Hang Yu1, Yuxiang Pan2, Mingming Dai3, Xiaoqi Wang4, Haibo Chen5. 1. Department of Cardiovascular Surgery Intensive Care Unit, The Second Affiliated Hospital of Hainan Medical College, Haikou City, Hainan Province, China. 2. Department of Critical Care Medicine, The Second Affiliated Hospital of Hainan Medical College, Haikou City, Hainan Province, China. 3. Department of Neurology Three Areas, The Second Affiliated Hospital of Hainan Medical College, Haikou City, Hainan Province, China. 4. Department of Cardiovascular Surgery, The Second Affiliated Hospital of Hainan Medical College, No. 368 Yehai Avenue, Longhua District, Haikou City, 570105, Hainan Province, China. WangXiaoqi767@163.com. 5. Department of Blood Transfusion, The Second Affiliated Hospital of Hainan Medical College, No. 368 Yehai Avenue, Longhua District, Haikou City, 570105, Hainan Province, China. chenhaibo2767@163.com.
Abstract
BACKGROUND: Mesenchymal stem cell (MSC)-derived exosomes play significant roles in ameliorating cardiac damage after myocardial ischemia-reperfusion (I/R) injury. Long non-coding RNA alpha-2-macroglobulin antisense RNA 1 (Lnc A2M-AS1) was found that might protect against myocardial I/R. However, whether Lnc A2M-AS1 delivery via MSC-derived exosomes could also regulate myocardial I/R injury remains unknown. METHODS: Exosomes were isolated by ultracentrifugation, and qualified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot. Hypoxia/reoxygenation (H/R) treatment in human cardiomyocytes was used to mimic the process of myocardial I/R in vitro. The viability and apoptosis of cardiomyocytes were detected using cell counting kit-8, flow cytometry, and Western blot assays. The contents of lactate dehydrogenase (LDH), malondialdehyde (MDA), and superoxide dismutase (SOD) were evaluated using corresponding commercial kits. The quantitative real-time polymerase chain reaction and Western blot were used to determine the expression levels of Lnc A2M-AS1, microRNA (miR)-556-5p, and X-linked inhibitor of apoptosis protein (XIAP). The binding interaction between miR-556-5p and Lnc A2M-AS1 or XIAP was confirmed by the dual-luciferase reporter, RIP and pull-down assays. RESULTS: Exosomes isolated from hMSCs (hMSCs-exo) attenuated H/R-induced apoptosis and oxidative stress in cardiomyocytes. Lnc A2M-AS1 was lowly expressed in AMI patients and H/R-induced cardiomyocytes. Besides, Lnc A2M-AS1 was detectable in hMSCs-exo, exosomes derived from Lnc A2M-AS1-transfected hMSCs weakened H/R-induced apoptosis and oxidative stress, and enhanced the protective action of hMSCs-exo on H/R-induced cardiomyocytes. Further mechanism analysis showed that Lnc A2M-AS1 acted as a sponge for miR-556-5p to increase XIAP expression level. Importantly, miR-556-5p overexpression or XIAP knockdown reversed the action of exosomal Lnc A2M-AS1 on H/R-induced cardiomyocytes. CONCLUSION: Lnc A2M-AS1 delivery via MSC-derived exosomes ameliorated H/R-induced cardiomyocyte apoptosis and oxidative stress via regulating miR-556-5p/XIAP, opening a new window into the pathogenesis of myocardial I/R injury.
BACKGROUND: Mesenchymal stem cell (MSC)-derived exosomes play significant roles in ameliorating cardiac damage after myocardial ischemia-reperfusion (I/R) injury. Long non-coding RNA alpha-2-macroglobulin antisense RNA 1 (Lnc A2M-AS1) was found that might protect against myocardial I/R. However, whether Lnc A2M-AS1 delivery via MSC-derived exosomes could also regulate myocardial I/R injury remains unknown. METHODS: Exosomes were isolated by ultracentrifugation, and qualified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot. Hypoxia/reoxygenation (H/R) treatment in human cardiomyocytes was used to mimic the process of myocardial I/R in vitro. The viability and apoptosis of cardiomyocytes were detected using cell counting kit-8, flow cytometry, and Western blot assays. The contents of lactate dehydrogenase (LDH), malondialdehyde (MDA), and superoxide dismutase (SOD) were evaluated using corresponding commercial kits. The quantitative real-time polymerase chain reaction and Western blot were used to determine the expression levels of Lnc A2M-AS1, microRNA (miR)-556-5p, and X-linked inhibitor of apoptosis protein (XIAP). The binding interaction between miR-556-5p and Lnc A2M-AS1 or XIAP was confirmed by the dual-luciferase reporter, RIP and pull-down assays. RESULTS: Exosomes isolated from hMSCs (hMSCs-exo) attenuated H/R-induced apoptosis and oxidative stress in cardiomyocytes. Lnc A2M-AS1 was lowly expressed in AMI patients and H/R-induced cardiomyocytes. Besides, Lnc A2M-AS1 was detectable in hMSCs-exo, exosomes derived from Lnc A2M-AS1-transfected hMSCs weakened H/R-induced apoptosis and oxidative stress, and enhanced the protective action of hMSCs-exo on H/R-induced cardiomyocytes. Further mechanism analysis showed that Lnc A2M-AS1 acted as a sponge for miR-556-5p to increase XIAP expression level. Importantly, miR-556-5p overexpression or XIAP knockdown reversed the action of exosomal Lnc A2M-AS1 on H/R-induced cardiomyocytes. CONCLUSION: Lnc A2M-AS1 delivery via MSC-derived exosomes ameliorated H/R-induced cardiomyocyte apoptosis and oxidative stress via regulating miR-556-5p/XIAP, opening a new window into the pathogenesis of myocardial I/R injury.
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