Novel MicroRNA Regulators of Atrial Natriuretic Peptide Production

Abstract

Atrial natriuretic peptide (ANP) has a central role in regulating blood pressure in humans. Recently, microRNA 425 (miR-425) was found to regulate ANP production by binding to the mRNA of NPPA, the gene encoding ANP. mRNAs typically contain multiple predicted microRNA (miRNA)-binding sites, and binding of different miRNAs may independently or coordinately regulate the expression of any given mRNA. We used a multifaceted screening strategy that integrates bioinformatics, next-generation sequencing data, human genetic association data, and cellular models to identify additional functional NPPA-targeting miRNAs. Two novel miRNAs, miR-155 and miR-105, were found to modulate ANP production in human cardiomyocytes and target genetic variants whose minor alleles are associated with higher human plasma ANP levels. Both miR-155 and miR-105 repressed NPPA mRNA in an allele-specific manner, with the minor allele of each respective variant conferring resistance to the miRNA either by disruption of miRNA base pairing or by creation of wobble base pairing. Moreover, miR-155 enhanced the repressive effects of miR-425 on ANP production in human cardiomyocytes. Our study combines computational, genomic, and cellular tools to identify novel miRNA regulators of ANP production that could be targeted to raise ANP levels, which may have applications for the treatment of hypertension or heart failure.

FIG 1

Schematic workflow for identification of NPPA-targeting miRNAs. In silico miRNA predictions were based on mirDIP. Evidence of expression in human atrial tissues was based on the work of Hsu et al. (15).

FIG 2

Four miRNAs predicted in silico to target the NPPA 3′ UTR reduce the luciferase activity produced by the firefly luciferase-NPPA 3′ UTR (WT-Luc) construct. The ratio of firefly luciferase activity to renilla luciferase activity was normalized to the ratio in COS-7 cells transfected with constructs encoding luciferase without the NPPA 3′ UTR and renilla. The effect of each miRNA was compared to that of a scrambled negative-control miRNA mimic (relative luciferase activity). miR-155 (A), miR-103 (B), miR-107 (C), and miR-105 (D) reduced the activity of the WT-Luc construct. Data are means ± SEMs (n = 6 biological replicates; two-tailed independent t test).

FIG 3

miR-155 interacts with the NPPA 3′ UTR in an rs61764044 allele-specific manner. (A) miR-155 reduced the activity of the WT-Luc construct but not the 155 Mut-Luc construct (containing a 6-nucleotide mutation at the predicted miR-155 seed binding site) or the rs61764044 Minor-Luc construct. The ratio of firefly luciferase activity to renilla luciferase activity was normalized to the ratio in COS-7 cells transfected with constructs encoding luciferase without the NPPA 3′ UTR and renilla. For each construct (WT-Luc, 155 Mut-Luc, or rs61764044 Minor-Luc), the effect of the miRNA was compared to that of a scrambled negative-control miRNA mimic (relative luciferase activity). (B) Anti-miR-155 increased the activity of the WT-Luc construct but not the 155 Mut-Luc construct or the rs61764044 Minor-Luc construct. The ratio of firefly luciferase activity to renilla luciferase activity was normalized to the ratio in COS-7 cells transfected with constructs encoding luciferase without the NPPA 3′ UTR and renilla. For each construct (WT-Luc, 155 Mut-Luc, or rs61764044 Minor-Luc), the effect of the anti-miR was compared to that of a scrambled negative-control anti-miRNA (relative luciferase activity). The rs61764044 Minor-Luc construct contains the minor alleles of both rs61764044 and rs5068. Data are means ± SEMs (n = 6 biological replicates; two-tailed independent t test).

FIG 4

miR-155 reduces and anti-miR-155 increases NPPA mRNA and secreted Nt-proANP protein levels in human cardiomyocytes. hESC-CMs (∼1 × 10⁵ per well) were transfected with miR-155, a scrambled negative-control miRNA mimic, anti-miR-155, or a scrambled negative-control anti-miRNA. Twenty-four hours later, cells were washed and incubated in 1 ml of medium. After an additional 24 h, cells and media were harvested. (A) NPPA mRNA levels in hESC-CMs transfected with miR-155 relative to cells transfected with negative-control miRNA mimic. (B) Nt-proANP protein levels (nanomoles/liter) in the medium of hESC-CMs transfected with miR-155 relative to negative-control miRNA mimic. (C) NPPA mRNA levels in hESC-CMs transfected with anti-miR-155 relative to negative-control anti-miRNA. (D) Nt-proANP protein levels in the medium of hESC-CMs transfected with anti-miR-155 relative to negative-control anti-miRNA. Data are means ± SEMs (n = 6 biological replicates; two-tailed independent t test).

