Abstract
PTEN/AKT signaling plays pivotal role in myocardial ischemia reperfusion injury (MIRI), and miRNAs are involved in the regulation of AKT signaling. This study was designed to investigate the interaction between miR-129 and PTEN in MIRI. A MIRI rat model and a hypoxia reoxygenation (H/R) H9C2 cell model were constructed to simulate myocardial infarction clinically. TTC staining, creatine kinase (CK) activity, TUNEL/Hoechst double staining, Hoechst staining and flow cytometer were used for evaluating myocardial infarction or cell apoptosis. miR-129 mimic transfection experiment and luciferase reporter gene assay were conducted for investigating the function of miR-129 and the interaction between miR-129 and PTEN, respectively. Real-time PCR and western blotting were performed to analyze the gene expression. Compared to the control, MIRI rats presented obvious myocardial infarction, higher CK activity, increased expression of caspase-3 and PTEN, decreased expression of miR-129, and insufficient AKT phosphorylation. Consistently, H/R significantly increased the apoptosis of H9C2 cells, concomitant with the downregulation of miR-129, upregulation of PTEN and caspase-3, and insufficient phosphorylation of AKT, while miR-129 mimic obviously inhibited the expression of PTEN and caspase-3, increased the AKT phosphorylation, and decreased the cell apoptosis. Additionally, miR-129 mimic obviously decreased the relative luciferase activity in H9C2 cells. To our best knowledge, this study firstly found that the low expression of miR-129 accelerates the myocardial cell apoptosis by directly targeting 3′UTR of PTEN. miR-129 is an important biomarker for MIRI, as well as a potential therapy target.
Highlights
PTEN/AKT signaling plays pivotal role in myocardial ischemia reperfusion injury (MIRI), and miRNAs are involved in the regulation of AKT signaling
TTC staining, creatine kinase (CK) activity, TUNEL/Hoechst double staining, Hoechst staining and flow cytometer were used for evaluating myocardial infarction or cell apoptosis. miR-129 mimic transfection experiment and luciferase reporter gene assay were conducted for investigating the function of miR-129 and the interaction between miR-129 and PTEN, respectively
hypoxia reoxygenation (H/R) significantly increased the apoptosis of H9C2 cells, concomitant with the downregulation of miR-129, upregulation of PTEN and caspase-3, and insufficient phosphorylation of AKT, while miR-129 mimic obviously inhibited the expression of PTEN and caspase-3, increased the AKT phosphorylation, and decreased the cell apoptosis
Summary
PTEN/AKT signaling plays pivotal role in myocardial ischemia reperfusion injury (MIRI), and miRNAs are involved in the regulation of AKT signaling. MIRI rats presented obvious myocardial infarction, higher CK activity, increased expression of caspase-3 and PTEN, decreased expression of miR-129, and insufficient AKT phosphorylation. Thereby, elucidation of the molecular mechanism of decreased AKT phosphorylation in MIRI is very important for the knowing of myocardial infarction as well as its therapy. Previous studies have found that higher expression levels of PTEN were, at least partially, the cause of insufficiency of AKT phosphorylation in myocardial cells in pathological state caused by MIRI [6]. Xing et al have demonstrated that PTEN is a target gene of miR-26a-5P [10] This suggested that miRNAs are involved in MIRI, at least partly, by regulating of PTEN. This was the first time to observe the interaction of miR-129 and PTEN and it will provide a novel target for MIRI therapy
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