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A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation
Published on: July 13, 2018
MiR-134-5p targeting XIAP modulates oxidative stress and apoptosis in cardiomyocytes under
Min Lu1, Xinglei Qin2, Jungong Yao1
1Department of Cardiologry, Henan Provincial People's Hospital, People's Hospital of Zhengzhou University, School of Clinical Medicine, Zhengzhou, Henan, China.
Abstract:
MicroRNA-134-5p (MiR-134-5p) has been proposed as a promising novel biomarker for the diagnosis of acute myocardial infarction (AMI). However, the biological role of miR-134-5p in ischemic cardiomyocytes has been little disclosed yet. Expression of miR-134-5p and X-linked inhibitor of apoptosis protein (XIAP) was detected using RT-qPCR and western blot. Oxidative stress and cell apoptosis were determined by enzyme-linked immunosorbent assays, 3-(4, 5-dimethylthiazole-2-y1)-2, 5-biphenyl tetrazolium bromide assay, flow cytometry, western blot, and terminal-deoxynucleoitidyl transferase-mediated nick end labeling (TUNEL). The interaction between miR-134-5p and XIAP was confirmed by luciferase reporter assay. Expression of miR-134-5p was upregulated in serum of AMI patients and hypoxia/reoxygenation (H/R)-induced cardiomyocytes (AC16 and HCM). MiR-134-5p downregulation could inhibit H/R-mediated release of lactic dehydrogenase enzyme (LDH) and malondialdehyde (MDA), and promote superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) levels. Meanwhile, cell viability was increased, while the apoptosis rate and TUNEL positive cells were inhibited by miR-134-5p downregulation in H/R-treated AC16 and HCM cells. Mechanically, XIAP was downregulated and targeted by miR-134-5p in H/R-induced cardiomyocytes in vitro. Overexpression of XIAP inhibited oxidative stress and cell apoptosis in H/R-treated AC16 and HCM cells, which was similar to miR-134-5p knockdown. Moreover, downregulation of XIAP could partially reverse the effect of miR-134-5p knockdown in H/R-induced cardiomyocytes. Knockdown of miR-134-5p protected cardiomyocytes from H/R-induced oxidative stress and apoptosis in vitro through targeting XIAP.
Insights
MicroRNA-134-5p (miR-134-5p) protects heart cells from damage after injury. Lowering miR-134-5p levels reduces oxidative stress and cell death by targeting XIAP, offering potential therapeutic strategies for acute myocardial infarction.
Area of Science:
- Cardiology
- Molecular Biology
- Biochemistry
Background:
- MicroRNA-134-5p (miR-134-5p) is a potential biomarker for acute myocardial infarction (AMI).
- The specific function of miR-134-5p in cardiomyocytes under ischemic conditions requires further elucidation.
Purpose of the Study:
- To investigate the biological role of miR-134-5p in cardiomyocytes subjected to hypoxia/reoxygenation (H/R) injury.
- To explore the underlying molecular mechanism involving X-linked inhibitor of apoptosis protein (XIAP).
Main Methods:
- Quantitative real-time PCR (RT-qPCR) and Western blot for gene and protein expression analysis.
- ELISA, MTT assay, flow cytometry, and TUNEL assay to assess oxidative stress and apoptosis.
- Luciferase reporter assay to confirm the interaction between miR-134-5p and XIAP.
Main Results:
- miR-134-5p expression was elevated in AMI patient serum and H/R-induced cardiomyocytes.
- Downregulation of miR-134-5p reduced cardiomyocyte injury markers (LDH, MDA) and apoptosis, while increasing protective enzymes (SOD, GSH-PX).
- XIAP was identified as a direct target of miR-134-5p, and its modulation mimicked or reversed miR-134-5p's effects on H/R-induced injury.
Conclusions:
- miR-134-5p exacerbates H/R-induced oxidative stress and apoptosis in cardiomyocytes.
- Targeting miR-134-5p or its downstream effector XIAP may represent a novel therapeutic approach for AMI treatment.
