Atrial natriuretic peptide (ANP), which is encoded by the NPPA
gene, is a cardiac hormone that exhibits diuretic, natriuretic, and vasorelaxant activities (1
). In the PARADIGM-HF clinical trial, inhibition of neprilysin (an enzyme that degrades natriuretic peptides) in combination with renin-angiotensin-aldosterone system blockade reduced the number of hospitalizations for congestive heart failure and deaths from cardiovascular causes (2
). The PARADIGM-HF trial has rekindled interest in the natriuretic peptide system as a therapeutic target and highlights the therapeutic importance of identifying factors that may increase endogenous natriuretic peptide levels.
Intravenous administration of ANP lowers blood pressure and induces natriuresis in animal models and in patients with heart failure (3
). Transgenic mice overexpressing ANP are hypotensive (4
), while ANP-deficient mice are hypertensive (5
). However, the role of ANP in blood pressure regulation in the general population has remained uncertain until recent population genetic studies revealed that the minor allele of a common genetic variant (rs5068) in NPPA
is associated with increased plasma ANP levels, lower blood pressure, and reduced risk of hypertension (6
). These findings support a direct role of ANP in blood pressure regulation in humans and illustrate that modulating ANP levels could be a clinically relevant approach to treating hypertension or heart failure. The rs5068 single nucleotide polymorphism (SNP) is located in the 3′ untranslated region (3′ UTR) of NPPA
, where it disrupts the target binding site of a microRNA (miRNA), miR-425 (7
Binding of more than one miRNA can independently or coordinately modulate the expression of any given mRNA (8
). However, identifying bona fide miRNAs that regulate an mRNA of interest has been challenging. Although computational prediction algorithms are helpful in generating lists of potential miRNA candidates, the lists often contain a substantial number of false-positive miRNAs, which do not actually regulate the target mRNA (10
). Due to the poor predictive accuracy of these algorithms, it is difficult to discern which of the candidate miRNAs warrant further experimental validation.
In the current study, we sought to identify additional miRNAs that target the NPPA 3′ UTR. We used a sequential screening strategy that involved (i) in silico prediction of miRNA candidates, (ii) prioritization of candidates based on (a) miRNA expression data in human atrial tissues and/or (b) the presence of common genetic variants within the predicted miRNA binding site that are associated with circulating ANP levels in human population genetics studies, (iii) experimental validation of the predicted interaction with NPPA mRNA using luciferase reporter assays, and (iv) confirmation of the effect of the miRNA on NPPA mRNA levels and ANP protein levels in human cardiomyocytes.
MATERIALS AND METHODS
In silico analysis.
The microRNA Data Integration Portal (mirDIP) (11
) was used to generate a list of miRNAs that are predicted in silico
to interact with the NPPA
3′ UTR (NM_006172). The mirDIP (version 1.1.2) was run with the default settings.
COS-7 cells (American Type Culture Collection) were cultured in Dulbecco's modified Eagle's medium (Life Technologies) supplemented with 10% fetal calf serum, 2 mM l
-glutamine, 200 U/ml penicillin, and 200 μg/ml streptomycin (Fisher Scientific). Human cardiomyocytes were differentiated from human embryonic stem cells (hESCs) as follows: hESCs from the WA07 (H7) cell line were cultured on Matrigel-coated tissue culture polystyrene plates and maintained in mTeSR1 medium (Stem Cell Technologies). hESC medium was refreshed every 24 h, and hESCs were passaged using dispase (Sigma-Aldrich) at confluence. Cardiac differentiation of hESCs was induced using small molecules as previously described (12
). Briefly, when hESCs maintained on Matrigel plates achieved confluence, cells were treated with CHIR99021 (Stemgent) in RPMI medium (Life Technologies) supplemented with Gem21 NeuroPlex without insulin (Gemini Bio Products) for 24 h (from day 0 to day 1). The medium was replaced with RPMI medium-Gem21–insulin at day 1. The cells were then treated with IWP4 (Stemgent) in RPMI medium-Gem21–insulin at day 3, and the medium was refreshed on day 5 with RPMI medium-Gem21–insulin. Cells were maintained in RPMI supplemented with Gem21 NeuroPlex (Gemini Bio Products) starting from day 7, with the medium changed every 3 days. Beating clusters were seen starting from day 10 of differentiation. Cardiomyocytes were harvested 5 to 10 days after the onset of beating, typically from day 15 to 20 of differentiation. For dissociation, cardiomyocytes were treated with collagenase A and B (Roche) in RPMI medium-Gem21 for 15 min. Afterward, the collagenase solution was removed from the cardiomyocytes and replaced with 0.05% trypsin-EDTA (Life Technologies) for a 3-min treatment to obtain single cells. Trypsin was neutralized with RPMI medium-Gem21, and cardiomyocytes were centrifuged and resuspended in RPMI medium-Gem21 for plating. Expression of cardiac troponin T was confirmed in all experiments. Cardiomyocytes were confirmed by sequencing to be homozygous for the rs5068, rs61764044, and rs5067 major alleles.
miRNA mimics and inhibitors.
