The Human Angiotensin II Type 1 Receptor +1166 A/C Polymorphism Attenuates MicroRNA-155 Binding*

The adverse effects of angiotensin II (Ang II) are primarily mediated through the Ang II type 1 receptor (AT1R). A silent polymorphism (+1166 A/C) in the human AT1R gene has been associated with cardiovascular disease, possibly as a result of enhanced AT1R activity. Because this polymorphism occurs in the 3′-untranslated region of the human AT1R gene, the biological importance of this mutation has always been questionable. Computer alignment demonstrated that the +1166 A/C polymorphism occurred in a cis-regulatory site, which is recognized by a specific microRNA (miRNA), miR-155. miRNAs are noncoding RNAs that silence gene expression by base-pairing with complementary sequences in the 3′-untranslated region of target RNAs. When the +1166 C-allele is present, base-pairing complementarity is interrupted, and the ability of miR-155 to interact with the cis-regulatory site is decreased. As a result, miR-155 no longer attenuates translation as efficiently as demonstrated by luciferase reporter and Ang II radioreceptor binding assays. In situ hybridization experiments demonstrated that mature miR-155 is abundantly expressed in the same cell types as the AT1R (e.g. endothelial and vascular smooth muscle). Finally, when human primary vascular smooth muscle cells were transfected with an antisense miR-155 inhibitor, endogenous human AT1R expression and Ang II-induced ERK1/2 activation were significantly increased. Taken together, our study demonstrates that the AT1R and miR-155 are co-expressed and that miR-155 translationally represses the expression of AT1R in vivo. Therefore, our study provides the first feasible biochemical mechanism by which the +1166 A/C polymorphism can lead to increased AT1R densities and possibly cardiovascular disease.

fluid homeostasis, aldosterone production, renal function, and contraction of vascular smooth muscle (1). Apart from these hemodynamic actions, Ang II is also capable of inducing a multitude of nonhemodynamic effects, such as the induction of reactive oxygen species, cytokines, stimulation of collagen synthesis, apoptosis, and hypertrophy (2)(3)(4)(5).
Most of the biological responses to Ang II are mediated by its interaction with a G protein-coupled receptor defined as the type 1 Ang II receptor, or AT 1 R (1). AT 1 R activation triggers a variety of signaling pathways, including tyrosine kinase-mediated phosphorylation, production of arachidonic acid metabolites, alteration of reactive oxidant species, and fluxes in intracellular Ca 2ϩ concentrations (2)(3)(4)(5). Therefore, the nonhemodynamic functions of Ang II are thought to play a central role not only in the etiology of hypertension in humans but also in the pathophysiology of cardiac hypertrophy and remodeling, heart failure, vascular thickening, and atherosclerosis (6 -9).
Because the expression level of the AT 1 R defines the biological efficacy of Ang II, it is important to understand the mechanisms by which AT 1 R density is regulated. Recently, our laboratory demonstrated that microRNA-155 (miR-155) specifically interacted with the 3Ј-untranslated region (3Ј-UTR) of the human AT 1 R (hAT 1 R) mRNA and inhibited the expression of the hAT 1 R in fibroblasts (10). miRNAs are produced endogenously in mammalian cells by specific RNA gene transcription or from introns during pre-mRNA splicing (11)(12)(13)(14). miRNAs are expressed as long hairpin-forming precursor RNAs that get processed into partially double-stranded RNAs in which one strand is the mature miRNA (i.e. 21-23 nucleotides). The mature miRNA is taken up by a protein complex that is similar, if not identical, to the RNA-induced silencing complex that supports RNA interference, and the miRNAbound complex functions to regulate translation (15)(16)(17)(18)(19)(20). It has recently been demonstrated that mRNA targets sites with as few as 7 bp of complementarity (designated as the "seed" sequence) to the miRNA 5Ј end are sufficient to confer regulation in vivo (21,22).
The hAT 1 R gene has been found to be highly polymorphic (23). In particular, a single nucleotide polymorphism (SNP) has been described in which there is an A/C transversion at position 1166 in the 3Ј-UTR of this gene. The increased frequency of the hAT 1 R ϩ1166 C-allele has been associated with essential hypertension (24,25), cardiac hypertrophy (26), aortic stiffness (27), myocardial infarction (28), and increased oxidative stress levels in human heart failure (29). However, the physiological significance of this polymorphism is uncertain because of its location within the noncoding region of the hAT 1 R gene. Because we previously demonstrated that miR-155 interacted with a specific cis-response element localized in the hAT 1 R 3Ј-UTR (10), we investigated whether there was a correlation between the ϩ1166 A/C SNP and the miR-155-binding site. Computer alignment revealed that the ϩ1166 A/C SNP occurs in the cis-response element where miR-155 was shown to interact. Therefore, the aim of the following study was to investigate whether the presence of the ϩ1166 C-allele decreased the ability of miR-155 to translationally regulate the expression of the hAT 1 R. Importantly, we demonstrate that the A3 C bp change results in augmented hAT 1 R expression because of the inability of miR-155 to inhibit translation of hAT 1 R mRNAs as efficiently.

