Abstract 15663: MicroRNA-144/451 Cluster Confers Protection against Simulated Ischemia/Reperfusion-Induced Cardiomyocyte Death via Activation of COX-2
Background: Among the identified microRNAs (miRs) thus far, ∼50% of mammalian miRs are clustered in the genome and transcribed as polycistronic primary transcripts. However, whether clustered miRs mediate non-redundant and cooperative functions remains poorly understood.
Methods and Results: Using luciferase-reporter assay, we first identified the promoter of miR-144/451 cluster was activated by GATA-4, a critical transcription factor in the heart. This result was further confirmed in AdGATA-4-infected adult cardiomyocytes in which miR-144/451 single transcript was upregulated by 2-fold, mature miR-144 and miR-451 were increased by 1.9-fold and 2.5-fold, respectively, compared with AdGFP-cells. To gain insights into the functional role of the miR-144/451 cluster in cardiomyocytes, we generated six adenoviral vectors bearing primary miR-144, miR-451 and miR-144/451 in sense or antisense direction to overexpress or knockdown miR-144 and miR-451 individually or in combination. We observed that ectopic expression of miR-144 and -451 individually augmented cell survivals by ∼15%, which was further improved by ∼28% in Ad.miR-144/451-infected cardiomyocytes, compared to GFP-cells upon simulated 1-h ischemia/3-h reperfusion (I/R). Accordingly, DNA fragmentation and Caspase-3 activity were greatly reduced. In contrast, knockdown of endogenous miR-144 and -451 revealed opposite effects. Furthermore, using luciferase reporter assay and western-blot analysis, we validated that both miR-144 and miR-451 targeted CUG triplet repeat-binding protein 2 (CUGBP2), a ubiquitously expressed RNA-binding protein, known to interact with COX-2 3′-UTR and inhibit its translation. Accordingly, protein levels of CUGBP2 were greatly reduced and COX-2 activity was increased by 1.5-, 1.7- and 2.5-fold in miR-144-, miR-451- and miR-144/451-overexpressing myocytes, respectively, compared to GFP-cells. Moreover, inhibition of COX-2 activity by either NS-398 or DUP-697 partially offset protective effects of the miR-144/451 cluster.
Conclusions: Our findings indicate that both partners of the miR-144/451 cluster confer protection against simulated I/R-induced cardiomyocyte death via targeting CUGBP2-COX-2 pathway, at least in part.
- © 2010 by American Heart Association, Inc.