and miR-335 inhibitor (Fig. 3E).

Based on the aforementioned results, TargetScan and miRTarBase were used to investigate the binding site of miR-335 (29,30). The overlapping analysis of the 196 differentially expressed mRNAs identified by TargetScan and the predicted single mRNA identified by miRTarBase indicated that only RASA1 interacted with miR-335 (Fig. S2B). The results indicated that miR-335 targets the 3′-UTR of the RASA1 mRNA (Fig. 4A). The schematic diagram of the relationship between miR-335 and RASA1 is shown in Fig. 4B. A dual-luciferase reporter assay was used to further verify this prediction, and the results indicated that miR-335 mimics decreased the luciferase activity of pGL3-RASA1-WT, whereas no effect on pGL3-RASA1-Mut was observed (Fig. 4C). In addition, it was demonstrated that overexpression of miR-335 significantly downregulated the expression level of RASA1 mRNA, whereas transfection with the miR-335 inhibitor had the opposite effect on the expression of RASA1 (Fig. 4D). The expression of RASA1 following transfection of HI/R-induced H9C2 cells with SNHG8 siRNA was also confirmed. The results demonstrated that SNHG8 siRNA treatment significantly downregulated the expression of RASA1 (Fig. 4E).

To investigate whether miR-335 and RASA1 mediated the protective effect of SNHG8 on HI/R-induced H9C2 cell injury, a series of experiments was performed. Knockdown of RASA1 protected HI/R-induced H9C2 cells by increasing cell viability and cell proliferation, and decreasing LDH release, whereas compared with RASA1 siRNA + HI/R group, there was no significant difference in cell viability, proliferation and LDH following treatment with SNHG8 siRNA and RASA1 siRNA (Fig. 5A-C). Moreover, following RASA1 silencing, SNHG8 did not further reduce apoptosis, again demonstrating that SNHG8 may act through RASA1 (Fig. 5D). Western blot analysis showed that, compared with the RASA1 siRNA group, there was no significant difference in Bax and Bcl-2 expression after combined treatment with RASA1 siRNA and SNHG8 siRNA (Fig. 5E).

The protective effect of SNHG8 combined with RASA1 overexpression plasmid on HI/R induced H9C2 cells was analyzed further. The data demonstrated that RASA1 overexpression reduced cell viability, whereas SNHG8 siRNA combined with RASA1 overexpression increased viability (Fig. S2C). These results demonstrated that lncRNA SNHG8 may regulate the expression of RASA1 and that RASA1 may mediate the effects SNHG8 on the damage to cells. These data indicated that SNHG8 may protect HI/R-induced H9C2 cell injury through the regulation of miR-335 and RASA1 expression.