MiR‐486a‐5p Alters Cell Survival in Ischemia‐Reperfusion Induced Myocardial Injury

Abstract

Previous work in our lab has shown that mesenchymal stem cells (MSC)‐derived exosomes protect cardiomyocytes from ischemia‐reperfusion (I/R) induced myocardial injury both in vitro and in vivo. We found that microRNA‐486a‐5p (miR‐486) was highly and selectively expressed in MSC‐derived exosomes (1686‐fold relative to MSC (p<0.001)). The role of miR‐486 in cell survival is still debated and miR‐486 has been reported to act as either a tumor suppressor or an oncogene. We hypothesized that miR‐486 upregulation will protect cardiomyocytes from I/R injury. We searched for miR‐486 gene targets using miRanda and Targetscan and found 1687 gene targets. We focused upon targets involving in the cell death and survival pathways, leaving 104 gene targets. Of these 104 targets, program cell death 4 (PDCD4), a tumor suppressor and PTEN, which negatively regulates the PI3K/Akt survival pathway, were chosen for further study based upon considerations of scores for mRNA‐miRNA interaction and the predicted targets. Luciferase reporter gene assays indicated that the 3′‐UTR of PDCD4 and PTEN are both targeted by miR‐486. A mimic and inhibitor (antagomir) of miR‐486 were used to assess its ability to modulate PDCD4, and PTEN expression and Akt activity. Although the mRNA levels of PDCD4 and PTEN were not altered by treatments, immunoblotting results showed a 0.5‐fold reduction in PDCD4 protein expression with miR‐486 mimic and no change in expression with antagomir (n=3). We did not observe a change in PTEN protein levels, and we also saw no change in pAkt/Akt ratio which is regulated downstream of PTEN. Simulated ischemia/reperfusion (sim I/R) was performed on H9c2 cells treated with miR‐486 mimic or scrambled control to measure the cytoprotective effect of miR‐486. H9c2 cells treated with the mimic of miR‐486 showed a significant increase in cell survival by 28–43% (p<0.001, n=4–6) and a reduction of 20% in LDH release compared to control cells (p<0.05, n=6). Western blot results with cell lysate after sim I/R showed a marked decrease in levels of PDCD4, cleaved‐caspase 3 and Bax in the group treated with miR‐486 mimic compared to control cells (p<0.05, n=3). These findings support that miR‐486 upregulation is anti‐apoptotic. Additional studies are needed to discern whether miR‐486 upregulation will reduce cell injury using rodent models, and how much exosomal miR‐486a‐5p contributes to the effects of exosomes and stem cells. Such efforts will lead to a new therapeutic approach for prevention damage of I/R injury.Support or Funding InformationNIH 5R01HL091478 (WKJ)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.