A mouse model of myocardial ischemia-reperfusion injury
C57BL/6 mice from Liaoning Changsheng Biotechnology Co., Ltd., aged between 8 to 10 weeks and weighing between 22 and 25 g, were selected for animal research. The left anterior descending artery (LAD) coronary artery of the mouse was occluded for 50 minutes with a 7 − 0 ligature and reperfused for 48 hours. The use of animals has been approved by the Ethics Committee of Harbin Medical University and complies with the Guidelines for the Care and Use of Laboratory Animals published by the National Institutes of Health.
Mice were injected intraperitoneally with 2,2,2-tribromoethanol (200 mg kg -1; Sigma, St. Louis, Missouri, USA) for anesthesia, and their chests were depilated. The mice were fixed on the operating table supine, and a proper amount of coupling agent was applied to the chest. The scanning probe was used to collect the left ventricular electrocardiogram through the chest wall ultrasound, and at the same time recorded the M-mode echocardiogram, and calculated the fractional shortening (FS) and ejection fraction (EF).
Cell culture and cell dosing
Following the procedure described in our previous study , cardiomyocytes isolated from 1–3 day old newborn mice (C57BL/6) were treated with hydrogen peroxide (100umol/L, #323381, Sigma-Aldrich, USA ) for 48 hours to simulate MIRI in vitro. On this basis, the cardiomyocytes were treated with GRP94 inhibitor (5umol/L, PU-WS13, #58687, Med Chem Express, USA) for 48 hours.
Construction and transfection of lncRNA siRNA and plasmid
The siRNA of lncRNA-AK138945 (5’-GAAGAGGUAUUGAAUGCUA-3’) was purchased from Ribobio (Guangzhou, Guangdong Province, China) and transfected into neonatal mice cardiomyocytes (NMC) according to the manufacturer’s instructions.
Cell counting kit-8 (CCK8, CK04-500T, Dojindo, Japan) was used to measure cell viability under different conditions. Cardiomyocytes were cultured in 96-well culture clusters (about 1*104 per well), and then the cells were transfected with lncRNA-AK138945 specific siRNA for 48 hours. The CCK8 reagent (20 µL) and DMEM (180 µL ) were added to each well and incubated for 1–4 hours. The absorbance was measured at 450 nm using an Infinite m200pro microplate spectrophotometer (Tecan, Salzburg, Austria). Read the absorbance at 490 nm in a spectrophotometer (BioTek, USA).
Terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL) staining was used to evaluate the apoptosis of cultured cardiomyocytes. Briefly, cardiac myocytes cultured on coverslips in 24-well plates were fixed in 4% paraformaldehyde. The TUNEL staining was done using the in situ cell death detection kit (Minneapolis, MN, USA) according to the manufacturer’s protocol. The numbers of TUNEL-positive cells and the total cells were counted under a confocal microscopy.
Live/Dead cell staining.
The cardiomyocytes were seeded on a cover glass of a 24-well culture plate. Forty-eight hours after transfection, live and dead cells were detected using the "Live/Dead Viability/Cytotoxicity Assay Kit" (Invitrogen, Shanghai, China) according to the manufacturer's instructions. A fluorescence microscope (Olympus, Tokyo, Japan) was used to acquire images.
The total protein extracted from NMVC or mouse left ventricular tissue samples was transferred to a nitrocellulose membrane (Life Science, Überlingen, Germany) using SDS-PAGE (10%-15%). The membrane was sealed with 5% skimmed milk, and GRP94 (Cat#20292, Cell Signaling Technology), Caspase9 (Cat#9504, Cell Signaling Technology), Caspase3 (Cat#9662, Cell Signaling Technology), Bcl2 (Cat#3498, Cell Signaling Technology), Bad ( Cat#A19595, ABclonal Technology) and β-actin antibody purchased from ZSGB-BIO (Beijing, China) were incubated overnight at 4°C. After that, the membrane was washed with PBST and incubated with a secondary antibody (Alexa Fluor, Molecular Probes, Eugene, OR, USA) in the dark for 50 minutes. Odyssey v1.2 software (LI-COR Biosciences, Lincoln, NB, USA) was used to quantify the obtained bands. The results were normalized to the level of β-actin.
Quantitative real-time PCR (qPCR)
Total RNA was isolated from myocardial tissue and neonatal cardiomyocytes using TRIzol reagent (Invitrogen, Camarillo, CA, USA). The RNA concentration was checked by NanoDrop 8000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). According to the manufacturer's instructions, it was reverse transcribed into cDNA using a reverse transcription kit (Dalian, China). The Thermocycler ABI 7500 fast real-time PCR system (Applied Biosystems, Carlsbad, California, USA) was used for real-time quantitative PCR. The primer sequence used is shown as below.
ZFAS1-forward, 5’-AGCGTTTGCTTTGTTCCC-3’, reverse5’-CTCCCTCGATGCCCTTCT-3’; AK138945-forward, 5’-AGCCCAGGAACAAATGCAGAA-3’, reverse, 5’-TCAACGTCACACTTCATGATGGA-3’; CDR1as-forward, 5’-TCTGCTCGTCTTCCAACATC-3’, reverse, 5’-AGATCAGCACACTGGAGAC-3’; β-ACTB-forward, 5’-ACTGCCGCATCCTCTTCCT-3’; reverse, 5’-TCAACGTCACACTTCATGATGGA-3’; miR-1a-3p-forward, 5’-GGCGTGGAATGTAAAGAA-3’, reverse, 5’-CGGCAATTGCACTGGATA-3’, miR-1a-3p-RT, 5’-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACATACATAC-3’; U6-forward, 5’-GCTTCGGCAGCACATATACTAAAAT-3’, reverse, 5’-CGCTTCACGAATTTGCGTGTCAT-3’, U6-RT, 5’-GCTTCGGCAGCACATATACTAAAAT-3’; FOXO3-forward, 5’-CCGTGAGCAAGCCGTGTACTG-3’, reverse, 5’-TATCCAGCAGGTCGTCCATGAGG-3’; GRP94-forward, 5’-ATGAAGGCACAAGCATACCAGACG-3’, reverse5’-TCATCTTCCTTAATCCGCCGCAAC-3’.
Dual-luciferase reporter gene assay
The HEK293T was transfected cells with miR-1a-3p (20µmol/L), blank, miR-1a-3p inhibitor, NC or NC inhibitor and plasmid (0.5µg). Forty-eight hours after transfection, luciferase activity was measured on a fluorometer (Promega, Madison, Wisconsin, USA) using Dual Luciferase Reporter Assay Kit (Promega, Madison, Wisconsin, USA).
Data And Statistical Analysis
The experimental data are expressed as mean ± standard error (mean ± SEM). The results of qRT-PCR and other results were statistically analyzed by one-way analysis of variance between the three groups and t-test between the two groups. GraphPad Prism software was used for chart production. P < 0.05 was used as the standard for statistically significant differences (*P < 0.05; **P < 0.01; ***P < 0.001).