Chemically modified, double-stranded RNAs designed to mimic endogenous mature miRNAs, as well as a scrambled negative-control miRNA mimic, were purchased from Life Technologies. Throughout this work, miRNA is used to indicate miRNA mimic when exogenously administered. Chemically modified single-stranded RNA designed to inhibit endogenous mature miRNAs (anti-miRs) and a single-stranded scrambled negative-control anti-miRNA were also purchased from Life Technologies.
Generation of luciferase-NPPA 3′ UTR reporter constructs.
The NPPA 3′ UTR sequence (299 bp) including the major alleles of rs5068, rs5067, and rs61764044 was PCR amplified from a deidentified human HapMap genomic DNA sample obtained from Coriell Repositories (Coriell Institute for Medical Research). The PCR product was cloned into the pIS0 vector (Addgene) 3′ of the sequence encoding firefly luciferase to generate the wild-type luciferase construct (WT-Luc). In addition, mutated NPPA 3′ UTR luciferase constructs were created by site-directed mutagenesis using the QuikChange II XL site-directed mutagenesis kit (Stratagene) and synthetic oligonucleotides (Life Technologies) according to the manufacturers' protocols. The synthetic oligonucleotides used to generate the 155 Mut-Luc construct were 5′-CCTCGCCTCTCCCACCCCATCGTGGCAATTTTAAGGTAGAACCTC-3′ and 5′-GAGGTTCTACCTTAAAATTGCCACGATGGGGTGGGAGAGGCGAGG-3′. The synthetic oligonucleotides used to generate the rs61764044 Minor-Luc construct were 5′-GCCTCTCCCACCCCACGCATTAAATTTTAAG-3′ and 5′-CTTAAAATTTAATGCGTGGGGTGGGAGAGGC-3′. The synthetic oligonucleotides used to generate the rs5067 Minor-Luc construct were 5′-GGTCTCTGCTGCATTCGTGTCATCTTGTTGC-3′ and 5′-GCAACAAGATGACACGAATGCAGCAGAGACC-3′. The synthetic oligonucleotides used to generate the 103/107 Mut-Luc construct were 5′-CTCCTGTCCCCTGGGGTCTCAACGCGATTTGTGTCATCTTGTTGC-3′ and 5′-GCAACAAGATGACACAAATCGCGTTGAGACCCCAGGGGACAGGAG-3′.
Transient transfection of luciferase constructs and miRNAs or anti-miRs into heterologous cells.
COS-7 cells were transfected with wild-type or mutated luciferase-NPPA 3′ UTR constructs using X-tremeGENE HP DNA transfection reagent (Roche) according to the manufacturer's protocol. Twenty-four hours after transfection of the constructs, mirVana miRNA mimics (5 nM), scrambled negative-control miRNA mimic (5 nM), anti-miRNA inhibitors (100 nM), or scrambled negative-control anti-miR (100 nM) were transfected using Lipofectamine RNAiMax (Life Technologies). (For miRNA transfection using two miRNA mimics, the total amount of transfected miRNA was held constant under all experimental conditions. For the experimental 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-miRNA cotransfection, the total amount of transfected anti-miR was held constant under all experimental conditions. 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.) After an additional 48 h, firefly and renilla luciferase activities in cell extracts were measured using the Dual-Luciferase reporter assay system (Promega).
Transient transfection of miRNAs or anti-miRNAs into hESC-CMs.
hESC-derived cardiomyocytes (hESC-CMs) were transfected with either miRNA mimic (50 nM), scrambled negative-control miRNA mimic (50 nM), anti-miRNA (100 nM), or scrambled negative-control anti-miRNA (100 nM) using Lipofectamine RNAiMax. (For miRNA cotransfection, the total amount of transfected miRNA was held constant under all experimental conditions. For the experimental 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.) After 24 h, cells were washed and incubated in serum-free medium for an additional 24 h. NPPA gene expression was measured using quantitative reverse transcription-PCR (qRT-PCR), and ANP protein production and secretion were assessed using an enzyme-linked immunosorbent assay (ELISA) (proANP 1-98; Biomedica Medizinprodukte GmbH & Co KG) to detect N-terminal-proANP (Nt-proANP) levels in culture medium.