EXPERIMENTAL PROCEDURES
Cell Culture-CHO cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT), 80 units/ml penicillin, 80 g/ml of streptomycin, and 0.0175 mg/ml L-proline (Sigma). Primary dermal arteriolar vascular smooth muscle cells (VSMCs) were cultured from punch biopsies as reported previously (30). These cells were grown in Ham's F-12/Dulbecco's modified Eagle's medium (1:1) supplemented with 10% fetal bovine serum, L-glutamine, 80 units/ml penicillin, and 80 g/ml streptomycin (Invitrogen). The human VSMCs were genotyped as described previously (24) and shown to be AA at position ϩ1166. All cells were maintained in a humidified atmosphere of 5% CO 2 at 37°C.
Constructs-An 883-bp fragment encompassing the entire hAT 1 R 3Ј-UTR was PCR-amplified utilizing the following sense (5Ј-CATGTTCGAAACCTGTCCATAAAG-3Ј) and antisense (5Ј-ATAAAATTATTTTATTTTAAAGTAAAT-3Ј) primers using standard procedures and a proofreading polymerase (Platinum Pfu, Invitrogen). A full-length hAT 1 R cDNA clone (10) was used as template. The PCR product was subcloned into the pCR2.1 vector following the manufacturer's protocol (Invitrogen). Plasmid DNA was subsequently isolated from recombinant colonies and sequenced to ensure authenticity. The hAT 1 R 3Ј-UTR inserts were removed from the pCR2.1 plasmid by EcoRI digestion. The fragments were gel-purified, filled in, and blunt end-ligated into a filled in HindIII site that is located downstream of the firefly luciferase reporter gene (pMIR-REPORT TM , Ambion). The authenticity and orientation of the inserts relative to the luciferase gene were confirmed by sequencing. The resulting plasmid was designated p3Ј-UTR-A. The reporter plasmid, p3Ј-UTR-C, was generated by utilizing the p3Ј-UTR-A plasmid as template, and an A3 C mutation was generated at position 86 of the hAT 1 R 3Ј-UTR, which is equivalent to the ϩ1166 A3 C SNP (24) using the QuikChange site-directed mutagenesis kit (Stratagene). Briefly, a forward mutagenic primer (5Ј-CACTACCAAATGAGCCT-TAGCTACTTTTCAG-3Ј) and a complementary reverse mutagenic primer (5Ј-CTGAAAAGTAGCTAAGGCTCATT-TGGTAGT-3Ј) were synthesized and utilized in PCR experiments as described by the manufacturer. The site of the "C" mutation is shown in boldface. The amplification reaction was treated with DpnI restriction enzyme to eliminate the parental template, and the remaining DNA was used for transformation. The presence of the A3 C mutation (i.e. equivalent to the ϩ1166 A/C SNP) was confirmed by dideoxy chain termination sequencing. A full-length hAT 1 R cDNA which encompassed the entire open reading frame and the 3Ј-UTR was PCR-amplified (31), and the product was subcloned into the eukaryotic expression vector, pCR3.1 (Invitrogen), and sequenced to ensure authenticity and proper orientation with respect to the cytomegalovirus (CMV) immediate-early promoter. This expression construct was designated pCR/hAT 1 R-A. The A3 C SNP was generated in this construct as described above. The mutant construct was designated pCR/hAT 1 R-C. The A3 C SNP corresponds to the ϩ1166 A/C SNP described previously (24). Transformed bacterial cultures were grown, and each reporter construct was purified using the PureLink TM Hipure Plasmid Maxiprep kit (Invitrogen).
Hybridization Prediction-The minimum free energy hybridization of miR-155 and the hAT 1 R 3Ј-UTR cis-response element harboring either the A-allele or C-allele were predicted by RNAHYBRID software (35).
