Male Sprague-Dawley (SD) rats (weighting 180-200 g) were provided by Peking University Laboratory Animal Center. All rats were kept at 22-24°C and 55-60% humidity on a 12 h light-dark cycle with free access to water and food. At the end of the study, all rats (220-270g) were anesthetized by an intraperitoneal injection of 50 mg/kg pentobarbital sodium, and then sacrificed by cervical dislocation. All animal experiments were conducted strictly in accordance with the National Institutes of Health guide for the care and use of Laboratory animals.
Establishment of the myocardial IR model in rats
Rats weighing 200-240g were used to establish the IR model. Briefly, rats were anesthetized with pentobarbital sodium (50 mg/kg, i.p.). The left anterior descending coronary artery (LAD) was ligated using 6-0 silk suture slipknot for 30 min, and then reperfused for 2 h. Myocardial ischemia was confirmed by the appearance of regional epicardial cyanosis over the myocardial surface and by arrhythmia. Successful reperfusion was confirmed by the disappearance of epicardial cyanosis and the production of epicardial hyperemia and arrhythmia (IR group). Rats undergoing thoracotomy without LAD ligation were considered as the Sham group.
One week after modeling, the hemodynamic parameters including left ventricular ejection fraction (LVEF), left ventricular systolic pressure (LVSP), left ventricular end-diastolic volume (LVEDV), left ventricular end-systolic volume (LVESV), left ventricular end-diastolic pressure (LVEDP), the maximum up rate of left ventricular pressure (+dP/dtmax), and the maximum down rate of left ventricular pressure (-dP/dtmax) were measured using a Vevo770 scanner (VisualSonics, Toronto, Canada).
Infarct size measurement
The infarct size was detected using 2,3,5-triphenyltetrazolium chloride (TTC) (Sangon, Shanghai, China) staining. Briefly, the ventricle was sliced into pieces with equal thickness. The slices were then incubated in 2% TTC for 15 min in the dark and fixed in 10% formaldehyde for 10 min. The infarct area was measured by an image analyzer. The infarct size was calculated as the ratio of the infarct area and total area (%).
Hematoxylin-Eosin (HE) staining
The ventricle was fixed in 4% formaldehyde overnight at 4°C. Followed by dehydration, vitrification, and paraffin-embedding, the tissue samples were cut into 5 μm-thick slices. The sections were then deparaffined in xylene, rehydrated in gradient ethanol, and stained with hematoxylin for 4 min and Eosin for 2 min. The histopathological changes were observed under a light microscope (400 ×).
Terminal dexynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining
Cell apoptosis was detected using a TUNEL kit (Beyotime, Shanghai, China). Briefly, the paraffin-embedded tissue sections were deparaffined in xylene, and rehydrated in gradient ethanol. The sections were then incubated with DNase-free Proteinase K for 20 min, with 3% hydrogen peroxide (in PBS) for 10 min, and with TUNEL mix for 60 min. After 30 min of incubation with Streptavidin-HRP, the apoptotic cells were visualized using diaminobenzidine, and re-stained with hematoxylin. The apoptotic cells were counted under a light microscope (400 ×) at five randomly selected fields.
Isolation of IR myocardial cells
The myocardial tissues at the ischemic site were collected and homogenated. The tissue homogenate was digested with collagenase IV (0.45 mg/ml) containing 0.1% trypsin and 15 μg/ml DNase I. After centrifugation, the residue (myocardial cells) was collected. Myocardial cells were cultured in RPMI 1640 medium (Gibco, USA) containing 15% FBS, and maintained in an incubator at 37°C with 5% CO2.
Cell transfection and grouping
The rno-miR-30c-5p mimics, rno-miR-30c-5p inhibitor, SIRT1 siRNA1-3 and the negative controls (mimics NC, inhibitor NC and si-NC) were purchased from Genepharma (Shanghai, China). IR myocardial cells were seeded into 24-well plates (1 × 105/well), and cultured until 80% confluence. Cells were then transfected with the above agents using Lipofectamine 3000. IR myocardial cells were randomly divided into 9 groups: IR (no treatment), inhibitor NC, rno-miR-30c-5p inhibitor, mimics NC, rno-miR-30c-5p mimics, si-NC + inhibitor NC, siRNA2 + inhibitor NC, siRNA2 + rno-miR-30c-5p inhibitor, and si-NC + rno-miR-30c-5p inhibitor group. After 48 h of transfection, cells were used for subsequent experiments.
Myocardial cells were washed with PBS twice and then stained with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) for 15 min in the dark. The apoptosis was detected by a flow cytometer (Beckman Coulter, USA).
