MiR‐486a‐5p Exerts Cardioprotection Against Ischemia Repefusion via Suppression of PDCD4
Authors: Phan Anh , Lynch Thomas , McGuinness Michael , Jones Walter K. 
Published: June 2021

Extracellular vesicle (EV) derived from mesenchymal stem cells (MSCs) have been demonstrated to be a critical component for cardioprotection against myocardial infarction triggered by stem cell therapy. EVs are nano‐size phospholipid bilayer particles that carry proteins, lipids, RNA, including microRNAs (miRs). Because EVs can deliver their cargo to other cells, they hold potential application for development as clinical therapeutics. Herein we tested the hypothesis that a specific MSC‐EV miR, miR‐486a‐5p, mediates some aspect of cardioprotective effect against ischemia/reperfusion injury (I/R).

Extracellular vesicles were isolated from B6/129‐derived MSCs using differential ultracentrifugation method. To characterize EV RNA, total RNA was isolated from MSC cells and EVs and used to synthesize libraries for small RNA deep sequencing. EVs and cell miRs were ranked based on enrichment in EV RNA (EV/cell levels). We found that miR‐486a‐5p is the most highly enriched miR in MSC‐derived EV RNA at level of 5853‐fold higher than in cells (Padj=1.52E‐59). The differential level of miR‐486‐5p between EVs and cell was validated by Taqman Advanced small RNA qRT‐PCR. We searched for miR‐486a‐5p gene targets using Targetscan7.0 and found 64 gene targets involving in cell death and survival pathways (Pathway Studio). Programmed cell death 4 (PDCD4) was chosen for further study based on weighted accumulative score++ (Targetscan7.0). To test whether miR‐486 can directly target PDCD4, luciferase assay was performed using a reporter vector containing PDCD4 3′‐UTR. 3′‐UTR of PTEN, a known target of miR‐486 was used as a control. When H9c2 cells transfected with the reporter vector were incubated with a range of miR‐486 mimic doses, luciferase activity was significantly reduced in a dose‐specific manner. Next, we determined whether transfection of H9c2 cells with miR‐486 mimic affected PDCD4 expression. The mimic transfection was efficient with a 200‐fold expression of miR‐486 mimic relative to the control. Whereas miR‐486 mimic transfection did not influence the expression of PDCD4 mRNA, PDCD4 protein was significantly suppressed (~60% downregulation, P<0.001 vs. control).

In H9c2 cardiomyocytes, in vitro experiments showed that treatment with miR‐486 mimic did not change cell survival in normoxic condition. However, treatment with 50 and 100 nM of mimic significantly reduced I/R mediated injury (increased cell viability (Hoechst‐PPI assay) and decreased lactate dehydrogenase leakage (P<0.01 vs. control) after simulated I/R (12h hypoxia with ischemia mimetic solution and 2h reperfusion). Moreover, TUNEL assay revealed miR‐486 mimic treatment significantly suppressed apoptosis. Western blotting analysis of cell lysates after simulated I/R showed that improved I/R outcome was associated with the inhibition of PDCD4 protein levels, and levels of its downstream effectors, cleaved caspase‐3 and Bax. In conclusion, these findings indicate that miR‐486‐5p can ameliorate I/R mediated damage through suppression of PDCD4 protein levels and inhibition of apoptosis in cardiomyocytes. Whether miR‐486a‐5p contributes to exosome‐mediated cardioprotection in vivo will be determined using our in vivo I/R model of coronary occlusion.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.