Abstract 13479: Identification of microRNAs that Target mRNA Encoding Atrial Natriuretic Peptide

Abstract

Introduction: The minor allele of a common variant (rs5068) in the 3’UTR of NPPA, the gene encoding atrial natriuretic peptide (ANP), is associated with increased plasma ANP levels, lower blood pressure, and reduced risk of hypertension. A microRNA (miR-425) expressed in the human heart binds to a sequence spanning rs5068 for the major but not the minor allele leading to reduced ANP biosynthesis. mRNAs typically contain multiple miR-binding sites, and binding of different miRs often coordinately regulates the function of any given mRNA. Hypothesis: We hypothesize that miRs, in addition to miR-425, target NPPA mRNA and that interference with miR-based silencing of NPPA mRNA using miR antagonists (antimiRs) could be used to increase ANP biosynthesis. Methods: Luciferase reporter assays were used to screen 10 miRs that are predicted in silico to interact with the NPPA 3’UTR. The expression levels of validated miRs in human atria were determined. Human cardiomyocyte progenitor cells (hCMPCs) or human embryonic stem cell (HUES 9)-derived cardiomyocytes (hESC-CMs) were transfected with antimiRs targeting the validated miRs, and the levels of NPPA mRNA were measured. Results: Of the 10 miRs predicted in silico to interact with the NPPA 3’UTR, we identified 3 miRs (miRs-103, -105, and -107) that reduced luciferase activity in cells containing a luciferase-NPPA 3’UTR construct (miR-103, p=0.034; miR-105, p=0.0009; and miR-107, p=0.021), but the miRs had no effect on luciferase activity in cells containing a luciferase construct in which the predicted miR-binding site in the NPPA 3’UTR was mutated. Using qRT-PCR, the three miRs were detected in human atria. Transfection of an antimiR directed against miR-103, miR-105, or miR-107 increased NPPA mRNA levels in hCMPCs (antimiR-103, p=0.024; antimiR-105, p=0.039; and antimiR-107, p=0.036). Similar results were observed in hESC-CMs. Conclusions: Our studies suggest that miRs-103, -105, and -107 regulate NPPA expression in human cardiomyocytes. AntimiRs directed against these miRs increased NPPA mRNA in human cardiomyocytes. AntimiR-based methods to increase ANP levels may represent a novel approach to the treatment of hypertension.