Transfection-Hsa-miR-155, a partially double-stranded RNA that mimics endogenous precursor miR-155, miRNA Negative Control 1 and miRNA Negative Control 2, which are random sequence controls, were obtained from Ambion. A "customer-defined" miRNA precursor molecule was synthesized (Ambion) that harbored a mutation in the miR-155 sequence and was designated mut-miR-155. Anti-miR-155, designed to inhibit endogenous miR-155 function, and anti-miR Negative Control 1 were also obtained from Ambion. Transfection of CHO cells with small RNAs was optimized utilizing Lipofectamine 2000 (Invitrogen) and a fluorescein-labeled double-stranded RNA oligomer designated BLOCK-iT TM fluorescent oligonucleotide (Invitrogen). Alternatively, VSMC transfection was optimized using the same fluorescent small RNA; however, magnet-assisted transfection was utilized as described by the manufacturer (IBA BioTAGnology, Gottingen, Germany; magnets were purchased separately from Engineered Concepts, Birmingham, AL). Once transfection conditions were optimized, CHO and/or VSMCs (approaching 100% transfection efficiency in both cell types) were transfected with the reporter or expression constructs described above and the appropriate miRNA precursor as indicated. After 48 h, cells were washed and lysed with Passive Lysis Buffer (Promega), and firefly luciferase and Renilla luciferase activities were determined using the dual-luciferase reporter assay system (Promega) and a luminometer. The relative reporter activity was obtained by normalization to the Renilla luciferase activity. In some experiments, transfected cells were utilized for real time PCR, radioreceptor binding assays, and/or immunoassays.
AT 1 Receptor Radioligand Binding Studies-Whole cell AT 1 receptor binding was measured as described previously (32). Briefly, 48 h after transfection, the cell medium was aspirated and replaced with monoiodinated [ 125 I-Sar 1 ,Ile 8 ]Ang II (2-3 ϫ 10 5 cpm; Peptide Radioiodination Service, Oxford, MS) in Hanks' balanced salt solution, 20 mM Hepes, 0.1% bovine serum albumin. After incubation at room temperature for 60 min, unbound ligand was removed by washing each well twice with 1 ml of ice-cold phosphate-buffered saline. Bound ligand was recovered by dissolving the protein in each well with 1 ml of 0.5 M NaOH, 0.01% SDS. Nonspecific binding was determined by performing the binding assay in the presence of 1 M unlabeled Ang II. The quantity of [ 125 I-Sar 1 ,Ile 8 ]Ang II present in each sample was determined using a Cobra ␥-spectrophotometer (Packard Bell, Palo Alto, CA). Protein content in wells was assessed using the Bio-Rad protein assay dye reagent (Bio-Rad). Values presented represent specific (total minus nonspecific) binding.
Real Time PCR-Total RNA was isolated from VSMCs and CHO cells using TRIzol (Invitrogen). The RNA was subsequently treated with RNase-free DNase I, and mature miR-155 was quantified utilizing the TaqMan microRNA assay kit for hsa-miR-155 (Applied Biosystems, Foster City, CA) as described previously (33). Briefly, 100 ng of total RNA was heated for 5 min at 80°C with 10 M of the 18 S rRNA antisense primer followed by cooling to room temperature. Three microliters of the 5ϫ RT primer was then added, and the cDNA was synthesized as described (34). Real time PCR (10 l total reaction) was performed as described using 1 l of a 1:50 dilution of cDNA. Gene expression was calculated relative to 18 S rRNA as described above and multiplied by 10 6 to simplify data presentation. Alternatively, total RNA was treated as described above; however, cDNA was synthesized using oligo(dT). The expression of hAT 1 R mRNA relative to 18 S rRNA was determined as described above. The hAT 1 R-specific primers used were as follows: sense primer, 5Ј-CACCATGTTTTGAGGTTGACTGAC-3Ј, and antisense primer, 5Ј-CAGGCTAGGGAGATTGCATTT-CTG-3Ј.
Immunoassay for ERK-Twenty four hours after transfection the VSMCs were washed and serum-starved for an additional 24 h. Serum-starved cells were stimulated with Ang II (1 M) for 5 min, washed with phosphate-buffered saline, and lysed with a concentrated buffer solution containing 250 mM Tris, pH 6.8, 8% SDS, 40% glycerol, 200 mM dithiothreitol, and 0.04% bromphenol blue (300 l/1 ϫ 10 6 cells). An aliquot of the supernatant was separated by 10% SDS-PAGE. Following transfer to nitrocellulose membrane and blocking with 5% nonfat milk, the blot was incubated with an antibody (1:2000) specific for phospho-ERK1/2 (Cell Signaling, Beverly, MA). The immunoblot was then incubated with a secondary antibody conjugated with horseradish peroxidase and visualized with ECL, and the autoradiograph was quantitated by densitometric analysis. The blots were subsequently stripped and reprobed with an ERK1/ 2-specific antibody (Cell Signaling) to normalize the level of phosphorylated ERK to total ERK.
