microRNA-130a-5p suppresses myocardial ischemia reperfusion injury by downregulating the HMGB2/NF-κB axis

Fig. 1. Identification of MIRI mouse models. a The electrocardiography of the MIRI mice, ischemia: 30 min, reperfusion: 120 min, n = 10. b HE staining revealed that the myocardial tissues were disrupted in the MIRI mice, × 200, n = 6. MIRI myocardial ischemia reperfusion injury, HE hematoxylin and eosin

Fig. 2. Overexpressed miR-130a-5p alleviates MIRI in mice. a RT-qPCR showed that the MIRI mice had a lower miR-130a-5p expression pattern, which was increased after overexpressing miR-130a-5p, n = 4. b, c The cardiac function and hemodynamics in mice were detected, n = 10. d ELISA results suggested notable increases in serum CK and cTnT levels from the MIRI mice, which were repressed by overexpressed miR-130a-5p, n = 10. e HE staining revealed that the disrupted myocardial tissues were improved with overexpressed miR-130a-5p, × 200, n = 6. f TUNEL assay revealed that the high apoptosis rates in cardiomyocyte in MIRI mice was declined by overexpressing miR-130a-5p, × 400, n = 6. g Western blot analysis demonstrated that the growth of Bax expression pattern and slowdown of Bcl-2 expression pattern in mice caused by MIRI were reversed by miR-130a-5p overexpression, n = 4. The images of g shown are cropped. Two-way ANOVA was used to determine data in panels c, g, and one-way ANOVA was employed to determine data in panels a, c, d, f. Tukey’s multiple comparisons test was applied for the post hoc test. **p < 0.01; ##p < 0.01. miR microRNA, MIRI myocardial ischemia reperfusion injury, RT-qPCR…

Fig. 3. Overexpressed miR-130a-5p alleviates MIRI-induced oxidative stress and mitochondrial disorder in mice. a Serum levels of LDH, MDA and SOD measured using kits, n = 10. b MMP, ATP, ROS and Ca2+ expression were tested with kits, n = 4. c Western blot analysis showed that the elevated protein levels of Cyt-c and AIF in MIRI mice were discouraged by miR-130a-5p overexpression, n = 4. The images of c shown are cropped. One-way ANOVA and Tukey’s multiple comparisons test were applied to determine the statistical significance. **p < 0.01; ##p < 0.01. miR mircoRNA, MIRI myocardial ischemia reperfusion injury, LDH lactate dehydrogenase, MDA malondialdehyde, SOD superoxide dismutase, MMP mitochondrial membrane potential, ATP adenosine triphosphate, ROS reactive oxygen species, Cyt-c Cytochrome C, AIF apoptosis inducing factor, ANOVA analysis of variance

Fig. 4. miR-130a-5p targets HMGB2. a TargetScan identified the binding site between miR-130a-5p and HMGB2 3′ untranslated region. b Dual-luciferase reporter gene assay verified the targeting relation between miR-130a-5p and HMGB2. c, d RT-qPCR and western blot analysis indicated that mRNA expression and protein level of HMGB2 in MIRI mice were both lowered by overexpressed miR-130a-5p, n = 4. The images of d shown are cropped. Two-way ANOVA was used to determine data in panel b and one-way ANOVA was employed to determine data in panels c, d. Tukey’s multiple comparisons test was applied for post hoc test. **p < 0.01; ##p < 0.01. miR microRNA, HMGB high mobility group box, RT-qPCR reverse transcription quantitative polymerase chain reaction, MIRI myocardial ischemia reperfusion injury, ANOVA analysis of variance

Fig. 5. Overexpressed HMGB2 inverts the protective effects of miR-130a-5p on MIRI mice. a, b RT-qPCR and western blot analysis revealed that the mRNA expression and protein level of HMGB2 were evidently promoted when HMGB2 was up-regulated, n = 4. c, d EF, FS, LVSP and ± dp/dtmax were declined while LVEDP was enhanced when HMGB2 was up-regulated, n = 10. e CK and cTnT levels in serum were increased with up-regulated HMGB2 as detected using ELISA kits, n = 10. f myocardial injury was further deteriorated with up-regulated HMGB2 as detected by TUNEL assay, × 200, n = 6. g TUNEL assay suggested that the apoptosis rate of cardiomyocytes was enhanced with up-regulated HMGB2, × 400, n = 6. h Western blot analysis indicated that the Bax expression pattern was enhanced and the Bcl-2 expression pattern was reduced upon upregulated HMGB2, n = 4. The images of b, h shown are cropped. Two-way ANOVA was adopted to determine data in panels c, d, h, and one-way ANOVA was employed to determine data in panels a, b, d, e, g. Tukey’s multiple comparisons test was applied for post hoc test. **p < 0.01. HMGB high mobility group box, miR mircoRNA, MIRI myocardial ischemia reperfusion injury, RT-qPCR reverse…

Fig. 6. miR-130a-5p targets HMGB2 to downregulate the NF-κB axis, mitigating inflammatory injury induced by MIRI. a Western blot analysis was applied to assess the p65 and IκBα protein levels and phosphorylation in myocardial tissues in MIRI mice, n = 4. b ELISA kits were utilized to determine the serum levels of TNF-α, IL-6 and IL-1β, n = 10. The images of a shown are cropped. One-way ANOVA and Tukey’s multiple comparisons test were applied to determine statistical significance. **p < 0.01; ##p < 0.01; &&p < 0.01. miR microRNA, HMGB high mobility group box, NF-κB nuclear factor kappa-B, MIRI myocardial ischemia reperfusion injury, IκB inhibitor of κB, ELISA enzyme-linked immunosorbent assay, TNF-α tumor necrosis factor-α, IL interleukin