Beneficial Effect of miR-25 Decoy Overexpression in a Murine Model of Heart Failure

Abstract

Introduction: Recently, our group found that miR-25 is a key microRNA that regulates SERCA2a and we showed anti-miR25 treatment enhanced cardiac contractility and function through SERCA2a restoration in murine heart failure model. However, the strong and stable suppression of specific microRNA activity would be essential to evaluate the therapeutic potential of such an approach. In this study, we constructed a miR-25 decoy t using TuD RNA (tough decoy RNAs) and miR-25 decoy activity was evaluated in various settings.

Methods: First, miR-25 decoy construct was applied to both isolated adult cardiomyocytes and H9C2 cardiomyoblasts. A pMirTarget vector containing SERCA2a 3’-UTR under luciferase was used to evaluate this decoy specificity. Second, we generated pressure-overload heart failure models in mouse by TAC operation. In the HF mice, AAV9-GFP (control) and AAV9-miR-25 decoy were delivered by tail vein injection. Two months following gene transfer, cardiac function was evaluated by echocardiography and invasive hemodynamics. Protein and RNA expression levels of SERCA2a and miR-25 expression level were confirmed by qRT-PCR analysis.

Results: First, we observed that we were able to achieve about 2-3 fold increase of SERCA2a expression by miR-25 decoy transfection in both H9C2 and in isolated adult cardiomyocytes. Also, similar expression pattern was confirmed in the heart of miR-25 decoy injected normal mice. Second, the HF model by TAC surgery was confirmed with echocardiography. Overall average FS (%) in HF was 37.77+/- 8.75 (n=10) and in non-surgery control mice was 62.51 +/- 3.42(n=4). After AAV9 injection, cardiac function of AAV9-miR-25 decoy injected mice was enhanced but AAV9-GFP injected mice showed severe cardiac dysfunction and dilation (AAV-GFP (n=6) vs AAV-miR-25 decoy (n=4)). Third, western blot analysis showed that SERCA2a expression was significantly restored in miR-25 decoy injected mice. In addition, we confirmed that miR-25 expression level was kept low by qRT-PCR analysis. Taken together, our data would indicate that using miR-25 decoy is an effective strategy for the long-term suppression of miR-25 and it may be a promising therapeutic target to restore SERCA2a and reverse the HF phenotype.