Animals and Treatment.

A total of 30 C57BL/J6 mice (8 weeks, 20–30 g) were obtained from the animal center of the People’s Hospital of Zhengzhou. All animals were kept in a light-controlled room under a 12-hour light/dark cycle and controlled temperature (23–25°C), and they had free access to food and water according to the U.S. National Research Council’s Guide for the Care and Use of Laboratory Animals. The research was approved by the Institutional Animal Care Committee at the People’s Hospital of Zhengzhou.

The left anterior descending coronary artery ligation model was used to establish our myocardial I/R model (Han et al., 2014). The occlusion was confirmed by balancing of the left ventricular myocardium below the suture. Mice were subjected to 45 minutes of transitory ligation followed by 3 hours of reperfusion. The control group only received a sham operation that used the same procedure except for the placement of the ligature. At 72 hours after surgery, the animals were sacrificed before experiments. The infarction volume was measured by staining with 2,3,5-triphenyltetrazolium chloride (Sigma-Aldrich, St. Louis, MO) and calculated with ImageJ software (U.S. National Institutes of Health, Bethesda, MD). H&E staining was used for the histologic analysis of myocardial tissues.

Cell Culture and Transfection.

H9c2 cardiomyocytes (American Type Culture Collection, Manassas, VA) were seeded on 96-well plates and incubated with 10% FBS Dulbecco’s modified Eagle’s medium (Sigma-Aldrich Co) at 37°C, 5% CO₂. To establish the hypoxia/reoxygenation (H/R) model, we cultured the cells in a 2.3% O₂, 5% CO₂, 92.7% N₂ hypoxic environment for 2 hours, and then cultured them at 37°C 5% CO₂ for 4 hours. Then the cells were transfected with miR-29a inhibitor or negative control (NC), as well as small interfering (siRNA) of SIRT1 (si-SIRT1) or si-NC (all above were purchased from GeneChem Corp., Shanghai, People’s Republic of China) using the lipo6000 reagent (Beyotime Biotechnology, People’s Republic of China) according to the manufacturer’s instructions.

Quantitative Reverse-Transcription Polymerase Chain Reaction.

Expression of miR-29a and SIRT1 was detected by quantitative reverse-transcription polymerase chain reaction (PCR). RNA was extracted with the TRIzol (Takara Biochemicals, Otsu, Japan) experimental method. The DNA was retrieved by stem-loop primers. Real-time PCR was performed after the DNA has been obtained. We designed a stem-loop reverse-transcription primer for MIR-29a: 5′-GTC​GTA​TCC​AGT​GCA​GGG​GTC​CGA​GTA​TTC​GCA​CTG​GAT​ACG​ACT​AAC​CG-3′, and a reverse transcription primer for U6: 5′-AAC​GCT​TCA​CGA​ATT​TGC​GGT-3′, which was synthesized by GeneChem Corp.

The PCR reactions were conducted using ABI 7500 Fast Fluorescent Quantitative PCR instrument (Applied Biosystems, Foster City, CA), and the SYBR-green I dye method was used to perform the experiment. The upstream and downstream primers of micro RNA miR-29a and internal reference gene U6 are to be shown in Table 1. The relative RNA levels were calculated by the 2^−ΔΔCq method. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control.

Western Blot Analysis.

The expression of SIRT1, nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), caspase-1, interleukin-1β (IL-1β), and the related pathways of endothelial nitric oxide synthase (eNOS) oxidative stress were detected by Western blot analysis. The treated cells were collected, centrifuged, and washed with PBS. Protein lysate, the protease inhibitor phenylmethylsulfonyl fluoride, and phosphatase inhibitor were added after washing. The cells were placed on ice for 40 minutes and centrifuged for 15 minutes at 4°C 12,000g. The supernatant was added to the SDS buffer and boiled in water for 5 minutes to make the protein sample.

After 10% SDS-PAGE electrophoresis, the protein samples were transferred to polyvinylidene fluoride membranes at 300 mA for 90 minutes and sealed with 5% skimmed milk powder at room temperature for 2 hours. The samples were then incubated with a primary antibody at 4°C overnight, followed by incubation with a corresponding secondary antibody at 37°C for 45 minutes.

The films were scanned using a Super Signal West Pico Chemiluminescent Substrate kit (Pierce/Thermo Fisher Scientific, Waltham, MA). The relative protein expression was quantified using Image-Pro Plus software (version 6.0; Media Cybernetics, Rockville, MD).

Dual Luciferase Assay.

The binding of miR-29a to SIRT1 was detected by double luciferase assay. Bioinformatic prediction between miR-29a and SIRT1 was obtained from the software Targetscan 7.2 (http://www.targetscan.org; Whitehead Institute for Biomedical Research, Cambridge, MA). Briefly, the wild-type (WT) SIRT1 3ʹ untranslated region or mutant (Mut) was amplified and subcloned into a pGL4.10 luciferase reporter vector. The cells were then cotransfected with either the vectors and the miR-29a mimics or NC using the Lipo6000 reagent. Luciferase assays were performed 48 hours after transfection using a Bright-Glo LuciferaseAssay System (Promega, Madison, WI). The luciferase activity was normalized to values of Renilla luciferase activity.

Detection of Cell Death.

For the apoptosis analysis, briefly, the cells were stained with Annexin V/propidium iodide double-staining kit (BD Biosciences, Woburn, MA) strictly according to the manufacturer’s introductions. Cell apoptosis was measured by flow cytometry (BD Biosciences).

Measurement of Serum and Cell Supernatant Factor Levels.

Commercial ELISA kits (all purchased from Abcam, Cambridge, MA) were used to detect the serum and cell supernatant levels of inflammatory factors IL-1, IL-6, and tumor necrosis factor-α (TNF-α) according to manufacturer’s instructions. Levels of oxidative factors malondialdehyde (MDA) and superoxide dismutase (SOD) were measured using the corresponding kits (Nanjing Jiancheng Bio-Technology, Nanjing, People’s Republic of China). Myocardial enzymes of 2-hydroxybutyrate dehydrogenase (HBDH), lactate dehydrogenase-1 (LDH), creatine kinase (CK), creatine kinase MB activity (CK-MB), and IMA were measured using an automatic biochemical analyzer (Olympus5400; Olympus, Tokyo, Japan).

Statistical Analysis.

All data were analyzed by SPSS 22.0 software (IBM, Armonk, NY). The measurement data are all expressed as mean ± S.D. The independent samples t test was used to measure the data between two groups. Comparisons among three or more groups were conducted using one-way ANOVA followed by Tukey post hoc test. P < 0.05 was considered statistically significant.