Accumulating evidence shows that long non-coding RNAs (lncRNAs) are widely involved in cellular processes of myocardial ischemia/reperfusion (I/R). The present study investigated the functions of lncRNA SNHG16 in myocardial I/R and the mechanism mediated by SNHG16. The myocardial I/R rat and cell model and hypoxia/reoxygenation injury (H/R) models of H9C2 cardiomyocytes were established to detect the expression of SNHG16. Cell Counting Kit-8, flow cytometric and western blot assays were conducted to detect cell viability, apoptosis and protein expression. Myocardial cell apoptosis was assessed by TUNEL staining. Dual-luciferase gene reporter was applied to determine the interaction between the molecules. The expressions of SNHG16 were upregulated in myocardial I/R injury models. Inhibition of SNHG16 relieved myocardial I/R injury
Myocardial ischemia/reperfusion injury (I/R) is the restoration of blood perfusion following ischemia, which causes metabolic dysfunction and aggravates the structural damage of myocardial cells, leading to cell death and infarction enlargement (
Long non-coding RNAs (LncRNAs) are a class of non-coding RNAs with a length of more than 200 nucleotide molecules (
LncRNA can regulate target gene expression through competitively binding microRNA (miRNA/miR) (
A total of 26 male SPF grade Sprague-Dawley (SD) rats (male rats are more resilient and their hormone levels change more steadily than female ones) weighing 200-250 g (7-8 week-old) were obtained from Beijing Experimental Animal Center and kept at 25±3˚C and 50-60% humidity in a room with a 12-h light/dark cycle. Experiments were conducted according to the Declaration of Helsinki. The Animal Care and Use Committee of Cangzhou Central Hospital (approval no. 2022-013-01z) approved all rat protocols and procedures. Rats were randomly divided into four groups, including sham (Sham group, n=6) and the reperfusion 24 h group, including I/R group, n=7; I/R + shNC, n=7 and I/R + shSNHG16, n=6).
SD rats were fasted for 12 h before surgery, anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg body weight) and fixed in an inverted position. Tracheotomy and intubation were performed and an artificial ventilator (frequency: 70 times/min; tidal volume: 20 ml; respiration ratio: 1:1) was connected. A needle electrode was inserted subcutaneously into the extremities to record the ECG and connect the BL420S biological function experimental system. The left anterior descending coronary artery was found between the pulmonary artery conus and the left atrial appendage. The anterior descending coronary artery was ligated with a 5/0 thread between the pulmonary artery conus 2-4 mm below the root of the left atrial appendage to block the coronary blood flow. Following ligation for 30 min, the ligation line was loosened and reperfusion for 120 min. The rats were allowed to regain consciousness during reperfusion period. At the end of experiment, the rats were anesthetized with 3% pentobarbital sodium (50 mg/kg) for approximately 10 min (until the rat was immobile) followed by sacrifice via cervical dislocation.
After 120 min of reperfusion, blood samples from each group were collected. The levels of lactate dehydrogenase (LDH) in the serum were detected by ELISA according to the manufacturer's instructions (cat. no. A020-2-2; Nanjing Jiancheng Bioengineering Institute).
At the end of reperfusion, the myocardium tissue was fixed with 10% neutral buffer formalin for 48 h. After processed in a series of graded ethanol and dimethyl benzene, the tissues were embedded in paraffin, and 4-5 µm sections were stained with stained with hematoxylin for 5 min and eosin for 3 min at room temperature, sealed and observed under light microscope (magnification, x200) and the results from 3 fields of each group were obtained.
Rat cardiomyocytes H9C2 were obtained from the cell bank of Shanghai Institute of Biology, Chinese Academy of Sciences. The H9C2 cells were cultured in high glucose DMEM (Gibco; Thermo Fisher Scientific, Inc.) medium containing 10% FBS and 1% penicillin-streptomycin at 37˚C in an atmosphere of 95% air and 5% CO2. The culture medium was changed every 3 days and the logarithmic growth cells were digested with 0.25% trypsin. For cell transfection, cells were seeded into 12-well plates at a density of 1.5x105 cells per well and transfected with the short hairpin (sh)SNHG16 and negative control (NC) shRNA-NC plasmid, miR-183 specific inhibitor (miR-183 antisense oligodeoxyribonucleotide, miR-183-ASO) and miR-NC, miR-183-mimics and mimics control, pcDNA3.1-forkhead box O1 (FoxO1) and pcDNA3.1 vectors were synthesized by Hanbio Biotechnology Co., Ltd. and infection was performed according to the manufacturer's manual at 37˚C. For each transfection, 1 µg of each construct was added to each well. At 24 h after transfection, the cells were collected and the depletion efficiency was validated through reverse transcription-quantitative (RT-q) PCR analysis. miRNA sequences were as follows: miR-183 mimics, 5'-UAUGGCACUGGUAGAAUUCACU-'3; mimics control, 5'-ACUACUGAGUGACAGUAGA-3'.