In Situ Hybridization-Locked nucleic acid (LNA) probes complementary to human mature miR-155 (5Ј-CCCCTATC-ACGATTAGCATTAA-3Ј) and scrambled negative control (5Ј-TTCACAATGCGTTATCGGATGT-3Ј) digoxigenin-labeled at the 5Ј position were purchased from Exiqon (Vedbaek, Denmark). Detection of RNAs by in situ hybridization utilizing oligonucleotide probes was performed as described previously (50). Briefly, human tissues were deparaffinized, treated with protease (30 min in 2 mg/ml of pepsin), washed in sterile water, and subsequently washed with 100% ethanol and air-dried. Hybridization was performed at 37°C overnight followed by a low stringency wash in 0.2ϫ SSC and 2% bovine serum albumin at 4°C for 10 min. The probe-target complex was visualized utilizing a digoxigenin antibody conjugated to alkaline phosphatase acting on the chromogen nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate. Nuclear fast red served as the counterstain.
Statistical Analysis-All data are reported as means Ϯ S.E. When comparisons were made between two different groups, statistical significance was determined using Student's t test. When multiple comparisons were made, statistical significance was determined using one-way analysis of variance followed by Tukey's post-test. All statistical analysis was performed using the software package Prism 4.0b (GraphPad Software, San Diego).

The hAT 1 R ϩ1166 A/C SNP Occurs in the miR-155-binding
Site-The hAT 1 R ϩ1166 A/C SNP has been associated with cardiovascular disease (23)(24)(25)(26)(27)(28)(29). However, the association of this polymorphism with cardiovascular disease is difficult to interpret because it is not known how a genetic variation in the noncoding region results in differences in hAT 1 R expression. As a result of our demonstration that miR-155 can interact with a specific cis-response element localized in the hAT 1 R 3Ј-UTR and inhibit the expression of this receptor (10), we investigated whether there was a relationship between the ϩ1166 A/C SNP and the miR-155-binding site. Computer alignment demonstrated that this polymorphism occurs in the middle of the miR-155 complementarity seed binding sequence (Fig. 1, A and B).
To determine whether the thermodynamics of binding between miR-155 and the hAT 1 R 3Ј-UTR cis-response element harboring either the A-or C-allele was distinct, the minimal free energy of hybridization was predicted (35). Computational modeling suggests that the presence of the C-allele leads to a decrease in free energy (Fig. 1, C and D), which would result in a destabilized miR-155/hAT 1 R 3Ј-UTR hybrid. Therefore, we hypothesize that when the ϩ1166 C-allele is expressed, hAT 1 R mRNAs would not fulfill the seed sequence target requirement, which in turn would result in augmented hAT 1 R expression levels because of the decreased binding of miR-155 with the hAT 1 R 3Ј-UTR cis-response element.
The hAT 1 R ϩ1166 C-allele Decreases Ability of miR-155 to Regulate Translation-To begin to test this hypothesis, the entire hAT 1 R 3Ј-UTR (i.e. 883-bp region) was subcloned immediately downstream from the firefly luciferase open reading frame, and the resulting construct was designated p3Ј-UTR-A ( Fig. 2A). This construct harbors the hAT 1 R ϩ1166 A-allele. In contrast, the mutant p3Ј-UTR-C construct expresses the hAT 1 R ϩ1166 C-allele ( Fig. 2A). To test the potency of miR-155 in regulating luciferase expression, either p3Ј-UTR-A or p3Ј-UTR-C was transfected into CHO cells with increasing concentrations of miR-155 or control nontargeting miRNAs (i.e. Con 1 or Con 2), and luciferase activities were determined.

hAT 1 R Polymorphisms and MicroRNA-155
CHO cells were utilized because they do not express miR-155, 3 and therefore, endogenous expression of this miRNA did not mask the effects of the transfected miR-155. Dose-response experiments demonstrated that relative luciferase activity was significantly decreased with as little as 1 nM of miR-155 in CHO cells co-transfected with the p3Ј-UTR-A construct (Fig. 2B). A maximal decrease was obtained in p3Ј-UTR-A-transfected cells with a concentration of 30 -50 nM miR-155. In contrast, the potency of miR-155 was dramatically reduced in CHO cells transfected with the p3Ј-UTR-C construct (Fig. 2B). For example, firefly luciferase activity of cells transfected with the p3Ј-UTR-C construct was not significantly reduced until co-transfected with 30 nM miR-155. Additionally, transfection of 50 nM miR-155 resulted in a maximal decrease of luciferase activity by only ϳ20% (Fig. 2B).
