H9c2 cell (catalog: GNR 5) were obtained from Cell bank of Chinese Academy of Sciences and cultured in DMEM (Gibco, CA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, CA, USA), 100 U/mL penicillin and 100 μg/mL streptomycin under 5% CO2 at 37 ℃ in a humidified atmosphere.
I/R model establishment
20 12-week-old female C57BL/6 mice were obtained from Charles River Laboratories and maintained in the specific pathogen free (SPF) environment with free access to water and food. The mice were randomly divided into two groups including sham and I/R group. All animal procedures were performed in accordance with institutional guidelines and approved by the Animal Studies Committee of Health Science Center, Peking University. The left thoracotomy was performed to expose the heart. Then, the left coronary artery (LCA) was ligated using a 6-0 silk suture. After occlusion for 30 mins, blood supply was restored for 2 hours by loosening the suture. After 2 hours reperfusion, the rats were sacrificed and the hearts were harvested. All rats underwent the same I/R procedure, while sham group experienced the surgical procedure without the ligation of left anterior descending coronary artery (LAD). After reperfusion, mice were anaesthetized by injection with 1% pentobarbital sodium (40mg/kg) intraperitoneally, .20 heart tissues come from the sham and I/R group were extracted for the further research.
TSA were purchased from National Institute for the Control of Pharmaceutical and Biological Products (>99% purity, Beijing, China). Cells were treated with TSA (0.5, 1, 5 μM) respectively 2 hours before inducing the hypoxia and during the hypoxia period.
MiR-124-5p mimic and mimic control as well as siRNA for AK003290 and the negative control were designed and synthesized by Genepharma (Shanghai, China). The wild and mutant region of AK003290 that was to be targeted by miR-124-5p were synthesized by Genepharma (Shanghai, China) and cloned into pGL3 luciferase reporter vectors (Promega, CA, USA).
The cultured H9c2 cells were digested with trypsin, washed with cold PBS and dual-stained with Annexin V-FITC/propidium iodide according to the manufacturer’s instructions. Cell apoptosis was detected by flow cytometry on a BD FACSCalibur (Becton Dickinson, NJ, USA).
Reverse transcription and quantitative realtime PCR
RNA extraction was performed with Trizol reagent (Invitrogen, CA, USA), precipitated with isopropanol, washed with 75% ethanol and dissolved in RNase free water. Reverse transcription was performed with 1 μg RNA using the cDNA transcription kit (Transgen, USA). 20 ng cDNA was used for qPCR to validate the expression of relative mRNA. It was determined by using SYBR green mix (Yisheng, Shanghai, China).
Cells were lysed on ice using a lysis buffer (Beyotime, Shanghai, China). The proteins were separated by centrifuging at 12000 g for 10 min at 4 ℃. Protein lysates were loaded with 5× loading buffer on the SDS-PAGE. After electrophoresis, the gel was transferred to PVDF membrane. Followed by blocking with skim milk for 1 hour, protein bands on the PVDF membrane were incubated with relative primary antibodies at 4 ℃ overnight and the corresponding secondary HRP antibody at room temperature for 2 hours. The blots were visualised by ECL chemiluminescence.
RNA Pull-down assay
Biotinylated miR-124-5p probe and the control probe were synthesized by Sangon Biotech (Shanghai, China). Probe-coated beads were generated by co-incubating the probe with streptavidin-coated beads (Invitrogen, CA, USA) at 25 ℃ for 2 h. H9c2 cells were collected, lysed, and incubated with AK003290 or miR-124-5p probes overnight at 4 ℃. Thereafter, the beads were eluted, and the complex was purified with TRIzol (Takara, Dalian, China). Then, the abundance of AK003290 and miR-124-5p was analyzed by qRT-PCR.
LDH and MDA detection
After reperfusion, the hearts were resected and homogenized in cold phosphate buffer. After centrifuging at 3000 rpm for 15 min, the supernatant was collected for measurement at -20 ℃. The levels of LDH as well as MDA were evaluated using commercial kits following manufacturer’s protocols (Jiancheng, Nanjing, China).
After I/R treatment, myocardial tissue or cardiomyocytes were collected and washed in PBS followed by co-culturing with 10 μM DCFH-DA (DCF-DA, Shanghai, Beyotime) at 37 ℃ for 20 min with gentle shaking in the dark. The mean fluorescence intensity (MFI) was evaluated using a flow cytometer (BD FACSCalibur, Becton Dickinson, NJ, USA).
After treatment, the transfected cells were incubated with 10 mM 5, 5’,6, 6’-tetrachloro-1, 1’,3, 3’- tetraethylbenzimidazolylcarbocyanine iodide (JC-1) (Beyotime, Shanghai, China) for 30 min at 37℃. Then, the fluorescence labeled cells were washed with PBS and analyzed by GraphPad Prism software. The ratio of fluorescence at 590 nm versus 530 nm emission was applied to measure the mitochondrial membrane potential (MMP).
Fluorescence in situ hybridization (FISH)
Alexa Fluor 555-labelled AK003290 probes were designed and synthesized by RiboBio (Guangzhou, China). The experiment was carried out with a Fluorescent In Situ Hybridisation kit (RiboBio, Guangzhou, China). The cells were seeded onto autoclaved glass slides at a density of 1×105 cells and cultured for 24 h. After fixing with 4% paraformaldehyde for 20 min followed by permeabilization with 0.5% Triton X-100 for 10 min, the cells were cultured at 37 ℃ overnight. Finally, the slides containing cells were incubated with DAPI to stain the cell nucleus and observed under a fluorescence microscope (Leica, Wetzlar, Germany).
The wild-type (WT) or mutant (mut) seed sequence at the predicted region in AK003290 and miR-124-5p were synthesized and cloned into the pGL3 Luciferase Reporter Vectors (Promega, CA, USA) at the KpnI and BamHI sites. H9c2 cells were co-transfected with either miR-124-5p mimic or mimic control, together with pGL3 vectors, which contained the WT or mut predicted binding regions of AK003290. TRL-SV40 plasmid (Promega, CA, USA) was also transfected as a normalizing control. The cells were harvested to detect of luciferase activity using Dual-Luciferase Assay kit (Promega, WI, USA) 48 h post-transfection.
SPSS 20.0 software (SPSS Inc., Chicago, IL, USA) were used to analyze all data for statistical significance. All the data are presented as the means ± SD. One-way ANOVA was used to assess the difference between multiple groups. Differences between two groups were analyzed by the Student’s t-test. P<0.05 was considered as statistical significance.