Transient transfection of NPPA cDNA expression plasmid and miR-105 or anti-miR-105 into heterologous cells.
A full-length human NPPA cDNA expression plasmid containing the rs5067 major allele (OriGene; catalogue no. SC122740) and a plasmid specifying renilla luciferase (as a transfection efficiency control) were transfected into COS-7 cells using X-tremeGENE HP DNA transfection reagent. Twenty-four hours after transfection of the constructs, miR-105 mimic (5 nM), scrambled negative-control miRNA mimic (5 nM), anti-miR-105 (100 nM), or scrambled negative-control anti-miRNA (100 nM) was transfected using Lipofectamine RNAiMax. Cells and culture medium were collected 24 h after miRNA or anti-miRNA transfection for measurement of renilla luciferase activity and secreted Nt-proANP levels by ELISA, respectively.
Measurement of mRNA and miRNA levels.
Total RNA was extracted from cultured cells using TRIzol (Life Technologies), and cDNA was synthesized using the high-capacity cDNA reverse transcription kit (Applied Biosystems) according to the manufacturer's protocol. NPPA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were measured by qRT-PCR using TaqMan assays (Applied Biosystems) in a 7500 Fast real-time PCR system (Life Technologies) according to the manufacturer's protocol. Relative changes in NPPA mRNA levels normalized to GAPDH mRNA levels were determined using the relative cycle threshold (CT) method. TaqMan miRNA reverse transcription and real-time assay kits (Applied Biosystems) were used to detect mature miRNAs and U6 small nuclear RNA (U6 snRNA). Relative changes in miRNA levels normalized to U6 snRNA levels were determined using the relative CT method.
Human population genetic analysis.
The midregional fragment of proANP (MR-proANP) was measured in plasma samples from the Malmö Diet and Cancer Study using an immunoluminometric assay (BRAHMS), as previously described (13
). Genotypes were generated using a genome-wide SNP microarray (Illumina Omni Express Exome; Illumina). MR-proANP showed right-skewed distributions and underwent natural logarithmic transformation (lnMR-proANP) before analysis. Genetic association was tested using linear regression with an additive genetic model adjusted for age and sex.
For all figures, data are presented as means ± standard errors of the means (SEMs) from 6 biological replicates (n = 6), and statistical significance was assessed by two-tailed independent t test and one-way analysis of variance (ANOVA) with Bonferroni post hoc testing, as appropriate. In all cases, a P value of <0.05 was considered statistically significant. t tests are reported without adjustments for multiple comparisons. Each experiment was repeated at least three times, with consistent results. Representative figures are presented in this work.
In this study, we used a multifaceted screening strategy that combined in silico
miRNA target prediction, miRNA expression data, human genetic association data, and cellular models to identify four miRNAs, in addition to miR-425, that target the NPPA
3′ UTR. Two of the four miRNAs (miR-155 and miR-105) were shown to have a functional role in modulating endogenous NPPA
mRNA levels and secreted Nt-proANP levels in human cardiomyocytes (Table 1
; see also Fig. S4 in the supplemental material). We observed that a genetic variant (rs61764044), which disrupts the miR-155 binding site, contributes to the ANP-raising and blood pressure-lowering effects of the previously published rs5068 variant (6
) by virtue of perfect linkage disequilibrium with rs5068. Similarly to miR-425 and miR-155, miR-105 interacts with a genetic variant (rs5067), specifically binding to the rs5067 major but not minor allele, consistent with the finding that the rs5067 minor allele is associated with increased plasma Nt-proANP levels in humans. Thus, by integrating computational, genomic, and cellular approaches, we have identified novel regulatory mechanisms controlling ANP levels and highlighted the potential of applying a similar integrated screening strategy to uncover miRNA regulators of other genes of interest.
