2.1. Establishment of mice I/R model
Male C57BL/6 mice (25–30 g) were obtained from Vital River Laboratory Animal Technology (Beijing, China) and adaptive feeding for a week at the suitable temperature and humidity. Then, mice were randomly divided into myocardial I/R group (n = 10) and Sham group (n = 10). The myocardial I/R operation were followed by previous research9. Briefly, mice were anesthetized (50 mg/kg pentobarbital sodium) and supine fixed on the operating table connected with the standard lead II electrocardiogram. The left thorax was cut to expose the heart and the left anterior descending (LAD) coronary artery was ligated by 7/0 sterile suture. Myocardial ischemia was induced by LAD ligation for 30 min followed by 120 min of reperfusion. Sham group mice underwent the same surgical procedures without LAD coronary artery ligation. The project was approved by the Ethics Committee of Lianyungang Hospital of traditional Chinese Medicine (No. LYGH2018026).
2.2. Primary cardiomyocytes (CMs) culture and H/R injury administration
Primary neonatal ventricular myocytes were acquired by enzymatic digestion of 1–4-d-old neonatal mice hearts as described8. The supernatant of each round of cell digestion and cultured with DMEM medium (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin liquid in 37℃ incubator containing 5% CO2. After centrifugation to remove the supernatant, the cells are resuspended and cultured in a culture flask. To mimic H/R, CMs were treated with 4 h of hypoxia (1% O2, 5% CO2, 94% Nitrogen) and then followed by 3 h reoxygenation (5% CO2, 21% O2, 74% Nitrogen) as described previously9.
2.3. Plasmids and Cell transfection
The ALKBH5-overexpression plasmids (pcDNA3.1-ALKBH5), vector controls (pcDNA.3.1-NC) and small interfering RNAs (siRNAs) (si-ALKBH5-1, si-ALKBH5-2 and si-NC) were designed and synthesized from GemmaPharma (Shanghai, China). Transfection of siRNAs and plasmids was performed using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. Cells were harvested at 48 hours for future analysis.
2.4. Western blotting
Cells total protein was extracted by RIPA lysis buffer after different treatments with protease inhibitor (Solarbio, Beijing), and protein concentration were quantified by BCA protein detection kit (Solarbio, Beijing). The proteins were separated using SDS-PAGE (Solarbio, Beijing) and transferred to the PVDF membranes (Millipore, USA) by an electroblot apparatus. Membranes were incubated at 4°C overnight with primary antibodies of ALKBH5 (1:1000, Abcam) and GAPDH (1:1000, Cell Signaling Technology). After washed three times with 0.1% TBST, membranes were incubated with secondary antibody (1:1000, Cell Signaling Technology) for 2 h at room temperature. Bands were visualized with an enhanced chemiluminescence (ECL) detection reagent (Millipore, USA). The intensity of the bands was quantified using Image J software.
2.5. Quantitative real-time PCR
Total RNA was separated from myocardial tissues or CMs using Trizol reagent (Invitrogen, Carlsbad, CA, USA), and then cDNA was synthesized using Transcriptor First Strand cDNA Synthesis Kit (Roche, USA). Real-time PCR was performed using SYBR Green PCR Master Mix (Takara, Dalian, China) on Applied Biosystems 7500. Amplification was performed as follows: a denaturation step at 94°C for 5min, followed by 40 cycles of amplification at 95°C for 30s, 60°C for 32s and 72°C for 30s. The relative expression levels were calculated by 2 −ΔΔCt method, and normalized to GAPDH mRNA. The primers were listed in supplementary Table S1.
2.6. Flow cytometric analysis
Cell apoptosis were analyzed by flow cytometric analysis. Briefly, CMs were collected after transfection 48 h, and incubated with Annexin V (BD Biosciences, San Jose, CA, USA) for 15 min. Then, 5 µl Annexin-FITC were added and incubated for 15 min at dark room temperature. Apoptosis rate were generated using flow cytometry with Modifit software (Verity Software House, Topsham, ME, USA).
2.7. Enzyme-linked immunosorbent assay (ELISA)
The markers of myocardial injury creatine kinase (CK) and lactic dehydrogenase (LDH), and oxidative stress related indicators superoxide dismutase (SOD), malonaldehyde (MDA) and glutathione peroxidase (GSH-px) were detected by ELISA. After treated with indicated processing, cells supernatants were collected to detect concentration of SOD, MDA and GSH-px on the basis of the manufacturer’s instructions.
2.8. RNA stability assay
CMs were seeded in six-well plates (1×105 cells per well) for 24 h. Then actinomycin D (Act D, Sigma) was added to 2 µg/ml at indicated time (0 h, 3 h and 6 h) before cell collection. RNA was extracted and real-time PCR were performed as described earlier.
2.9. Total m6A quantification
The total m6A mRNA levels were determined using an m6A methylation quantification kit (EpiGentek, USA) according to the manufacturer’s protocol. Briefly, total RNA was extracted and coated on assay wells (200 ng per well). After that, m6A antibody in solution was added into each well. The m6A levels were detected at wavelength of 450 nm absorbance.
2.10. MeRIP-qPCR
Total RNA were isolated from CMs after ALKBH5 overexpression or knockdown, and treated with genomic DNA purification reagents (Sigma). After fragmentation, the mRNA was incubated with primary antibody of m6A with a Magna MeRIP™ m6A kit (#17-10499, Millipore, MA, USA). The m6A bound RNA was then detected through qRT-PCR.
2.11. RNA immunoprecipitation assay
RNA immunoprecipitation (RIP) was performed using Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, MA, USA) according to the manufacturer’s protocol. CMs were collected and lysed in complete RIP buffer comprising protease inhibitor cocktail and RNase inhibitor. Anti-m6A antibody or anti-ALLKBH5 antibody was incubated with RIP buffer containing magnetic bead conjugated with indicated antibody or control IgG for 1.5 h at 4 ℃. The purified RNA was subjected to qRT-PCR to determine the binding target.
2.12. Analysis of publicly available datasets
SRAMP ( http://www.cuilab.cn/sramp) tools was utilized to predicting potential m6A modification sites of SIRT1 mRNA.
2.13. Statistical analysis
All the experiments were performed three times at least. The data were expressed as the mean ± S.D. SPSS software (version 18.0, IBM Corp., Armonk, NY, USA) and GraphPad Prism (version 8, GraphPad Software, La Jolla, CA, USA) were used to analysis the statistical results. Differences between groups were estimated using a t-test. The comparisons of multiple groups were performed by one-way ANOVA and then an LSD-t test. P༜0.05 were considered statistically significant.