Animals
Sixty male Sprague-Dawley (SD) rats (weighting 180-200 g) were supplied by Peking University Laboratory Animal Center. All rats were kept at 22-24°C and 55-60% humidity on a 12 h light-dark cycles. Water and food were free to access. All animal experiments were conducted strictly in accordance with the National Institutes of Health guide for the care and use of Laboratory animals.
After the study, all rats were euthanized. The right hand held the rat tail and pull it back, and the left thumb and forefinger pressed down firmly on the mouse head at the same time. The external force was used to dislocate the cervical spine of the rat, and the spine and the brain were disconnected.
Establishment of the rat myocardial ischemia-reperfusion (IR) model
The left anterior descending (LAD) coronary artery was ligated to establish IR rat model as described in the preivious study 13,14. Firstly, rats were anesthetized with pentobarbital sodium (50 mg/kg, i.p.). LAD of coronary artery in IR group was occluded by using 6-0 silk suture slipknot for 30 min and then reperfused for 2 h. For the Sham-operation group, an identical procedure was performed without the LAD ligation.
Hemodynamic examination
To evaluate the cardiac function, changes of 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 and recorded after a week of IR model by using an 8-channel polygraph system.
Myocardial infarct size determination
Myocardial infarct size was assessed using triphenyltetrazolium chloride (TTC, Sangon, China) staining. At one week after IR, rats were killed, and the hearts were obtained. Subsequently, the ventricular tissue was sliced into 5 pieces of equal thickness. The slices were then incubated in 2% TTC for 15 min in dark and fixed in 10% formaldehyde for 10 min. The area of infarction tussues was determined by an image analyser. Infarct size was calculated as the ratio of the ventricular infarction areato ventricular total area × 100%.
Hematoxylin-Eosin (HE) Staining
After a week of IR, the hearts were collected and fixed in 4% formaldehyde overnight at 4°C. The paraffin-embedded tissues were cut into 5 μm thick slices after dehydration and vitrification. The paraffin sections were deparaffined in xylene and rehydrated in gradient ethanol. Subsequently, the tissue sections were stained with HE dye. Finally, tissues histopathological changes were analyzed by a light microscope (400 ×) and images were acquired.
Terminal dexynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay
At one week after IR, the heart was collected from rat. After washing twice time, the heart was embedded in paraffin and then heart sections were stained. Apoptotic cells were stained brown due to the binding of dUTP enzyme to their fragmented DNA. Apoptotic cells were counted for five fields per section under a magnification (400×) by a blinded manner.
Cardiacmyocyte cell culture
The myocardial tissues of the ischemic part were collected. The myocardial tissue was minced and then centrifuged for 5 min. Then, the tissue pieces were digested by using collagenase IV (0.45 mg/ml), 0.1% trypsin and 15 μg/ml DNase I. After centrifugal, myocardial cells were collected. Cardiomyocytes in Sham and IR groups were cultured in RPMI 1640 medium (Gibco, USA) containing 15% FBS. The myocardial cells (2.5 × 107 cells/well) were planted into 6-well plates and cultivated in CO2 incubator.
Cell transfection and grouping
Myocardial cells (1 × 105) of IR group were planted into 24-well plates. After 24 hours of cell adherence, medium was removed. The miR-30c mimics, miR-30c inhibitor, SIRT1 siRNA and the negative control (NC mimics, NC inhibitor and control siRNA) were supplied by Genepharma (Shanghai, China). To further investigate whether SIRT1 is involved in miR-30c function on myocardial cells, SIRT1 siRNA1, SIRT1 siRNA2, SIRT1 siRNA3 and control siRNA were separately transfected into myocardial cells by using Lipofectamine® 3000 reagent. And the transfected myocardial cells were randomly assigned to 5 groups: Control group (no-treated group), si-NC (treated with control siRNA), siRNA1 group (treated with SIRT1 siRNA1), siRNA2 group (treated with SIRT1 siRNA2) and siRNA3 group (treated with SIRT1 siRNA3). SIRT1 siRNA2 with the best transfection effect was selected for subsequent experiments.
The miR-30c mimics, miR-30c inhibitor, SIRT1 siRNA2 and their corresponding negative control (NC mimics, NC inhibitor and control siRNA2) were transfected into myocardial cells by Lipofectamine® 3000 reagent. The transfected myocardial cells were assigned to 9 groups: IR group (no-treated group), inhibitor NC group (treated with NC inhibitor), miR-30c inhibitor group (treated with miR-30c inhibitor), mimics NC group (treated with NC mimics), miR-30c mimics group (treated with miR-30c mimics), siRNA2 NC + miR-30c inhibitor NC group (treated with control siRNA2 and NC inhibitor), siRNA2 + miR-30c inhibitor NC group (treated with SIRT1 siRNA2 and NC inhibitor), siRNA2 + miR-30c inhibitor group (treated with SIRT1 siRNA2 and miR-30c inhibitor) and siRNA2 NC + miR-30c inhibitor group (treated with control siRNA2 and miR-30c inhibitor).