analysis revealed that as many as 494 miRNAs are predicted to target the NPPA
3′ UTR. Because miRNA-mRNA interactions predicted by multiple algorithms appear to provide greater specificity without excessive loss of sensitivity (14
), miRNAs predicted to interact with the NPPA
3′ UTR by at least 3 of the algorithms were given priority. To further narrow down the list of miRNAs for subsequent experimental validation, we focused on miRNAs that are expressed at abundant levels in human atrial tissues (15
), where the target gene NPPA
is highly expressed. This strategy identified miR-155 as a novel miRNA that interacted with the NPPA
3′ UTR, as confirmed by luciferase reporter assays, and decreased NPPA
mRNA and secreted Nt-proANP protein levels in hESC-CMs. Although miR-103 and miR-107 were also identified as miRNAs that can target the NPPA
3′ UTR based on luciferase reporter assays, neither the miRNAs nor their anti-miRNAs altered NPPA
expression in hESC-CMs, the cell type of interest (Table 1
). Examination of the levels of miR-103 and miR-107 in COS-7 cells and hESC-CMs transfected with the respective miRNA mimics showed that compared to the endogenous miRNA levels in cells transfected with the negative control, there were similar levels of overexpression of the transfected miRNAs in COS-7 cells (see Fig. S5C and D in the supplemental material) and in hESC-CMs (see Fig. S6C and D in the supplemental material). (COS-7 cells and hESC-CMs transfected with miR-425, miR-155, and miR-105 also showed similar levels of overexpression of each of the miRNAs compared to cells transfected with the negative control [see Fig. S5 and S6].) As such, differences in the responses obtained for miR-103 and miR-107 in the COS-7 cells and hESC-CMs are most likely not due to differences in the levels of overexpression of these transfected miRNAs in the two cell types. A role for miR-103 and miR-107 in regulating endogenous NPPA
expression in the heart therefore remains unproven and requires further investigation. Others have also reported instances in which an miRNA was shown to target a 3′ UTR of interest in luciferase reporter assays but had no effects on endogenous target mRNA/protein levels in the cell type of interest (20
). It has been proposed that target secondary structure (21
) and the presence of RNA-binding proteins (23
), the levels of which may vary between different cell types, can influence the ability of miRNAs to regulate target mRNAs and interfere with the ability of miRNAs to directly interact with the 3′ UTR of target mRNAs.
In addition to prioritizing miRNAs that are highly expressed in human atria, we leveraged human population genetic data to investigate whether any of the candidate miRNAs were predicted to target variants in the NPPA
3′ UTR that are associated with plasma ANP levels in humans. This approach led to the identification of miR-105, which was validated to interact with rs5067 in an allele-dependent manner. The rs5067 minor allele disrupted base pairing between miR-105 and the NPPA
3′ UTR, which could underlie the observed association of the rs5067 minor allele with higher plasma Nt-proANP levels in humans. The cause of the failure of miR-105 to decrease NPPA
mRNA and protein levels in hESC-CMs is unclear and warrants further investigation, but a potential explanation is that the levels of miR-105 in cardiomyocytes, albeit low (Table 1
), may at endogenous levels already exert a maximal effect on the target NPPA
mRNA. It has been reported that the threshold for saturation of miRNA activity varies for different miRNAs, independent of the expression levels of the miRNA (25
). In this model, introducing more miR-105 into the hESC-CMs would not have any further effect, but inhibiting endogenous miR-105 would have an effect on NPPA
expression by relieving the repression mediated by endogenous miR-105. In COS-7 cells transfected with an NPPA
cDNA expression plasmid containing the rs5067 major allele, we observed that cotransfection of miR-105 reduced secreted Nt-proANP protein levels (see Fig. S7A in the supplemental material), while the opposite effect was obtained with cotransfection of anti-miR-105 (see Fig. S7B). These results suggest that the repressive effect of overexpressing miR-105 on ANP production can be revealed when the target NPPA
mRNA is also overexpressed, providing support for the possibility of saturation.
While miR-155 was predicted to target the NPPA
3′ UTR and was expressed in human atria, we discovered the rs61764044 variant in the miR-155 binding site only when additional sequencing identified the variant. The perfect correlation between rs5068 and rs61764044 was not known at the time of the prior studies of rs5068 (6
). The finding of an allele-specific negative regulatory effect of miR-155 in the current study indicates that rs61764044 contributes to the association between rs5068 and plasma ANP levels, as well as blood pressure. Furthermore, our results highlight the importance of having a complete catalogue of genetic variation. In this model, the presence of the coinherited minor alleles prevents both miR-425 and miR-155 from binding to the NPPA
3′ UTR, resulting in increased ANP production, lower systolic and diastolic blood pressures, and reduced risk of hypertension in humans. The rs61764044 minor allele creates a G-U wobble base pairing in the miR-155 seed binding site. Studies have shown that the presence of even a single G-U wobble in the seed binding region impairs miRNA-mediated repression of the mRNA target (16–18
). In the case of the FGF20
3′ UTR, for example, a similar disruption of a seed binding site was reported for the T allele of rs12720208, which introduces a G-U wobble base pairing in the miR-433 seed binding site and disrupts the ability of miR-433 to repress FGF20
The observation that cotransfection of miR-425 and miR-155 resulted in greater NPPA
gene repression in hESC-CMs than did transfection of either miRNA alone has implications for the therapeutic potential of using combinations of anti-miRNAs to increase ANP levels. In fact, coinheritance of the rs5068 and rs61764044 minor alleles in humans can be viewed as an experiment of nature that demonstrates the physiologic impact of these ANP-raising mechanisms on blood pressure and suggests therapeutic opportunities to increase ANP levels with potential benefits in hypertension or heart failure. Recent studies have implicated the therapeutic potential of miR-155 inhibition in suppressing cardiac hypertrophy and heart failure (28
). Moreover, miR-155 levels were also reported to be increased in human hearts with hypertrophy compared to nonhypertrophic controls and increased miR-155 levels were correlated with depressed cardiac function and increased wall thickness (29
). Our study is the first to report that miR-155 can target the NPPA
3′ UTR to directly regulate ANP levels in human cardiomyocytes. While miR-155 is a multifunctional miRNA with multiple targets (30
), its effects on human NPPA
expression could contribute to an effect on cardiac hypertrophy and heart failure.
