Abstract 18229: Vacuolar Protein Sorting-Associated Protein 4A (VPS4A), a Target of miR-16, in Fibroblasts
Members of the miR-16 family are up-regulated during ischemia and heart failure. The goal of these studies was to identify novel miR-16 mediated signaling pathways that may play a role in cardiovascular disease. We used bioinformatics to identify single nucleotide polymorphisms (SNPs) in binding sites of miR-16 using dbSNP and the exome sequencing project. Target genes with a SNP in a miR-16 binding site included cingulin 1 (CGNL1), zinc finger protein with CRAB and SCAN domains 1 (ZKSCAN1), uncoupling protein2 (UCP2), vacuolar protein sorting- associated protein 4A (VPS4A), and a disintegrin and metalloproteinase with a thrombospondin motif 1 (ADAMTS-1). The role of VPS4A in the heart is not known. VPS4A mRNA expression was greater in primary rat cardiac fibroblasts (0.41±0.02) compared to primary rat cardiac myocytes (0.21± 0.03) (n=3-4, p=0.003). Up-regulation of VPS4A in HEK 293T cells decreased cell number by 30% and 41% at 48h and 72h, respectively, compared to cells transfected with a control scrambled mimic (n=6, p<0.01). Thus VPS4A was further studied for its direct interaction with miR-16. Overexpression of a miR-16 mimic decreased VPS4A protein expression by 38% and 52 % at 48h and 60h, respectively, compared to cells transfected with a control scrambled mimic (n=3, p<0.01). Conversely, inhibiting endogenous miR-16 with a miR-16 inhibitor mimic increased VPS4A protein 1.5 fold (n=3, p<0.01). miR-16 decreased luciferase activity of a VPS4A-3’UTR promoter construct; however the presence of rs16958754 in the miR-16 binding site inhibited the interaction (n=10, p<0.01). In sum, we identified VPS4A as a novel downstream target of miR-16 in fibroblasts. This data supports a working model in which upregulation of miR-16 with heart failure decreases VPS4A, and promotes fibroblast proliferation. Work is underway to determine if the SNP rs16958754 may be protective under certain conditions in which limiting fibroblast proliferation may be beneficial.
- © 2013 by American Heart Association, Inc.