To investigate the importance of the SNP occurring in the middle of the miRNA and the hAT 1 R mRNA target seed complementarity sequence, a mutant miR-155 precursor was synthesized which compensated for the hAT 1 R ϩ1166 C-allele by placing a complementary "G" residue in the miRNA sequence (Fig. 3A). However, by generating the mutant miR-155, a base pair mismatch was created within the hAT 1 R ϩ1166 A-allele and the seed sequence of the mutant miR-155 (Fig. 3A). Identical transfection experiments were performed as described above; however, in these experiments, the mutant miR-155 was utilized (Fig. 3B). In contrast to the results obtained utilizing miR-155, mutant miR-155 demonstrated an increased potency at inhibiting relative luciferase activity in CHO cells transfected with the p3Ј-UTR-C construct and decreased for cells transfected with the p3Ј-UTR-A construct. Taken together, these results demonstrate that at least 7 bp of contiguous complementarity is needed between miR-155 and its hAT 1 R 3Ј-UTR target site to maximally inhibit luciferase activity and that a base pair mismatch within the seed sequence diminishes the ability of miR-155 to inhibit translation.
To validate the above results, expression constructs, which result in the synthesis of hAT 1 R mRNAs that harbor either the ϩ1166 A-allele (i.e. phAT 1 R-A) or the ϩ1166 C-allele (i.e. phAT 1 R-C) (Fig. 4A), were co-transfected with 50 nM miR-Con, miR-155, or mutant miR-155, and hAT 1 R levels were quantitated by performing radioreceptor binding assays. Importantly, only miR-155 significantly inhibited the expression of the hAT 1 R synthesized in CHO cells transfected with the 3 T. S. Elton, unpublished observations.

FIGURE 1. The hAT 1 R ؉1166 A/C SNP occurs in the miR-155-binding site.
A, complementarity between miR-155 and the hAT 1 R 3Ј-UTR site targeted (70 -90 bp downstream from the hAT 1 R stop codon). The ϩ1166 A/C SNP corresponds to the nucleotide 86 bp downstream from the hAT 1 R stop codon (in boldface). The binding of miR-155 to the hAT 1 R 3Ј-UTR target site fulfills the requirement of a 7-bp seed sequence of complementarity at the miRNA 5Ј end (21,22) when the ϩ1166 A-allele is expressed. B, complementarity between miR-155 and the hAT 1 R 3Ј-UTR harboring the ϩ1166 C-allele. If the ϩ1166 C-allele is expressed then the seed sequence requirement would not be met. C, computational modeling of the interaction between miR-155 and the hAT 1 R 3Ј-UTR cis-response element ϩ1166 A/C SNP was performed utilizing the RNAHYBRID software (35). Hybridization of miR-155 and hAT 1 R mRNA harboring the A-allele. D, hybridization of miR-155 and hAT 1 R mRNA harboring the C-allele. Arrows highlight the polymorphic site. Calculated mfe, minimal free energy, is shown.

FIGURE 2. The hAT 1 R ؉1166 A/C SNP decreases miR-155 effectiveness.
A, schematic representation of the firefly luciferase (f-luc) reporter constructs utilized. The p3Ј-UTR-A plasmid harbors the full-length hAT 1 R 3Ј-UTR (i.e. 883 bp) in the 5Ј33Ј orientation with respect to the firefly luciferase open reading frame. The "A" represents the A-allele at position ϩ1166. The p3Ј-UTR-C plasmid also contains the hAT 1 R 3Ј-UTR however this plasmid harbors a point mutation to represent the ϩ1166 C-allele. B, CHO cells were co-transfected with p3Ј-UTR-A or p3Ј-UTR-C, pRL-CMV, and either miR-Controls or miR-155 at the concentrations indicated. Forty eight hours after transfection, luciferase activities were measured. Firefly luciferase activity was normalized to Renilla luciferase expression, and mean activities Ϯ S.E. from five independent experiments are shown (*, p Ͻ 0.01 versus CHO cells transfected with p3Ј-UTR-C at each concentration shown). phAT 1 R-A plasmid (Fig. 4, B and C). In contrast, the mutant miR-155 could only inhibit hAT 1 R synthesis in CHO cells transfected with phAT 1 R-C expression construct. To investigate whether transfection of miR-155 and/or mutant miR-155 resulted in the degradation of hAT 1 R mRNAs, real time PCR experiments were performed. The results of these experiments demonstrated that these miRNAs did not decrease hAT 1 R protein levels by mediating increased mRNA degradation (Fig. 4, D  and E). Taken together these results again support the hypothesis that if miR-155 is co-expressed with hAT 1 R mRNAs, which harbor the ϩ1166 C-allele, enhanced hAT 1 R protein synthesis may result.
