Abstract
Objectives To explore the effects of miR-16-5p and PTPN4 on the apoptosis and autophagy of AC16 cardiomyocytes after hypoxia/reoxygenation treatment. Methods AC16 cells were divided into the control group (NC), hypoxia/reoxygenation group (H/R), knockdown miR-16-5p negative control group (NC inhibitor), knockdown miR-16-5p group (miR-16-5p inhibitor), overexpression miR-16-5p negative control group (NC mimics), overexpression miR-16-5p group (miR-16-5p mimics), silent PTPN4 negative control group (sh-NC), silent PTPN4 group (sh-PTPN4), and silent PTPN4 + knockdown miR-16-5p group (sh-PTPN4 + miR-16-5p inhibitor). Real-time fluorescent quantitative PCR (RT-qPCR) and western blotting (WB) were used to measure the expression level of miR-16-3p, miR-16-5p, protein tyrosine phosphatase nonreceptor type 4 (PTPN4), and autophagy-related proteins (beclin-1, LC3 II/I, and P26) in AC16 cells. The apoptosis level of AC16 cells in each group was measured by flow cytometry and TUNEL. The dual-luciferase reporter gene experiment was also used to verify the targeting relationship between miR-16-5p and PTPN4. Results After H/R treatment, the levels of myocardial injury markers including LDH and CK-MB in AC16 cells were increased significantly (P < 0.05), and the levels of cell apoptosis and autophagy also increased significantly (P < 0.05). The level of miR-16-3p in AC16 cells did not change significantly after H/R treatment, whereas the level of miR-16-5p was increased significantly (P < 0.05). After miR-16-5p was knocked down, the levels of LDH and CK-MB in AC16 cells treated with H/R were significantly reduced (P < 0.05), and the rates of cell apoptosis and autophagy were also significantly reduced (P < 0.05). miR-16-5p negatively regulated the expression level of PTPN4 protein in AC16 cells (P < 0.05), and the dual-luciferase reporter gene experiment confirmed that PTPN4 was the downstream target of miR-16-5p. Silencing of PTPN4 significantly increased the damage of AC16 cells induced by H/R treatment (P < 0.05), but simultaneously inhibiting the expression of PTPN4 and miR-16-5p reversed the protective effect of miR-16-5p knockdown on AC16 cells (P < 0.05). Conclusions The expression of miR-16-5p is upregulated in AC16 cells after H/R treatment and the knockdown which can protect AC16 cells from H/R-induced cell damage that may be due to its regulation on the expression of PTPN4.
Highlights
Acute myocardial infarction is a common cardiovascular disease [1]
After AC16 cells were treated with H/R, the levels of Lactate Dehydrogenase (LDH) and CK-MB in the cells increased significantly (P < 0.05), as shown in Figures 1(a) and 1(b). e results of flow cytometry and TUNEL showed that the apoptosis rate of AC16 cells was significantly increased after H/R treatment (P < 0.05), as shown in Figures 1(c) and 1(d)
Cardiomyocytes are prone to apoptosis and autophagy after H/R can cause heart tissue damage, resulting in a decrease in heart volume, and can affect the contractile function of the heart [16]. erefore, exploring the mechanism to alleviate H/R-induced cardiomyocyte apoptosis and autophagy is clinically important
Summary
Acute myocardial infarction is a common cardiovascular disease [1]. When it occurs, long-term ischemia and hypoxia can lead to interstitial fibrosis of myocardial cells [2], which in turn can induce left ventricular remodeling and heart failure. Previous studies have shown that the abnormal expression of various miRNAs is associated with acute myocardial infarction [7]. Previous studies have shown that miR-16 has a particular role in promoting cardiovascular endothelial injury [10]. The study by Wang et al [13] showed that miR-16-5p could increase the viability of cardiomyocytes and promote the growth of vascular endothelial cells by targeting IRS1. It has been shown that silencing of PTPN4 can induce the apoptosis of glioblastoma [14], but fewer studies focus on the role of PTPN4 in myocardial infarction
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