Up-regulation of miRNA-221 inhibits hypoxia/reoxygenation-induced autophagy through the DDIT4/mTORC1 and Tp53inp1/p62 pathways.
Timely reperfusion in acute myocardial infarction has improved clinical outcomes but the benefits are partially offset by ischemia-reperfusion injury (I/R). MiRNA regulates mRNA of multiple effectors within injury and survival cell signaling pathways. We have previously reported the protective effects of miRNA-221 in I/R injury. The purpose of this study was to explore the mechanisms underlying cardioprotection of miR-221. Myoblast H9c2 and neonatal rat ventricular myocytes (NRVM) were subjected to 0.2% O2 hypoxia followed by 2 h of re-oxygenation (H/R). In gain-and-loss function studies through transfections of miR-221 mimic (miR-221) and inhibitor (miR-221-i), the protective effects of miR-221 were confirmed as assessed by increased cell metabolic activity (WST-1) and decreased LDH release. Autophagy was assessed by GFP-LC3 labeling of autophagosome formation, LC3 and p62 measurements. Co-immuno-precipitation and specific gene cloning and function were used to identify the pathways underpinning miR-221 effects. MiR-221 significantly reduced H/R injury in association with inhibition of autophagy. Underlying mechanisms include (1) down-regulation of Ddit4 (disinhibiting the mTORC1/p-4EBP1 pathway) which inhibits autophagosome formation (2) down-regulation of Tp53inp1 (with reduced Tp53inp1/p62 complex formation) which inhibits autophagosome degradation. In conclusion, miRNA-221 exerts cytoprotective effects in hypoxia-reoxygenation injury in association with alterations in autophagic cell injury. Mir-221 may constitute is a novel therapeutic target in the treatment of cardiac I/R injury.
Huashan Hospital, Fudan University, Shanghai, China.
Qiying
Q
Chen
Cardiovascular Research Institute, National University Health System, Singapore; Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
Yue
Y
Zhou
Cardiovascular Research Institute, National University Health System, Singapore; Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; Cardiac Department, National University Health System, Singapore; Christchurch Heart Institute, Department of Medicine, University of Otago-Christchurch, Christchurch, New Zealand.
A Mark
AM
Richards
Cardiovascular Research Institute, National University Health System, Singapore; Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. Electronic address: mdcwp@nus.edu.sg.
Peipei
P
Wang
D000818:Animals; D001343:Autophagy; D002478:Cells, Cultured; D000076222:Mechanistic Target of Rapamycin Complex 1; D035683:MicroRNAs / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000378:metabolism*; D015428:Myocardial Reperfusion Injury / Q000378:metabolism* / Q000473:pathology; D009687:Nuclear Proteins / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D015854:Up-Regulation
C469992:Ddit4 protein, mouse; C546122:MIRN221 microRNA, mouse; D035683:MicroRNAs; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; D014157:Transcription Factors; C518507:tumor protein 53-induced nuclear protein 1, mouse; D000076222:Mechanistic Target of Rapamycin Complex 1; D058570:TOR Serine-Threonine Kinases
Autophagy; Ddit4; Ischemia/reperfusion; Tp53inp1; miRNA-221