MicroRNA-155 Regulates Human Angiotensin II Type 1 Receptor Expression in Fibroblasts*

A large number of studies have demonstrated that the expression of the angiotensin II type 1 receptor (AT1R) is regulated predominantly by post-transcriptional mechanisms. Recently, it has been suggested that 10% of human genes may be regulated, in part, by a novel post-transcriptional mechanism involving microRNAs (miRNAs). miRNAs are small RNAs that regulate gene expression primarily through translational repression. The aim of this study was to determine whether miRNAs could regulate human AT1R expression. Luciferase reporter assays demonstrated that miR-155 could directly interact with the 3′-untranslated region of the hAT1R mRNA. Functional studies demonstrated that transfection of miR-155 into human primary lung fibroblasts (hPFBs) reduced the endogenous expression of the hAT1R compared with non-transfected cells. Additionally, miR-155 transfected cells showed a significant reduction in angiotensin II-induced extracellular signal-related kinase 1/2 (ERK1/2) activation. Furthermore, when hPFBs were transfected with an antisense miR-155 inhibitor, anti-miR-155, endogenous hAT1R expression and angiotensin II-induced ERK1/2 activation were significantly increased. Finally, transforming growth factor-β1 treatment of hPFBs resulted in the decreased expression of miR-155 and the increased expression of the hAT1R. In summary, our studies suggest that miR-155 can bind to the 3′-untranslated region (UTR) of hAT1R mRNAs and translationally repress the expression of this protein in vivo. Importantly, the translational repression mediated by miR-155 can be regulated by physiological stimuli.

dominantly regulated by post-transcriptional mechanisms (reviewed in Ref. 7). For example, Ang II (8), cAMP stimulating agents (9), and estrogens (10,11) decrease rat AT 1A R expression by stimulating rat AT 1A R mRNA decay. In contrast, insulin (12), low density lipoprotein (13), and progesterone (11) up-regulate receptor expression by decreasing AT 1A R mRNA decay rates. Additionally, rat AT 1A R expression is regulated by translational mechanisms via cytosolic proteins that interact with the 5Ј-UTR of the receptor mRNA (10, 14 -18). Finally, it has also been demonstrated that the human AT 1 R (hAT 1 R) 5Ј-UTR harbors an internal ribosome entry site, which allows this mRNA to be translated during physiological conditions when cap-dependent translation is inhibited (19).
Recently it has been demonstrated that microRNAs (miRNAs) may provide an additional post-transcriptional mechanism by which protein expression can be regulated. miRNAs are produced endogenously in mammalian cells by specific RNA gene transcription or from introns during pre-mRNA splicing (20 -23). miRNAs are expressed as long hairpin-forming precursor RNAs that get processed to 21-23 nucleotide RNA molecules that regulate the stability or translational efficiency of target mRNAs (24 -29). In animals, miRNAs usually control gene expression through partial complementary elements in the 3Ј-UTRs of their target mRNAs (26 -30). The functional importance of miRNAs is evidenced by the many biological processes in which they are implicated, including developmental timing, cell proliferation, apoptosis, metabolism, cell differentiation, and morphogenesis (31,32). Currently, it is not known whether miRNAs play a role in regulating components of the renin-angiotensin system. Therefore, in this study, we have examined the hypothesis that hAT 1 R expression can be regulated by miRNAs binding to specific target sequences harbored in the 3Ј-UTR of hAT 1 R mRNAs. Experimental analyses demonstrate, for the first time, that miR-155 can regulate the expression of the hAT 1 R in primary human lung fibroblasts.