Cells were seeded into 96-well plates at a density of 0.25x104/well to assess cell proliferation. After 24 h of cell adherent growth, each well was replaced with 100 µl DMEM complete culture medium, 10 µl CCK-8 reagent was added and then placed in an incubator for dark incubation for 2 h. The D value of each well at 450 nm wavelength was measured with an enzyme-linked immunodetection and the cell growth curve was plotted.
H9C2 cells suspension of each group was collected and centrifuged at 1,000 x g for 5 min at room temperature, washed with PBS, stained in Annexin V-FTTC in the presence of 50 µg/ml RNase A and then the cells were incubated at room temperature for 10-15 min in the dark. A FACScan (Becton Dickinson) was used and the apoptotic rate (early + late apoptosis) was detected and analyzed using CytExpert version 2.0 software (Beckman Coulter, Inc.).
Apoptosis of H9C2 cells was detected by TUNEL staining for 60 min at 37˚C and then stained with DAPI for 5 min at 37˚C. The percentage of apoptotic cells was calculated by dividing TUNEL-positive cells by DAPI-positive cells.
The putative interacting sites between SNHG16 and miR-183 and between miR-183 and FOXO1 were predicted using StarBase version 2.0 (
Tissue and cells (seeded into a 6-well plate at a density of 4x104 cells/well and cultured for 24 h) were collected to extract total RNA using TRIzol® reagent (Thermo Fisher Scientific, Inc.). RNA was reversely transcribed into cDNA using the cDNA Reverse Transcription kit (Thermo Fisher Scientific, Inc.) or MicroRNA Reverse Transcription kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. The expression levels were determined using the SYBR PremixEx Taq II kit (Takara Biotechnology Co., Ltd.) with GAPDH as an internal control on ABI 7500 RT-PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The qPCR thermocycling conditions were: Initial denaturation at 95˚C for 10 min; followed by 40 cycles of 95˚C for 15 sec and 64˚C for 30 sec. The relative gene expression was analyzed by using the 2-ΔΔCq method (
Total protein was isolated from spinal cord samples and cells using a protein extraction kit (Bio-Rad Laboratories, Inc.). The total protein concentration was determined by the BCA method, the proteins (50 µg per lane) were separated via 8% SDS-PAGE. Proteins were then transferred onto PVDF membranes. The membranes were blocked using 5% skimmed milk for 2 h at room temperature, the primary antibody was incubated overnight and the HRP-conjugated secondary antibody was incubated at 1:5,000 for 2 h. Protein bands were developed with BeyoECL Plus (Beyotime Institute of Biotechnology). ImageJ software (National Institutes of Health, v1.8) was used to analyze the gray value for a gel imaging system for imaging. Western blotting was performed using the following antibodies: Bax (cat. no. ab32503; 1:1,000), Bcl-2 (cat. no. ab32124; 1:1,000), cleaved Caspase 3 (cat. no. ab32042; 1:1,000) and β-actin (cat. no. ab8227; Abcam) antibodies were purchased from Abcam, FOXO1 (cat. no. 2880; 1:1,000) was purchased from Cell Signaling Technology.
Statistical analysis software SPSS 21.0 (IBM Corp.) was used to complete the data sorting and analysis. Values are expressed as the mean ± standard deviation from at least three independent experiments. The differences between the two groups were compared using a Student's t-test, whereas the differences among several groups were compared using a one-way ANOVA with a post-hoc Tukey's test. P<0.05 was considered to indicate a statistically significant difference.
