MiR-27b promotes H₂O₂-induced cardiomyocyte apoptosis by suppressing the PPAR-γ/Bcl-2 signaling pathway to aggravate myocardial ischemia-reperfusion injury

Oxidative stress-induced cardiomyocyte apoptosis is a central event in myocardial ischemia-reperfusion (MIR) injury. MicroRNA-27b (miR-27b) is aberrantly expressed in this process, yet its role and regulatory mechanism in MIR injury remain to be elucidated. This study aims to clarify the molecular mechanism by which miR-27b regulates H₂O₂-induced cardiomyocyte apoptosis via the PPAR-γ/Bcl-2 signaling pathway, thereby providing a novel target for intervention in MIR injury.

① Healthy male C57BL/6J mice (10 to 12 weeks old, weighing 24 to 30 g) were randomly divided into a MIR group and a sham operation group, with 6 animals in each group. The MIR group underwent ligation of the left anterior descending coronary artery for 45 min followed by reperfusion to induce myocardial injury, while the Sham group underwent the identical surgical procedure without ligation. The heart was harvested 24 h postoperatively, and the entire heart was sectioned and stained with TTC for 30 min to assess infarct size. The ischemic myocardial tissue from the anterior wall of the left ventricle was used for total RNA extraction. ② Primary neonatal mouse ventricular cells (PNMVCs) were isolated from 1-to 3-day-old mice and transfected for 24 h with miR-27b mimics or anti-miR-27b oligonucleotides (AMO-miR-27b). The cells were subsequently treated with 100 μmol/L hydrogen peroxide (H₂O₂) for 24 h to induce cardiomyocyte apoptosis. Transfected cardiomyocytes were treated with the PPAR-γ antagonist T0070907 (10.0 nmol/L), and apoptosis was assessed 48 h later. Cell experiments were divided into normal control group, H₂O₂ model group, miR-27b mimic transfection+H₂O₂ group, AMO-miR-27b transfection+H₂O₂ group, AMO-miR-27b+H₂O₂+T0070907 group, and negative control (NC) +H₂O₂ group. RT-qPCR was performed to measure the expression levels of miR-27b and PPAR-γ. Cell viability was assessed using MTT assay. Cell apoptosis was quantified by TUNEL assay combined with DAPI staining. Western blotting was employed to detect the expression of PPAR-γ, Bcl-2, Bax, and Cyt-c, and other related proteins.

① Compared with the Sham group, the MIR group exhibited significantly larger infarct size in TTC-stained heart sections, and upregulated miR-27b expression in the ischemic myocardial tissue of the left ventricular anterior wall (P<0.05). ② Compared to the normal control group, miR-27b expression levels were significantly upregulated in H₂O₂-treated PNMVCs (P<0.01). Overexpression of miR-27b in PNMVCs induced cardiomyocyte damage, as evidenced by decreased cell viability[(48.75±5.55)% vs (87.20±3.52)%, P<0.01] and increased cell apoptosis [(41.08±2.84)% vs (1.75±0.29)%, P<0.01], accompanied by reduced expression of PPAR-γ at both mRNA (P<0.01) and protein levels (P<0.01). In contrast, inhibition of miR-27b expression enhanced PPAR-γ expression at both mRNA (P<0.05) and protein levels (P<0.01). MTT assay showed that AMO-miR-27b attenuated H₂O₂-induced decrease in cell viability [(79.46±6.41)% vs (60.29±4.83)%, P<0.01]; TUNEL assay further demonstrated that after AMO-miR-27b transfection, H₂O₂-induced cardiomyocyte apoptosis was significantly reduced [(19.60±2.95)% vs (42.96±5.80)%, P<0.01]. H₂O₂ treatment caused significantly decreased Bcl-2 levels (P<0.01) and elevated Bax levels (P<0.01) in cardiomyocytes, which were reversed by AMO-miR-27b. Furthermore, cytoplasmic Cyt-c protein levels were significantly increased in cardiomyocytes after H₂O₂ exposure (P<0.01), and the downregulatory effect of AMO-miR-27b on cytoplasmic Cyt-c (P<0.05) and its protective effect against cardiomyocyte apoptosis (P<0.01) were abolished by the PPAR-γ antagonist T0070907.

MiR-27b is highly expressed in myocardial tissues and cardiomyocytes after oxidative stress injury, which exacerbates tissue damage and cell apoptosis. Suppression of the PPAR-γ/Bcl-2 signaling pathway may be a primary mechanism underlying this effect, suggesting that targeted inhibition of miR-27b may be a potential novel strategy for MIR injury intervention.