Abstract 13117: Vacuolar Protein Sorting-Associated Protein 4a (VPS4a), Uncoupling Protein 2 (UCP2) and ADAM Metallopeptidase with Thrombospondin Type 1 motif (ADAMTS1) are Novel Targets of Mir-16 In End-Stage Heart Failure
We used a genetic approach to identify novel microRNA-mediated pathways in end-stage heart failure. An initial bioinformatics approach identified 1305 SNPs in 3'UTR miRNA Target Sites using dbSNP and the exome sequencing project, (ESP5500). The rationale was that the combined presence of a SNP, a miRNA binding site and miRNA that was regulated in the processes leading to heart failure may lead to the identification of new druggable pathways. From this list of 1305 SNPs, we carefully prioritized 126 SNPs based on miRNAs and pathways analyses. Initial genotyping using a Sequenom iPlex platform confirmed the SNPs in the miR-16 binding region of VPS4A (rs16958754, MAF=0.009), ADAMTS1 (rs12140, MAF=0.063), and UCP2 (rs59564714, MAF=0.001) in a cohort of end stage heart failure samples from the University of Minnesota and Royal Brompton and Harefield Trust (n=984). The second goal of this study was to test if VPS4A, ADAMTS1 and UCP2 were valid targets of miR-16. To validate VPS4A, ADAMTS1 and UCP2 as downstream targets of miR-16, experiments were performed in 293HEK cells. Direct interaction of miR-16 with VPS4A and UCP2 was assessed by Luciferase reporter assays. Luciferase activity was significantly decreased by 25% and 31% in cells transfected with VPS4A (n=5, p<0.01) and UCP2 (n=5, p<0.01) in the presence and absence of miR-16. Overexpression of miR-16 (mimic) also led to a significant decrease in VPS4A mRNA transcript (control 1.01 ± 0.1 vs VPS4A 0.42 ± 0.02*; n=3, p<0.01), in ADAMTS1 (control 1.01 ± 0.06 vs ADAMTS1 0.67 ± 0.01*; n=3, p<0.01) and UCP2 (control 1.06 ± 0.02 vs UCP2 0.78 ± 0.08; n=3, p=0.08) as assessed by qPCR 48h posttransfection. Further functional validation of these targets is in progress. In conclusion, using a combined bioinformatics and molecular approach we identified VPS4A, ADAMTS1 and UCP2 as novel targets of miR-16.
- © 2012 by American Heart Association, Inc.