Mir-155 Regulates hAT 1 R Expression in Primary Human VSMCs-It is well accepted that AT 1 Rs are expressed on endothelial cells, VSMCs, fibroblasts, and monocytes/macrophages (1,3). To investigate whether mature miR-155 was also expressed in these same cell types, in situ hybridization experiments were performed utilizing several paraffin-embedded, formalin-fixed human tissues from the lung, placenta, and cervix. These tissues were used for analysis because the former two tissues were known to contain many Ang II-sensitive cells, whereas few such cells should be present in the cervix. In situ hybridization experiments demonstrated that the smooth muscle layer of the bronchiole wall as well as the epithelium that lines the lumen and smooth muscle and endothelium of the arterioles in the lung express miR-155 (Fig. 5, A, C and D). Additionally, miR-155 expression was also evident diffusely in the placental endothelium (Fig. 5F). The mature miR-155 was localized in the cytoplasm and tended to concentrate around the nuclear membrane (Fig. 5C). MiR-155 expression was not evident in the cervical tissue (data not shown). Finally, the hybridization signal was not evident in tissue samples hybridized with a scrambled control probe (Fig. 5, B and E).
Because the AT 1 R and miR-155 are co-expressed in VSMCs, we next investigated whether the endogenously expressed miR-155 regulated hAT 1 R expression. Therefore, primary human VSMCs were transfected with an antisense RNA oligonucleotide complementary to miR-155 (i.e. anti-miR-155 at 20 nM final concentration) or a scrambled antisense control (i.e. anti-miR-Control at 20 nM final concentration), and the transfected cells were then subjected to radioreceptor binding assays. This concentration of miRNA was utilized because this value was within the physiological range of mature miR-155 expressed in primary human VSMCs (data not shown). Importantly, VSMCs transfected with anti-miR-155 demonstrated increased hAT 1 R levels (42 Ϯ 3%, p Ͻ 0.01) (Fig. 6A). To investigate whether the anti-miR-155-mediated increase in hAT 1 R density also resulted in enhanced Ang II-induced signal transduction, VSMCs were transfected as described above and subsequently activated with 0.1 M Ang II for 5 min, and phospho-ERK1/2 levels were determined (Fig. 6B). These experiments demonstrated that transfection of anti-miR-155 not only augmented hAT 1 R expression but also enhanced Ang II-induced signaling via the hAT 1 R (33.5 Ϯ 4.2%, p Ͻ 0.01). These results suggest that miR-155 expression can decrease hAT 1 R density and, therefore, decrease the biological efficacy of Ang II.

DISCUSSION
In this study, we have analyzed the potential role of the hAT 1 R ϩ1166 A/C SNP in modulating the expression of AT 1 Rs. Because this polymorphism occurs in the 3Ј-UTR of the hAT 1 R gene. the biological ramifications of this mutation have always been questionable. However, with the recent discovery of miRNAs that post-transcriptionally repress mRNA through cis-regulatory sites in 3Ј-UTRs (15)(16)(17)(18)(19)(20), polymorphisms in this region, which occur in predicted miRNA-binding sites, may be deleterious (36). In support of this conclusion, naturally occurring polymorphisms in miRNA-binding sites have been implicated in Tourette syndrome (37), papillary thyroid cancer (38), and muscularity in sheep (39). In the three examples cited, these polymorphisms either created a new target site for a miRNA or led to improved thermodynamics of binding between the miRNA and the mRNA target (i.e. increase in free energy), which in turn resulted in decreased protein expression levels because of the decreased translatability of each mRNA target described.
Systematic target site mutation experiments demonstrated that at least two classes of miRNA targets exist (20 -22). One class of target sites shows perfect Watson-Crick complementarity for at least 7 bp (i.e. seed site) starting at the 1st or 2nd bp of the 5Ј end of the miRNA. Furthermore, this class of miRNA does not require significant base pairing to the miRNA 3Ј end to FIGURE 3. A mutation in miR-155 can restore its ability to inhibit the hAT 1 R ؉1166 C-allele. A, schematic representation of the location of the mutation introduced in miR-155 when it was chemically synthesized. This mutation eliminates a complementary bp in the miR-155 seed sequence when the hAT 1 R ϩ1166 A-allele is present. The mutation in mut-miR-155 restores the complementary bp in the miR-155 seed sequence when the hAT 1 R ϩ1166 C-allele is present. B, CHO cells were co-transfected with p3Ј-UTR-A or p3Ј-UTR-C, pRL-CMV, and either miR-Controls or mutant miR-155 at the concentrations indicated. Forty eight hours after transfection, luciferase activities were measured. Firefly luciferase activity was normalized to Renilla luciferase expression, and mean activities Ϯ S.E. from five independent experiments are shown (*, p Ͻ 0.01 versus CHO cells transfected with p3Ј-UTR-A at each concentration shown).

