MicroRNA-15a/b are up-regulated in response to myocardial ischemia/reperfusion injury

Abstract

Objective: Several studies have indicated that miR-15a, miR-15b and miR-16 may be the important regulators of apoptosis. Since attenuate apoptosis could protect myocardium and reduce infarction size, the present study was aimed to find out whether these miRNAs participate in regulating myocardial ischemia reperfusion (I/R) injury.

Methods: Apoptosis in mice hearts subjected to I/R was detected by TUNEL assay in vivo, while flow cytometry analysis followed by Annexin V/PI double stain in vitro was used to detect apoptosis in cultured cardiomyocytes which were subjected to hypoxia/reoxygenation (H/R). Taqman real-time quantitative PCR was used to confirm whether miR-15a/15b/16 were involved in the regulation of cardiac I/R and H/R.

Results: Compared to those of the controls, I/R or H/R induced apoptosis of cardiomyocytes was significantly increased both in vivo (24.4% ± 9.4% vs. 2.2% ± 1.9%, P < 0.01, n = 5) and in vitro (14.12% ± 0.92% vs. 2.22% ± 0.08%). The expression of miR-15a and miR-15b, but not miR-16, was increased in the mice I/R model, and the results were consistent in the H/R model.

Conclusions: Our data indicate miR-15 and miR-15b are up-regulated in response to cardiac I/R injury, therefore, down-regulation of miR-15a/b may be a promising strategy to reduce myocardial apoptosis induced by cardiac I/R injury.

Keywords: Apoptosis; Ischemia/Reperfusion injury; Myocardial reperfusion injury; miR-15a/b.

Figure 1.. Myocardial infarction induced by I/R through occlusion of LAD. Mice I/R models were established by occluding the LAD coronary artery for 30-min and followed by 24-h reperfusion. (A): middle transverse section of the heart stained with TTC and Evans blue (white–gray area represents area of necrosis, blue area represents normal perfusion zone, brick-red area represents ischemic zone); (B): the surgery protocols for I/R group and control group; (C): TUNEL assay was performed in infarct border zone (blue-positive indicates all the nuclei, green-positive indicate apoptotic nuclei, × 400); (D), the proportion of apoptotic cells (24.4% ± 9.4% vs. 2.2% ± 1.9%, compared with sham group, P < 0.01, n = 5). I/R: ischemia reperfusion; LAD: left anterior descending coronary artery.

Figure 2.. H/R induced cell apoptosis and death. (A): cell viability detected by trypan blue (the percentage of trypan blue-negative cells, n = 3); (B): cells were labeled by Annexin V-FITC and PI (upper right represent late phrase apoptotic cells, lower right represent early phrase apoptotic cells, upper left represent necrotic cells, lower left represent normal cells); (C): the percentage of lower right region cells plus upper right region cells, H/R induced significant increase of cell apoptosis compared with control (P < 0.01, n = 3). H/R: hypoxia/reoxygenation; PI: propiduim iodide.

Figure 3.. Quantitative, real-time PCR (Taqman) displaying miR-15a, miR-15b, miR-16 fold change in mice I/R model group (A), and in cardiomyocytes H/R model group (B) compared with control (normalized to U6), repectively, ^*P < 0.05.