Apoptosis assay
After 48 h of transfection, the myocardial cells were collected. Myocardial cells were washed twice with PBS and then stained by using Annexin V-FITC and propidium iodide (PI) for 15 min in dark. Finally, myocardial cells apoptosis was detected by a flow cytometer (Beckman Coulter, USA).
Enzyme linked immunosorbent assay (ELISA)
The protein samples from myocardial cells and tissues were extracted and immediately placed on ice. The levels of inflammatory factors including TNF-α, IL-1β and IL-6 were detected using their correaponding ELISA kits (Thermo Fisher Scientific, USA).
Real-Time fluorogenic PCR assays
Total RNA was extracted from myocardial cells and tissues by using TRIZOL (Invitrogen, USA). Then, total RNA was reverse-transcribed into cDNA by a Reverse Transcription Kit (ThermoScientific, USA) and measured by using qRT-PCR (Bio-Rad, USA) with SYBR Green Mixture (Roche, Switzerland). Primers used for qRT-PCR analysis were as follows: miR-30c 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 were respectively used as internal controls of SIRT1 and miR-30c.
Western blot analysis
Total proteins from myocardial cells and tissues were extracted using RIPA lysis buffer (Beyotime, China) according to themanufacturer’s protocol. 50 μg of protein samples were separated by 10% SDS-PAGE and then transferred onto polyvinylidene difluoride membrane. After blocked with 5% non-fat milk for 2 h, the membranes were incubated with the primary antibody (Bax, 1:1000, 14796; Bcl-2, 1:1000, 4228s; IκBα, 1:500, #4814; p-IκBα, 1:500, #2859; SIRT1, 1:1000, #2310, Cell signal, USA; NF-κB p65, 1:1000, SAB4502610; p-NF-κB p65, 1:1000, SAB4301496, Sigma Aldrich, USA; caspase-3, 1:1000, sc-271759; β-actin, 1:1000, sc-517582, Santa Cruz, USA) with light shaking overnight at 4℃. After washing three times, peroxidase-labeled secondary antibody was used to incubate membranes for 2 h at room temperature. Finnally, the protein bands were visualized with ECL system (Thermo, USA).
Dual luciferase reporter gene assay
TargetScan was used to predict the targeted relationship between SIRT1 and miR-30c. We amplified 3ʹ-untranslated region (3ʹ-UTR), containing miR-30c binding site of SIRT1 and then cloned 3ʹ-UTR fragment into pmirGLO luciferase vector (Promega, USA) to construct wild pmirGLO-WT-SIRT1-3ʹ-UTR (SIRT1-wt) and mutant pmirGLO-MUT-SIRT1-3ʹ-UTR (SIRT1-mut). For luciferase assay, the reporter vectors with miR-30c mimics and miR-30c mimics NC were respectively co-transfected into myocardial cells by Lipofectamine 3000 Reagent. Then, the myocardial were grouped as follows: MT + miR-30c mimics group (treated with SIRT1-mut and miR-30c mimics), MT + NC group (treated with SIRT1-mut and miR-30c mimics NC), WT + miR-30c mimics group (treated with SIRT1-wt and miR-30c mimics) and WT + NC group (treated with SIRT1-wt and miR-30c mimics NC). After 48 h of transfection, the luciferase activity was measured using a dual luciferase kit (Promega).
RNA immunoprecipitation (RIP) assay
The RIP assay was used to isolate target RNA-protein complexes by Magna RIP Kit (Millipore, USA). Breifly, myocardial cells were lysed in lysis buffer. Then, anti-Ago2 and IgG bound beads were added into cell lysates at 4°C for 2 h. After washed with PBS, the RNA-protein-beads complexes were isolated using Trizol reagent. Finally, the expressions of miR-30c and SIRT1 were measured by q-PCR.
Statistical analysis
All data were analyzed using SPSS 22.0 Statistical Software (Chicago, IL). Quantitative results were expressed as mean ± SD. Statistical differences were determined by using Student’s t test for two groups, or a one-way ANOVA followed by Tukey’s post hoc test. The significant level of the statistical analysis was set at p<0.05.