Mouse MI model
8-week-old male C57BL/6 mice (n = 56), provided by Shenzhen People’s Hospital, Second Clinical Medical College of Jinan University were applied in this research. Mouse model of MI was constructed by ligation of the left anterior descending (LAD) coronary artery. At first, 50 mg/kg ketamine and 30 mg/kg pentobarbital sodium were used to anesthetize C57BL/6 mice (n = 8). Under aseptic conditions, thoracotomy was carried out. A silk suture was utilized to ligate the left coronary artery, and then the incision was sutured. Mice (n = 8) in the Sham group underwent the same procedures except coronary artery ligation, and they were used as controls. Three days later, all mice were sacrificed and myocardial tissues were obtained. The approval of animal studies was obtained from the Animal Care and Utilization Committee of Shenzhen People’s Hospital, Second Clinical Medical College of Jinan University.
Cell culture and treatment
DMEM medium which included 10% FBS, 3.7 g/L sodium bicarbonate, 110 mg/L sodium pyruvate and 4.5 g/L D-glucose was utilized to culture the primary cardiomyocytes (extracted from mouse MI model) and H9C2 cells (obtained from ATCC (Manassas, VA) ) at the temperature of 37 °C with 5% CO2. In order to simulate myocardial ischemia, H9C2 cells were subjected to anoxic treatment. Specifically, H9C2 cells were kept growing in an incubator containing 94% N2, 5% CO2, and 1% O2 to induce hypoxia injury [31].
Cell transfection
Plasmids including pcDNA3.1/BC002059, sh-BC002059 (sh-BC002059#1/#2), miR-19b-3p mimics, miR-19b-3p inhibitor, pcDNA3.1/ABHD10 and their corresponding NC plasmids were all obtained from Shanghai GenePharma Co., Ltd. (Shanghai, China). Cells seeded in 24-well plates were transfected with these plasmids by Lipofectamine 2000 (Invitrogen).
RNA extraction and qRT-PCR
TRIzol reagent was added for extracting the total RNA which was then reversely transcribed into cDNA. Subsequently, SYBR Green PCR Kit and the ABI 7500 Fast Real-Time PCR system were employed for PCR detection. The level of RNAs was calculated based on the 2−ΔΔCt method. U6 or GAPDH served as endogenous control.
Annexin V-FITC/PI double-labeled flow cytometry
On the basis of the instruction of user manual, Annexin V-FITC Apoptosis Kit was utilized to detect cell apoptosis. At first, cardiomyocytes were subjected to re-suspending in binding buffer after being digested by EDTA-free trypsin and rinsed by phosphate-buffered saline (PBS) separately. After that, Annexin V- FITC and PI were utilized to stain cells in a dark room at least 15 min at room temperature. Finally, flow cytometry was adopted to estimate cell apoptosis. The formula of cell apoptosis rate was as follows: Early and later apoptotic cell number/Total cell number.
Nucleus and cytoplasm isolation assay
Consistent with supplier’s instruction, Nuclei Isolation Kit: Nuclei Ez Prep was used to achieve the isolation of nucleus and cytoplasm. Trizol reagent was then utilized to extract the RNAs in the separated fragments. GAPDH and U6 were used as endogenous controls.
Luciferase reporter assay
The pmirGLO luciferase reporter plasmids which contained the sequences of BC002059-WT/ABHD10 3’UTR-WT or BC002059-Mut/ABHD10 3’UTR-Mut were synthesized. Afterwards, these plasmids were respectively transfected with miR-19b-3p mimics or NC mimics into cells. In the final step, luciferase reporter assay system was used to test the luciferase activity.
TUNEL assay
The rTdT solution was used to cultivate the cells in a dark room at room temperature for one hour. Next, PBS and DAPI were adopted to rinse and stain the cells by turn. Eventually, with the help of a fluorescence microscope, the TUNEL-stained cells were observed.
Trypan blue staining
Cell viability was evaluated by utilizing trypan blue staining assay kit (Beyotime, China). After transfection, primary cardiomyocytes and H9C2 cells were cultivated in a 6-well plate (1 × 105 cells per well) (Thermo Fisher Scientific, USA) at 37 °C for 24 h. Subsequently, PBS and the kit solution were separately used to wash and fix the collected cells, which were counted with a microscope (Nikon, Japan). Finally, cell viability was calculated based on the formula: (the number of viable cells/the number of total cells) × 100%.
Western blot
RIPA lysis buffer was added to extract the total proteins from the collected cells. Protein concentration was measured by BCA kit. Gel electrophoresis was used to separate the protein specimens which were then moved to the PVDF membrane. After that, the first antibodies and secondary antibodies were co-cultured with the proteins. Finally, protein bands were analyzed using ECL kit.
Measurement of serum CK-MB and LDH
Creatine Kinase Activity Assay Kit and LDH colorimetric assay kit were utilized to estimate the serum CK-MB and LDH separately according to the protocols of suppliers. The experiment was conducted on the automatic biochemical analyzer.
Quantification of infarct size
The tissues were sliced into two-mm thick slices. 10% formalin was used to fix tissues after they were cultivated by 2% 2, 3, 5-TTC solution for ten minutes at the temperature of 37 °C. The infarct size was measured by the use of Image-Pro Plus 6.0 software.
RIP
As per manufacturer’s instructions, RNA Immunoprecipitation Kit was utilized to conduct RIP assay. Briefly speaking, cells were first lysed in lysis buffer. Next, lysed cells were incubated with Anti-IgG and anti-Ago2 conjugated with magnetic beads. The precipitated RNAs were isolated and quantified by qRT-PCR to assess the enrichment of target genes.
Statistical analysis
Each independent experiment was carried out three times. Statistical analyses were conducted via SPSS18.0 statistical software package and GraphPad Prism. All quantitative data were presented in the form of mean ± standard deviation (SD). Student’s t-test, one-way or two-way ANOVA was implicated in analyzing the statistics differences between two groups or among multiple groups. Data differences represented to be statistically significant upon P < 0.05.