Enzyme linked immunosorbent assay (ELISA)
The myocardial cells and tissues were homogenated and maintained on ice. The levels of inflammatory factors including TNF-α, IL-1β and IL-6 were detected using specific ELISA kits (Thermo Fisher Scientific, USA) in accordance with the manufacturer’s instructions.
Quantitative real-time PCR
Total RNA was extracted from myocardial cells and tissues using TRIZOL (Invitrogen, USA). Total RNA was then reverse-transcribed into cDNA using a Reverse Transcription Kit (Thermo Fisher Scientific, USA). qRT-PCR was performed on a PCR instrument (Bio-Rad, USA) using SYBR Green Mixture (Roche, Switzerland). Primers were shown as follows: rno-miR-30c-5p F: 5′-GGGGTGTAAACATCCTACAC-3′, R: 5′-GTGGAGTCGGCAATTGCACT-3′; U6 F: 5′-GCTTCGGCAGCACATATACTAAAAT-3′, R: 5′-CGCTTCAC GAATTTG CGTGTCAT-3′; SIRT1 F: 5′-AAGGAGCAGATTAGTAAGC-3′, R: 5′-TAGAGGATAAGGCGTCAT-3′; GAPDH F: 5′-GACGGCCGCATCTTCTTGT-3′, R: 5′-CACACCGACCTTCACCATTTT-3′. GAPDH and U6 with stable expression were used as internal controls of SIRT1 and rno-miR-30c-5p, respectively.
Total protein was extracted from myocardial cells and tissues using RIPA lysis buffer (Beyotime, Shanghai, China). The protein samples (50 μg) were separated by 10% SDS-PAGE and then transferred onto polyvinylidenedifluoride membrane. After blocked with 5% skim milk in TBST for 2 h, the membrane was incubated with specific primary antibody (anti-Bax, 1:1000, 14796; anti-Bcl-2, 1:1000, 4228s; anti-IκBα, 1:500, #4814; anti-p-IκBα, 1:500, #2859; anti-SIRT1, 1:1000, #2310, Cell signal, USA; anti-NF-κB p65, 1:1000, SAB4502610; anti-p-NF-κB p65, 1:1000, SAB4301496, Sigma Aldrich, USA; anti-caspase-3, 1:1000, sc-271759; anti-β-actin, 1:1000, sc-517582, Santa Cruz, USA) overnight at 4°C. After washed with TBST for three times, the membrane was incubated with horseradish peroxidase (HRP)-labeled secondary antibody for 2 h at 25°C. The protein bands were visualized using a HRP kit and quantified by an ECL system (Thermo Fisher Scientific, USA).
The targets of rno-miR-30c-5p were predicted using TargetScan 7.1 (http://www.targetscan.org/vert_71/). A total of 1249 transcripts containing 1835 sites were predicted (Table S1). A target gene SIRT1 (ENST00000212015.6) was selected due to its important role in myocardial IR injury (Table S2).
Dual luciferase reporter gene (DLR) assay
DLR assay was used to identify the targeting relationship between SIRT1 and rno-miR-30c-5p. The fragment of SIRT1, containing the binding site was amplified and cloned into pmirGLO luciferase vector (Promega, USA) to construct wild pmirGLO-WT-SIRT1-3ʹ-UTR (SIRT1-Wt) and mutant pmirGLO-MUT-SIRT1-3ʹ-UTR (SIRT1-Mt). Myocardial cells were co-transfected with SIRT1-Wt/Mt and rno-miR-30c-5p mimics/mimics NC using Lipofectamine 3000. Myocardial cells were randomly divided into 4 groups: SIRT1-Mt + rno-miR-30c-5p mimics, SIRT1-Mt + mimics NC, SIRT1-Wt + rno-miR-30c-5p mimics, and SIRT1-Wt + mimics NC group. After 48 h of transfection, the luciferase activity was measured using a dual luciferase kit (Promega).
RNA immunoprecipitation (RIP) assay
RIP assay was performed using a Magna RIP Kit (Millipore, USA). Briefly, myocardial cells were lysed in lysis buffer. The cell lysate was then incubated with anti-Ago2 or IgG-coated beads at 4°C for 2 h. After washed with PBS, the RNA-protein-beads complexes were isolated using Trizol. The expression of rno-miR-30c-5p and SIRT1 was measured by qRT-PCR.
Three independent repetitions were conducted for each sample. Data were expressed as mean ± standard deviation (SD), and analyzed using SPSS 22.0 Statistical Software (Chicago, IL). Differences among multi-groups were analyzed by one-way ANOVA followed by Tukey’s post hoc test. Differences between two groups were analyzed by Student’s t test. The level of statistical significance was set at p < 0.05.