Transcriptomics: Open Access
Open Access
ISSN: 2329-8936
ISSN: 2329-8936
Peipei Wang
National University of Singapore, Singapore
Posters & Accepted Abstracts: Transcriptomics
Timely reperfusion in acute myocardial infarction has improved clinical outcomes but gains are partially offset by ischemiareperfusion injury (I/R). miRNA regulate multiple gene/protein effectors within injury and survival cell signaling pathways, therefore are potential therapeutic targets in the treatment of I/R injury. The purpose of our study was to establish a highly efficient approach to select miRNA targets and demonstrate their underlying mechanisms. Combined with isolated heart preparation and miRNA array technique, we pre-selected a panel of 20 miRNA targets from cardioprotection-induced changes rather than by revering disease-induced changes in I/R. Functional screening with gain and loss of function studies through transfections of miRNA mimic and inhibitor was performed. In a hypoxia re-oxygenation (H/R) model of myoblast H9c2 and neonatal rat ventricular myocytes (NRVM), the protective effects of miR-221, -150, -206, -184, and -140 were confirmed by increased cell metabolic activity (WST-1) and decreased LDH release. Among them, multiple predicted gene targets of miRNA-221 were validated by RT-qPCR, Western Blot and Luciferase reporter assay. Autophagosome formation was assessed by GFP-LC3 labeling and apoptosis by annexin V staining. Immuno-precipitation and specific gene cloning and function were used to identify the pathways underpinning miR-221 protective mechanisms. miR-221 significantly reduced H/R injury due to inhibition of H/R-induced autophagy and apoptosis in through (1) down-regulation of Ddit4 (disinhibiting the mTORC1/p-4EBP1 pathway) (2) down-regulation of Tp53inp1 (with reduced Tp53inp1/ p62 complex formation) and (3) decrease of pro-apoptotic effectors of Bmf and Bim. Through inducing favorable changes in autophagy and apoptosis, miRNA-221 is a promising therapeutic target in the treatment of cardiac I/R injury.
Email: mdcwp@nus.edu.sg