Abstract 11394: MicroRNA-128 Regulates Isl-1 Via Nkx-2.5/gsh-2 Competition During Cardiac Development
Background and Objective: Isl-1 plays a central role in cardiac development. However, the precise mechanism(s) of its regulation are unknown. We examined transcriptional/posttranscriptional mechanisms involved in regulating Isl-1 in embryonic hearts.
Methods and Results: Our data revealed Isl-1 expression was significantly higher in embryonic heart than that in adult heart. Bioinformatics predicted the 3’UTR of Isl-1 harbors conserved miR-128 target sites. Q-PCR showed miR-128 was inversely correlated with Isl-1 expression in embryonic and adult heart suggesting its role in regulation of Isl-1 expression. Luciferase assay confirmed miR-128 targeted Isl-1 by direct binding on its 3’UTR. Inhibition of miR-128 increased Isl-1 protein in cardiomyocytes, whereas its overexpression decreased Isl-1 levels. Therefore, Isl-1 is confirmed as a target of miR-128. Additionally, deletion analysis of the 5'-flanking sequence of miR-128 genes (miR-128-1, miR-128-2) revealed the major responsive elements responsible for miR-128-1 and miR-128-2 were highly conserved Nkx-2.5/Gsh-2-binding motifs. Deletion of Nkx-2.5/Gsh-2-binding motifs completely abolished promoter activation. Furthermore, EMSA showed Nkx-2.5/Gsh-2 recognized these motifs and ChIP verified the in vivo binding of Nkx-2.5/Gsh-2 on these motifs. Luciferase assay showed overexpression of Gsh-2 significantly increased miR-128 promoter activity while co-transfection of Nkx-2.5 dramatically decreased promoter activity by replacing Gsh-2 via competing with Gsh-2 for the same motif (Fig. 1).
Conclusions: miR-128-targeted Isl-1 activated its downstream target Nkx-2.5, establishing a negative feedback loop by competing with Gsh-2 at the binding motif to repress miR-128 expression in embryonic heart.
- © 2012 by American Heart Association, Inc.