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, Carlsbad, CA) supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT), 80 units/ml penicillin, 80 g/ml streptomycin, and 0.0175 mg/ml L-proline (Sigma). Human pulmonary fibroblasts (hPFB) were established in primary culture from enzyme-dispersed tissue fragments adhered to Primaria culture plates of human neonatal lung tissue obtained at autopsy and were the generous gift of Dr. Daren L. Knoell (Departments of Pharmacy and Internal Medicine, Ohio State University). Pulmonary fibroblasts were maintained in a 1:1 mixture of Ham's F12 and Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (HyClone), 80 units/ml penicillin, and 80 g/ml streptomycin and Fungizone TM (Invitrogen). Cells were rendered quiescent by a 48 h exposure to HEPES-buffered Dulbecco's modified Eagle's medium containing 0.5% fetal bovine serum and penicillin-streptomycin-Fungizone TM . Cells were used for ϳ7-10 passages before replacement with fresh early passage stocks. 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-CATGT-TCGAAACCTGTCCATAAAG-3Ј) and antisense (5Ј-ATAAAAT-TATTTTATTTTAAAGTAAAT-3Ј) primers using standard procedures and a proofreading polymerase (Platinum Pfu, Invitrogen). A fulllength hAT 1 R cDNA clone (19) was used as template. The PCR product was subcloned into the pCR TM 2.1 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 pCR TM 2.1 plasmid by EcoRI digestion. The fragments were gel-purified, filled in, and bluntend-ligated into a filled-in HindIII site which is located downstream of the firefly luciferase ( f-luc) reporter gene (pMIR-REPORT TM , Ambion). The authenticity and orientation of the inserts relative to the luciferase gene were confirmed by sequencing. The resulting plasmids were designated, pMIR/883/5Ј33Ј and pMIR/883/3Ј35Ј, respectively. Transformed bacterial cultures were grown, and each reporter construct was purified by using PureLink TM Hipure plasmid Maxiprep kit (Invitrogen). The expression plasmid pMIR/883/5Ј33Ј/⌬miR-155 was generated by utilizing the pMIR/883/5Ј33Ј plasmid as template and deleting the miR-155 7-bp seed binding site (Fig. 1B) harbored in the hAT 1 R 3Ј-UTR using the QuikChange site-directed mutagenesis kit (Stratagene). Briefly, a forward mutagenic deletion primer (5Ј-CTTCAC-TACCAAATGGCTACTTTTCAGAAT-3Ј) and a complementary reverse mutagenic deletion primer (5Ј-ATTCTGAAAAGTAGC-CATTTGGTAGTGAAG-3Ј), where the desired sequence to be deleted (i.e. AGCATTA) was missing from the middle of the primer, were synthesized and utilized in a PCR experiment as described by the manufacturer. The amplification reaction was treated with DpnI restriction enzyme to eliminate the parental template and the remaining DNA was used for transformation. The deletion of the AGCAUUA miR-155 seed binding sequence was confirmed by dideoxy chain termination sequencing.
Transfection-The following partially double-stranded RNAs that mimic endogenous precursor miRNAs, hsa-miR-124a-1, hsa-miR-155, hsa-miR-365, and miRNA Negative Control #1 were obtained from Ambion. Additionally, the following anti-miR miRNA inhibitors (anti-miR-124a-1, anti-miR-155, anti-miR-365-1, and anti-miR Negative Control #1), designed to inhibit endogenous miRNAs, were also obtained from Ambion. These RNA-based inhibitors are chemically modified to increase their stability and to improve their activity. Transfection of CHO and hPFB 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). CHO cells were transfected with the luciferase reporter constructs described above (1 g), pRL-CMV (50 ng, Promega) and the appropriate miRNA precursor using Lipofectamine 2000. After 48 h, cells were washed and lysed with passive lysis buffer (Promega) and f-luc and Renilla luciferase (r-luc) activities were determined using the dual-luciferase reporter assay system (Promega) and a luminometer. The relative reporter activity was obtained by normalization to the r-luc activity. Alternatively, hPFB cells were cotransfected with pRL-CMV and either specific pre-miR mRNAs or anti-miR miRNA as described above. After 48 h, cells were either utilized for luciferase, real-time PCR, and radioreceptor binding or immunoassay assays. AT 1 Receptor Radioligand Binding Studies-Whole cell AT 1 receptor binding was measured as described previously (33). 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 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-miRNA certified FirstChoice human total RNA survey panel was purchased from Ambion, the RNA was subsequently treated with RNase-free DNase I, and cDNA was synthesized from 1 g of total RNA using gene-specific primers to miR-155 precursor and 18 S rRNA as described (34). The expression of miR-155 precursor relative to 18 S rRNA was determined using SYBR green real-time quantitative PCR assay as described (35). Relative gene expression was calculated as 2 Ϫ(CTmiR-155-CT18S rRNA) . Relative gene expression was multiplied by 10 6 to simplify data presentation. An experiment was conducted to validate that the expression of the mature miR-155 correlated to the miR-155 precursor (data not shown). The TaqMan microRNA assay (Applied Biosystems, Foster City, CA) was used to quantify the expression of the mature miR-155 (36). Alternatively, RNA was isolated from hPFBs using TRIzol (Invitrogen) and 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-CACCAT-GTTTTGAGGTTGACTGAC-3Ј; antisense primer, 5Ј-CAGGCTAGG-GAGATTGCATTTCTG-3Ј.
