MicroRNA-383-5p Regulates Oxidative Stress in Mice with Acute Myocardial Infarction through the AMPK Signaling Pathway via PFKM

Figures

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  Figure 1. PFKM is a target gene of miR-383-5p. (a) The predicted binding sites of miR-383-5p on the 3′UTR sequence of PFKM gene. (b) The luciferase activity of PFKM-3′UTR-wt and PFKM-3′UTR-mut; ∗P < 0.05 vs. the NC group; UTR: untranslated region; wt: wild type; mut: mutant type; miR-383-5p: microRNA-383-5p; PFKM: phosphofructokinase muscle-type; NC: negative control. The results of luciferase activity were regarded as measurement data, expressed as mean ± SD and analyzed using the t-test, and the experiment was repeated 3 times.

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  Figure 2. miR-383-5p expresses poorly while PFKM expresses highly in H2O2-induced H9C2 cells. (a, b) The expression of miR-383-5p and PFKM in normal H9C2 cells and H2O2-induced H9C2 cells, H9C2 cells detected by RT-qPCR. (c, d) The protein expression of PFKM in normal H9C2 cells and H2O2-induced H9C2 cells examined by western blot analysis, with band intensity assessed. (e) and (f) The representative images of flow cytometry using Annexin V-FITC and PI staining. (g) Statistical analysis of apoptosis rate detected via flow cytometry. ∗P < 0.05 vs. the control group. The data were expressed as mean ± SD. Data at different time points were compared using repeated measurements ANOVA. The experiment was repeated 3 times. miR-383-5p: microRNA-383-5p; PFKM: phosphofructokinase muscle-type.

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  Figure 3. Upregulation of miR-383-5p suppresses PFKM. The myocardial tissues used for following detections were, respectively, treated with sham, AMI+PBS, AMI+miR-383-5p agomir, and AMI+miR-383-5p agomir NC. (a) miR-383-5p expression of myocardial tissue of mice by RT-qPCR. (b, c) Protein expression of PFKM in the myocardial tissue of mice by western blot analysis. (d) SOD1 and SOD2 expression by western blot. (e, f) The mRNA expression results of SOD1 and SOD2 in the three groups were determined by real-time PCR. ∗P < 0.05 vs. the sham group; #P < 0.05 vs. the AMI+PBS group; the results of miR-383-5p expression, PFKM mRNA, and protein expressions were regarded as measurement data, presented by mean ± SD and analyzed by one-way ANOVA. There were 10 mice in each group, and the experiment was repeated 3 times; miR-383-5p: microRNA-383-5p; PFKM: phosphofructokinase muscle-type; AMI: acute myocardial infarction.

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  Figure 4. miR-383-5p reduces the effects of oxidative stress through the AMPK signaling pathway. The myocardial tissues used for following detections were, respectively, treated with sham, AMI+PBS, AMI+miR-383-5p agomir, and AMI+miR-383-5p agomir NC. (a, b) Protein expression of AMPK and p-AMPK, in the myocardial tissue of mice by western blot analysis. (c) MDA kit was used to detect the expression level of MDA in the 4 groups. (d) Flow cytometry was used to detect ROS levels in the 4 groups of myocardial tissues. ∗P < 0.05 vs. the sham group; #P > 0.05 vs. the AMI+PBS; AMPK and p-AMPK protein expressions were regarded as measurement data, presented by mean ± SD and analyzed by one-way ANOVA. There were 10 mice in each group, and the experiment was repeated 3 times; miR-383-5p: microRNA-383-5p; AMPK: AMP-activated protein kinase; p-AMPK: phosphorylated AMP-activated protein kinase; MDA: malondialdehyde; ROS: reactive oxygen species.

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  Figure 5. Overexpression of miR-383-p increases the cardiac function in mice. The myocardial tissues used for following detections were, respectively, treated with sham, AMI+PBS, AMI+miR-383-5p agomir, and AMI+miR-383-5p agomir NC. (a) Representative photographs in mouse echocardiography. (b) Cardiac function index EF of mice. (c) Cardiac function index FS of mice. There were 10 mice in each group, and the experiment was repeated 3 times; miR-383-5p: microRNA-383-5p; EF: ejection fraction; FS: fraction shortening.