SNHG16 has been reported to be a significant regulator in multiple cancers (
To verify the efficiency of knockdown SNHG16 in H/R-induced injury cardiomyocytes, the present study transfected shSNHG6 into H9C2 cells before treatment with H/R. As shown in
StarBase version 2.0 (
To investigate whether SNHG6 aggravated H/R-induced myocardial apoptosis through regulating miR-183, rescue experiments were conducted in H9C2 cells. As shown in
The downstream target genes of miR-183 were predicted using online analysis tools, including TargetScan (
To further explore the effect of FOXO1 on SNHG16, H9C2 cells were transfected with pcDNA3.1-FOXO1 and shSNHG16. The RT-qPCR results showed that inhibition of SNHG16 could reduce the expression of FOXO1 and FOXO1 expression is significantly increased following overexpression of FOXO1 (
Cardiomyocyte H/R injury is a classic model to simulate the pathological and physiological processes of myocardial I/R. Short-time ischemia and reperfusion of myocardial cells can cause dysfunction of tissue cell function metabolism, aggravation of structural and functional damage and even irreversible damage (
A previous study indicates that silenced SNHG16 represses Ang II-imposed cardiac hypertrophy (
In summary, the experimental data from the present study indicated the function and mechanism of lncRNA SNHG16 in regulating myocardial I/R injury in rats and the H/R injury in H9C2 cells. The present study also showed that inhibition of SNHG16 could improve myocardial I/R injury by regulating the miR-183/FOXO1 axis, which could be a promising therapeutic agent for myocardial I/R injury.
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
TG and ZX initiated and designed the present study, analyzed and interpreted the results and wrote the manuscript. JX, YY and JL performed various experiments. TG and ZX confirm the authenticity of all the raw data. All authors read and approved the final manuscript.
Experiments were conducted according to the Declaration of Helsinki. The Animal Care and Use Committee of Cangzhou Central Hospital (approval no. 2022-013-01z) approved all rat protocols and procedures.
Not applicable.
The authors declare that they have no competing interests.
Inhibition of SNHG16 relieves myocardial I/R injury
Knockdown of SNHG16 alleviates H/R induced cardiomyocyte apoptosis. (A) SHNG16 mRNA expression was detected by quantitative PCR. (B) CCK-8 assay to detect H/R-induced cardiomyocyte viability. (C) Apoptosis was examined by flow cytometric analysis using Annexin V/PI assay. (D) Western blot detected the expression of apoptosis-related proteins Bax, Bcl-2 and Cleaved Caspase-3. The data are from three individual experiments and are shown as mean ± SD. ***P<0.001. H/R, hypoxia/reoxygenation; OD, optical density; sh, short hairpin; NC, negative control.
LncRNA SNHG16 target miR-183 in H9C2 cells. (A) The predicted binding site of SNHG16 and miR-183. (B) Dual-luciferase reporter detected the relative luciferase activity of WT-SNHG16 and MUT-SNHG16 following transfection of miR-183 mimic or NC. (C) The SNHG16 expression was detected by quantitative PCR. The data are from three individual experiments and are shown as mean ± SD. **P<0.01, ***P<0.001. Lnc, long non-coding; miR, microRNA; WT, wild-type; MUT, mutant-type; NC, negative control; ns, not significant.
miR-183 inhibitor rescues the effect of shSNHG16 on H/R-induced cardiomyocyte apoptosis. (A) The expression of miR-183 was detected by quantitative PCR. (B) Cell viability changes in four groups. (C) Apoptosis rates of cells assayed by flow cytometry. (D) The relative protein expression of Bcl-2, Bax and cleaved-Caspase-3 was detected by western blotting. The data are from three individual experiments and are shown as mean ± SD. *P<0.05, **P<0.01, ***P<0.001. miR, microRNA; sh, short hairpin; H/R, hypoxia/reoxygenation; NC, negative control.
miR-183 targets FOXO1 in H9C2 cells. (A) The predicted miR-183 binding sites in the 3'-UTR of FOXO1. (B) Luciferase activity was detected to evaluate the binding potential between FOXO1 and miR-183. (C) Quantitative PCR and (D) western blotting analysis were used to measure the expression of FOXO1. The data are from three individual experiments and are shown as mean ± SD. ***P<0.001. miR, microRNA; FOXO1, forkhead box O1.
Upregulation of FOXO1 can rescue the effect of sh-SNHG16 on H/R induced cardiomyocyte apoptosis. (A) The expression of FOXO1 was detected by quantitative PCR. (B) Cell viability was elevated using the CCK-8 assay. Cell apoptosis rate was determined by (C) flow cytometry and (D) TUNEL. The data are from three individual experiments and are shown as mean ± SD. **P<0.01, ***P<0.001. FOXO1, forkhead box O1; sh, short hairpin; H/R, hypoxia/reoxygenation; NC, negative control; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.