From our multitiered screening strategy, we observed that of the 14 candidate miRNAs (including miR-425) that we tested in our NPPA
3′ UTR luciferase reporter assay, only 5 of them (miR-425, miR-155, miR-105, miR-103, and miR-107) were able to reduce the luciferase activity of the WT NPPA
3′ UTR luciferase reporter construct. Examination of the predicted binding sites of these 5 miRNAs revealed that the binding sites of 4 of these miRNAs (miR-425, miR-105, miR-103, and miR-107) are located close to each other in the NPPA
3′ UTR (see Fig. S1A in the supplemental material). As mRNA secondary structure has been reported to affect the ability of miRNAs to target mRNAs (21
), it is reasonable to speculate that this region of the NPPA
3′ UTR may contain secondary structure elements that increase the accessibility of these miRNA binding sites, thereby facilitating the interaction between these miRNAs and the NPPA
3′ UTR. Additional work investigating the secondary structure of the NPPA
mRNA will be required to confirm this possibility. Moreover, out of these 5 miRNAs that reduced the luciferase activity of the WT NPPA
3′ UTR luciferase reporter construct, we found that only 3 of them (miR-425, miR-155, and miR-105) were able to modulate ANP levels in hESC-CMs. A unique characteristic shared by these 3 miRNAs that is not seen for miR-103 and miR-107 is that miR-425, miR-155, and miR-105 all target sequences in the NPPA
3′ UTR that include genetic variants that are associated with plasma ANP levels in humans. Our findings suggest that in attempts to narrow down the list of candidate miRNAs obtained from computational prediction algorithms, it is worthwhile to consider prioritizing the miRNAs which target genetic variants that are associated with phenotypes of interest. Such an approach may help increase the likelihood that the miRNA will have biological relevance in regulating the endogenous levels of its target mRNA.
Several limitations of the present study merit consideration. We chose to experimentally validate a subset of predicted NPPA
-targeting miRNAs based on their expression levels in human atrial tissues. It remains likely that there are other miRNAs, expressed at lower levels in human atria, that can interact with the NPPA
3′ UTR and regulate ANP levels. We have not examined primary human cardiomyocytes because of technical challenges, including difficulties associated with obtaining human cardiac tissue, low cardiomyocyte yields from the isolation process, and inability to maintain the viability of isolated cardiomyocytes for an extended period of time (31
). However, hESC-CMs have been well characterized and exhibit structural and functional properties of native human cardiomyocytes (32
). Gene expression profiling studies have also shown that gene expression patterns of cardiac transcription factors and cardiac structural markers (including NPPA
) in hESC-CMs are consistent with gene expression patterns in human cardiac tissues (34–36
). Moreover, miRNA profiling has demonstrated that known cardiomyocyte-specific miRNAs are expressed in hESC-CMs and exhibit the expected expression pattern (34
). These observations suggest that hESC-CMs are a biologically relevant model for studying the effect of miRNAs on NPPA
mRNA levels and ANP production in the human heart.
In conclusion, we identified miR-155 and miR-105 as novel regulators of ANP production. These miRNAs target sequences including genetic variants that are associated with plasma ANP levels in humans. Our findings suggest the potential for miRNA-targeted therapies to increase ANP levels, which could supplement current therapy that reduces natriuretic peptide degradation. Further experiments in animals examining the in vivo effects of anti-miRNAs in derepressing ANP production (which lie beyond the scope of this study) would help to characterize phenotypic responses that will further expand our understanding of the clinical applications of such treatments.