hAT 1 R Polymorphisms and MicroRNA-155
be functional. Two independent experimental approaches have suggested that ϳ30% of all human 3Ј-UTRs may be regulated via this class of miRNAs (40,41). In contrast, the second class of miRNAs has imperfect 5Ј matches but compensates via substantial base pairings in the 3Ј end of the miRNA (22).
Computer alignment of the hAT 1 R 3Ј-UTR sequence with the miR-155 sequence demonstrated that the ϩ1166 A/C SNP occurs in the miR-155 cis-regulatory binding site (Fig. 1, A and  B). The interaction between miR-155 and the hAT 1 R 3Ј-UTR harboring the A-allele would fit the first miRNA classification because there is a 7-bp region of complementarity between the 5Ј end of miR-155 and the hAT 1 R mRNA target site. In contrast, if an hAT 1 R mRNA that harbors the ϩ1166 C-allele is expressed, the complementary seed site is interrupted, and the thermodynamics of the miRNA:mRNA duplex would be significantly altered (i.e. a decrease in free energy) (Fig. 1, C and D). Thus, unlike the three examples described above (36 -38), the presence of the ϩ1166 C-allele would decrease the ability of miR-155 to interact with the cis-regulatory site located in the hAT 1 R 3Ј-UTR. As a consequence, it would be expected that aberrantly high levels of the hAT 1 R would be synthesized.
In support of this hypothesis we demonstrated that when the hAT 1 R cis-response element harboring the C-allele was present in luciferase mRNAs, the ability of miR-155 to inhibit luciferase activity was significantly attenuated (Fig. 2B). When identical experiments were performed utilizing mutant miR-155, which restored perfect Watson-Crick complementarity, luciferase activity was decreased to levels ( Fig.  3B) that were comparable with experiments utilizing the hAT 1 R cis-response element harboring the A-allele and miR-155 (Fig. 2B). To further demonstrate that the presence of the ϩ1166 C-allele can influence hAT 1 R density, expression constructs that produced hAT 1 R mRNAs containing either the A-or C-allele were co-transfected with miR-155 or mut-miR-155. These experiments again demonstrated that when seed sequence complementarity was not fulfilled, regardless of whether miR-155 or mutant miR-155 was utilized, hAT 1 R levels were always higher than the levels obtained when perfect complementarity was present between the miRNA and the hAT 1 R cis-response element (Fig. 4, B and C). Taken together, these results suggest that the presence of the ϩ1166 C-allele would lead to higher levels of hAT 1 R if miR-155 were co-expressed with hAT 1 R mRNAs.
Because it is well accepted that human endothelial cells and VSMCs express the AT 1 R (1, 3), we utilized in situ hybridization experiments to demonstrate that miR-155 was also expressed in these cell types in vivo (Fig. 5). Importantly, this is the first demonstration that miR-155 and the AT 1 R are coexpressed in VSMCs and endothelial cells. . Mir-155 seed sequence complementarity is needed to inhibit hAT 1 R expression. A, schematic representation of the hAT 1 R expression constructs utilized in transfection experiments. The phAT 1 R-A plasmid harbors a full-length hAT 1 R cDNA in the 5Ј 3 3Ј orientation with respect to the CMV promoter. The "A" represents the A-allele at position ϩ1166. The phAT 1 R-C plasmid also contains the full-length hAT 1 R cDNA; however, this plasmid harbors a point mutation to represent the ϩ1166 C-allele. B, CHO cells were co-transfected with phAT 1 R-A, pRL-CMV, and either miR-Controls, miR-155, or mutant miR-155 at 50 nM final concentration. C, alternatively, identical experiments were performed utilizing the phAT 1 R-C expression construct. Forty eight hours after transfection, luciferase activities were measured. Firefly luciferase activity was normalized to Renilla luciferase expression, and mean activities Ϯ S.E. from five independent experiments are shown. B, *, p Ͻ 0.01 versus mut-miR-155; C, *, p Ͻ 0.01 versus miR-155. Identical experiments were performed as described above; however, total RNA was isolated from transfected CHO cells and subjected to real time PCR experiments to quantitate the expression of hAT 1 R mRNA in cells transfected with either phAT 1 R-A (D) or phAT 1 R-C expression construct (E).
