Down-regulation of microRNA-320 suppresses cardiomyocyte apoptosis and protects against myocardial ischemia and reperfusion injury by targeting IGF-1

Abstract

Insulin-like growth factor-1 (IGF-1) is an important regulator of cardiomyocyte homeostasis and cardiac structure, and the prosurvival and antiapoptotic effects of IGF-1 have been investigated. However, the effect of microRNA-320 (miR-320) in ischemia and reperfusion (I/R) by targeting IGF-1 is rarely discussed. We investigated the role of miR-320 in I/R injury. A total of 192 healthy female Wistar rats were divided into eight groups (n = 24). Rat heart I/R model was established. Hemodynamics, infarct size weight (ISW), heart function, and rat cardiomyocyte apoptosis were measured. Hypoxia-reoxygenation (H/R) in rat cardiomyocyte was used to simulate the I/R process. The mRNA levels of miR-320 and IGF-1, and proteins levels of IGF-1, IGF-1R, p-IGF-1R, p-ASK1, p-JNK, p-p38, Bcl-2, Bax and Caspase-3 were measured. In vivo inhibition of miR-320 expression significantly increased IGF-1 and IGF-1R mRNA levels, elevated the absolute values of SBP, DBP, MAP, ± dp/dtmax, LVEF and LVFS, decreased ISW, LVESD and LVEDd and the number of TUNEL positive cells, lowered the levels of p-ASK1, p-JNK, p-p38, Bax and Caspase-3 and increased expression of Bcl-2 compared to the I/R + NC group. Compared to H/R + NC group in vitro, miR-320 inhibition increased IGF-1 mRNA levels, inhibited cardiomyocyte apoptosis, down-regulated p-ASK, p-JNK, p-p38, Bax and Caspase-3 levels, and up-regulated Bcl-2 level. MiR-320 inhibition target elevated IGF-1 mRNA and protein levels, suppress early cardiomyocyte apoptosis of I/R, and inhibited ASK1-JNK/p38 pathway, which provides a new target for clinical study of I/R injury.

Keywords: apoptosis rate; cardiomyocytes; hypoxia-reoxygenation; ischemia-reperfusion; microRNA-320.

Figure 1. MiR-320 targets IGF-1 gene

A. MiR-320 binding sites in the 3′UTR region of IGF-1 gene detected by TargetScan. B. Dual-luciferase reporter gene system showing miR-320 targeting IGF-1 gene. Different lowercase letters indicate statistically significant differences (P < 0.05) with the same letter indicating no difference (P > 0.05). Note: miR-320, microRNA-320; IGF-1, Insulin-like growth factor 1; UTR, untranslated region; wt, wild type; mut, mutant type.

Figure 2. Fluorescence detection of 48h in myocardial cells transfected with virus

A. myocardial cells transfected with miR-320 inhibitors overexpression; B. myocardial cells transfected with miR-320 mimics; C. myocardial cells transfected with ad-IGF-1.

Figure 3. MiR-320 and IGF-1 mRNA and protein expressions in myocardial tissue

A–B. Real time-PCR detecting miR-320 and IGF-1 mRNA expressions. C–F. Western blots detecting IGF-1 protein expression, IGF-1R and p-IGF-1R levels. Different lowercase letters indicate statistically significant differences (P < 0.05) with the same letter indicating no difference (P > 0.05). Note: miR-320, microRNA-320; IGF-1, Insulin-like growth factor 1; PCR, polymerase chain reaction.

Figure 4. MiR-320 and IGF-1 mRNA and protein expressions in rat myocardial cells

A–B. Real time-PCR detecting miR-320 and IGF-1 mRNA expressions. C–F. Western blots detecting IGF-1 protein expression, IGF-1R and p-IGF-1R levels. Different lowercase letters indicate statistically significant differences (P < 0.05) with the same letter indicating no difference (P > 0.05). Note: miR-320, microRNA-320; IGF-1, Insulin-like growth factor 1; PCR, polymerase chain reaction.

Figure 5. Hemodynamic changes during ischemia and reperfusion in each group

Hemodynamic changes during I/R in the sham, I/R, IR + NC, I/R + miR-320 mimics + ad-IGF-1, I/R + miR-320 inhibitor and I/R + ad-IGF-1 groups. Note: I/R, ischemia/reperfusion; NC, negative control; IGF-1, insulin-like growth factor 1.

Figure 6. Myocardial cell apoptosis rate and apoptosis-related proteins

A–B. Cell apoptosis rate detected by TUNEL assay. C–F. Bcl-2, Bax and Caspase-3 protein expressions detected by western blotting. Different lowercase letters indicate statistically significant differences (P < 0.05) with the same letter indicating no difference (P > 0.05). Note: TUNEL, transferase-mediated deoxyuridine triphosphate-biotin nick end labeling; Bcl-2, B-cell lymphoma 2; I/R, ischemia/reperfusion; NC, negative control; IGF-1, Insulin-like growth factor 1.

Figure 7. Rat myocardial cell apoptosis rate and apoptosis-related proteins

A–B. Cell apoptosis rate detected by Annexin V/Propidium iodide apoptosis assay. C–F. Bcl-2, Bax and Caspase-3 protein expressions detected by western blotting. Different lowercase letters indicate statistically significant differences (P < 0.05) with the same letter indicating no difference (P > 0.05). Note: Bcl-2, B-cell lymphoma 2; H/R, hypoxia/reoxygenation; NC, negative control; IGF-1, Insulin-like growth factor 1.

Figure 8. Western blot analysis of p-ASK1, p-JNK and p-p38 levels in myocardial tissue

Different lowercase letters indicate statistically significant differences (P < 0.05) with the same letter indicating no difference (P > 0.05). Note: p-ASK1, phosphorylated-apoptosis signal-regulating kinase 1; p-JNK, phosphorylated-c-Jun N-terminal kinase; I/R, ischemia/reperfusion; NC, negative control; IGF-1, Insulin-like growth factor 1.

Figure 9. Western blot analysis of p-ASK1, p-JNK and p-p38 levels in rat myocardial cells

Different lowercase letters indicate statistically significant differences (P < 0.05) with the same letter indicating no difference (P > 0.05). Note: p-ASK1, phosphorylated-apoptosis signal-regulating kinase 1; p-JNK, phosphorylated-c-Jun N-terminal kinase; H/R, hypoxia/reoxygenation; NC, negative control; IGF-1, Insulin-like growth factor 1.