Abstract 16495: A New Approach to Select Novel Potential Therapeutic Mirna Targets Effectively in the Cardiac Protection of Ischemia/Reperfusion (i/r) Injury
Modulation of miRNAs is a potential therapeutic strategy in cardiac ischemic injury. Therefore a highly efficient approach to select potential therapeutic miRNA targets is needed. We have assessed selection of microRNAs based on differential dysregulation in ischemia-reperfusion (I/R) in the comparison of presence and absence of cardioprotective drugs.
METHODS: Isolated rat hearts undergoing a I/R protocol were treated with 50 μM NaHS or 10 nM urocortin (UCN) in controlled experiments. Hearts were collected for miRNA array analysis (Exiqon). Up-regulated miRNAs were selected for high throughput functional screening. After transfection with 25 nmol of selected miRNA mimics, H9c2 and neonatal rat ventricular myocytes (NRVM) were subjected to 15 and 8 hours 0.2% O2 hypoxia / 2 hours re-oxygenation. Cell viability (WST) and injury (LDH release) were measured. RISK-loaded miRNAs from specific immunoprecipitation of protein Agonaute-2 were measured by qPCR.
RESULTS: NaHS or UCN treatment doubled cardiac functional recovery compared with Control and concomitantly reduced injury. In spite of differing protective mechanisms, miRNA dysregulation exhibited similar patterns (Fig. left). From 22 commonly up-regulated miRNAs, 11 miRNAs were selected for intervention. 6 of 11 miRNAs were cardioprotective in H9C2 cells and 4 of them further confirmed in NRVM as assessed by WST and LDH (Fig. Right: LDH release). Mimic trasfection increased the selected miRNA by ~ 1000 fold; while RISK-loaded miRNAs were increased ~ 20-100 fold negatively correlated to baseline expression. We further validated ~20 predicted apoptosis-related mRNA target genes by qPCR. miRNA-125b and -221 significantly down-regulated genes of BAK1, Bmf, Bcl-2-L-11, Hif1an, Tnfsf1b, Traf6, Tp53inp and Ddit4. This study demonstrated an approach to selecting miRNA targets from cardioprotection combined with functional screening to enable us to zoom-in novel miRNAs with therapeutic potential.
Author Disclosures: Y. Zhou: None. Q. Chen: None. K. Lew: None. A.M. Richards: None. P. Wang: None.
- © 2014 by American Heart Association, Inc.