FIG 5

miR-155 interacts with the rs61764044 major allele and acts together with miR-425 to further suppress NPPA mRNA and ANP protein levels in human cardiomyocytes. (A and B) Allele-specific effects of miR-425 plus miR-155 (A) and anti-miR-425 plus anti-miR-155 (B) on the luciferase activity produced by the WT-Luc and rs61764044 Minor-Luc constructs in COS-7 cells. The rs61764044 Minor-Luc construct contains the minor alleles of rs61764044 and rs5068. (C) NPPA mRNA levels in hESC-CMs transfected with the indicated miRNAs relative to scrambled negative-control miRNA mimic. (D) Nt-proANP protein levels in the medium of hESC-CMs transfected with the indicated miRNAs relative to scrambled negative-control miRNA mimic. For miR-425 plus miR-155 cotransfection, the total amount of transfected miRNA was held constant under all experimental conditions (10 nM total concentration in COS-7 cells, 100 nM total concentration in hESC-CMs). For the conditions labeled with “miR-425” or “miR-155,” the total concentration of transfected miRNA was held constant by the addition of the scrambled negative-control miRNA mimic. For anti-miR-425 plus anti-miR-155 cotransfection, the total amount of transfected anti-miRNA was held constant under all experimental conditions (200 nM total concentration in COS-7 cells). For the experimental conditions labeled with “anti-miR-425” or “anti-miR-155,” the total concentration of transfected miRNA was held constant by the addition of the scrambled negative-control anti-miRNA. Data are means ± SEMs (n = 6 biological replicates). P values are from a two-tailed independent t test. *, P < 0.001 versus miR-425, and #, P < 0.001 versus miR-155, by one-way ANOVA with Bonferroni post hoc testing.

FIG 6

miR-105 interacts with the NPPA 3′ UTR in an rs61764044 allele-specific manner. (A) miR-105 reduced the activity of the WT-Luc construct but not the rs5067 Minor-Luc construct. The ratio of firefly luciferase activity to renilla luciferase activity was normalized to the ratio in COS-7 cells transfected with constructs encoding luciferase without the NPPA 3′ UTR and renilla. For each construct (WT-Luc or rs5067 Minor-Luc), the effect of the miRNA was compared to that of a scrambled negative-control miRNA mimic (relative luciferase activity). (B) Anti-miR-105 increased the activity of the WT-Luc construct but not rs5067 Minor-Luc construct. The ratio of firefly luciferase activity to renilla luciferase activity was normalized to the ratio in COS-7 cells transfected with constructs encoding luciferase without the NPPA 3′ UTR and renilla. For each construct (WT-Luc or rs5067 Minor-Luc), the effect of the anti-miRNA was compared to that of a scrambled negative-control anti-miRNA (relative luciferase activity). Data are means ± SEMs (n = 6 biological replicates; two-tailed independent t test).

FIG 7

Anti-miR-105 increases NPPA mRNA and secreted Nt-proANP protein levels in human cardiomyocytes. hESC-CMs (∼1 × 10⁵ per well) were transfected with miR-105, a scrambled negative-control miRNA mimic, anti-miR-105, or a scrambled negative-control anti-miRNA. Twenty-four hours later, cells were washed and incubated in 1 ml of medium. After an additional 24 h, cells and medium were harvested. (A) NPPA mRNA levels in hESC-CMs transfected with miR-105 relative to negative-control miRNA mimic. (B) Nt-proANP protein levels in the medium of hESC-CMs transfected with miR-105 relative to negative-control miRNA mimic. (C) NPPA mRNA levels in hESC-CMs transfected with anti-miR-105 relative to negative-control anti-miRNA. (D) Nt-proANP protein levels in the medium of hESC-CMs transfected with anti-miR-105 relative to negative-control anti-miRNA. Data are means ± SEMs (n = 6 biological replicates; two-tailed independent t test).