To validate that the levels of miR-155 were physiologically relevant in VSMCs, we demonstrated that miR-155 "knockdown" experiments resulted in augmented hAT 1 R levels and enhanced Ang II-induced signaling (Fig. 6). Taken together, these results suggest that, dependent upon the cell type, miR-155 translational repression of hAT 1 R expression may be more or less pronounced contingent upon the relative expression levels of both miR-155 and hAT 1 R mRNAs and whether the genotype of a given individual is AA, AC, or CC at position ϩ1166.
The increased frequency of the hAT 1 R ϩ1166 C-allele has been associated with essential hypertension (24,25), cardiac hypertrophy (26), aortic stiffness (27), myocardial infarction (28), and increased oxidative stress levels in human heart failure (29). Additionally, it has been demonstrated that patients carrying the hAT 1 R CC genotype had enhanced vascular reactivity (42)(43)(44) and increased renovascular sensitivity to Ang II (45,46). Although others have been unable to confirm these findings (reviewed in Ref. 22), divergent results in many of these studies may be attributable to differences in age, sex, ethnic origin, or various nongenomic and environmental influences on the pathway between the hAT 1 R SNP and physiology.
It has been hypothesized that the association of the C-allele with cardiovascular disease can be accounted for if AC and/or CC subjects have increased AT 1 R activity. To begin to investigate this hypothesis, several investigators (46,47) have measured hAT 1 R-binding sites (B max ) and Ang II affinity (K d ) on platelets isolated from patients with the AA and CC genotypes. Neither study showed any difference in hAT 1 R number or affinity. However, with the low numbers of hAT 1 Rs per platelet (ϳ4 -6) (48,49), and because of the fact that platelets do not express detectable levels of mature miR-155, 3 it is not surprising that these investigators did not observe any differences in hAT 1 R number. Unfortunately, no studies have been published FIGURE 5. In situ detection of miR-155 in paraffin-embedded, formalin-fixed human tissues. A, representative example of the distribution of miR-155 after in situ hybridization analysis with an LNA miR-155-specific probe in a section of unremarkable human lung. Note that signal (blue due to the action of the alkaline phosphatase conjugate on the chromogen nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate) is evident in both the smooth muscle layer of the bronchiole wall (large arrow) as well as the epithelium that lines the lumen (small arrow). The signal was not evident in the serial section (4 m away) when the scrambled LNA miRNA probe was utilized (B). C, high magnification near the bronchiole lumen where the signal in the epithelium (small arrow) and smooth muscle layer (large arrow) can be seen. Note that the signal is present in the cytoplasm and tends to concentrate around the nuclear membrane. D, arterioles in the lung also demonstrated signal (small arrow, endothelium; large arrow, smooth muscle cells). No signal was evident in the serial section when the scrambled probe was used (E). F, miR-155 detection was evident in an unremarkable human placenta in the endothelium; note the cytoplasmic localization and concentration around the nuclear membrane (arrow). FIGURE 6. Anti-miR-155 enhances hAT 1 R expression and Ang II-induced signaling in VSMCs. VSMCs were transfected with the anti-miRNA oligonucleotides as indicated; 48 h after transfection, the cells were utilized as follows. A, AT 1 R radioreceptor binding assays were performed as described under "Experimental Procedures." The data have been normalized for protein and transfection differences, and data represent specific binding. The values are shown as percent of maximal specific binding of mock-transfected VSMCs and represent the mean Ϯ S.E. from four independent transfection experiments (*, p Ͻ 0.01 versus mock-transfected cells). B, Ang II-induced phospho-ERK1/2 experiments were performed utilizing serum-starved, transiently transfected cells as described under "Experimental Procedures." A representative immunoblot is shown. Results are representative of four independent experiments. C, quantitation of Ang II-(1 M for 5 min) induced ERK1/2 phosphorylation was determined by densitometry. Values are expressed as a percent of the maximal phosphorylation of ERK1/2 in response to Ang II in mocktransfected cells and represent the mean Ϯ S.E. from four independent transfection experiments (*, p Ͻ 0.01).
investigating hAT 1 R density on blood vessels isolated from patients with the AA, AC, or CC genotypes. Therefore, future studies in our laboratory will examine the potential correlation between the hAT 1 R ϩ1166 genotype, receptor number, and miR-155 expression levels.
In conclusion, we demonstrate that the hAT 1 R ϩ1166 SNP occurs in a cis-regulatory element that is recognized by miR-155. When the ϩ1166 C-allele is expressed, base-pairing complementarity between miR-155 and the hAT 1 R mRNA is reduced. As a consequence, miR-155 can no longer attenuate translation as efficiently and hAT 1 R density increases. Taken together, our study provides the first feasible biochemical mechanism by which the ϩ1166 A/C polymorphism can lead to increased AT 1 R densities and possibly cardiovascular disease.