Immunoassay for Extracellular Signal-related Kinase (ERK)-Twenty-four hours after transfection the hPFB cells 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 ERK1/2specific antibody (Cell Signaling) to normalize the level of phosphorylated ERK to total ERK.
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 analyses were performed using the software package Prism 4.0b (GraphPad Software, San Diego, CA).

MiR-155 Interacts with the 3Ј-UTR of the hAT 1 R mRNA-
Bioinformatic approaches have been utilized to identify potential miRNA targets (reviewed in Ref. 37). An on-line search of the miRBase Target data base (38) demonstrated that 9 -16 putative miRNA target sites were harbored in the 3Ј-UTR of the AT 1 R mRNA depending upon the species analyzed. For this study we have focused on three miRNAs (i.e. miR-124a, -155, and -365) that may target the hAT 1 R 3Ј-UTR since these sites are conserved, to various degrees, across species (Fig. 1). There is only a single target site for each miRNA studied, and the target site of miR-155 and miR-365 is overlapping (Fig. 1, A-C). To experimentally validate the computational data, the hAT 1 R 3Ј-UTR (i.e. 883 bp) was subcloned downstream of the f-luc open reading frame ( Fig.  2A). This reporter construct (pMIR/883/5Ј33Ј) was cotransfected in the CHO cell line with pRL-CMV (to normalize for transfection differences) and a control non-targeting RNA oligonucleotide (miR-Control) or miR-124a, -155, or -365 precursor RNA oligonucleotides (Fig. 2B).
Identical control experiments were also performed utilizing a reporter construct (pMIR/883/3Ј35Ј) in which the hAT 1 R 3Ј-UTR was cloned in the opposite orientation. Interestingly, the relative luciferase activity was only markedly diminished (61.3 Ϯ 4.2%) in cells cotransfected with the pMIR/883/5Ј33Ј construct and miR-155 (50 nM final concentration, Fig. 2B). To test the potency of the miRNAs, pMIR/883/5Ј33Ј was cotransfected into CHO cells with increasing concentrations of each specific RNA oligonucleotide and luciferase activities were determined. Dose response experiments demonstrated that relative luciferase activity was significantly decreased with as little as 1 nM miR-155 and a maximal decrease was obtained with a 50 nM concentration of this RNA (Fig. 2C). In contrast, increasing concentrations of miR-Control (Fig.  2C) or miR-124a or -365 had no effect on luciferase activity even when the concentration of these RNAs was 100 nM (data not shown).
To demonstrate that miR-155 interacts with a specific target sequence localized in the hAT 1 R 3Ј-UTR, an additional reporter construct was generated in which the 7 bp "seed" sequence (i.e. AGCAUUA), which is complementary to the 5Ј-end of miR-155 (Fig.  1B), was deleted using PCR. The resulting construct, pMIR/883/5Ј33Ј/ ⌬miR-155, was cotransfected into CHO cells as described above and luciferase activity measured. Importantly, miR-155 could no longer decrease luciferase activity of the new reporter construct (Fig. 2D). Taken together, these results indicate that of the three potential miR targets identified in the hAT 1 R 3Ј-UTR by bioinformatics analyses, only miR-155 can interfere with luciferase mRNA translation via direct interaction with the hAT 1 R 3Ј-UTR.
miR-155 Decreases hAT 1 R Expression on Human Pulmonary Fibroblasts-We have previously demonstrated that hPFB endogenously express the hAT 1 R (33). Therefore, hPFBs were transfected with the appropriate miRNAs, and AT 1 R levels were quantitated by performing radioreceptor binding assays. AT 1 R binding assays demonstrated that only hPFBs transfected with miR-155 showed a significant reduction in the expression of hAT 1 Rs (48.3 Ϯ 6.2%) compared with controls (Fig. 3A). To investigate whether miR-155 targeted hAT 1 R mRNA for degradation, real-time PCR experiments were performed on RNA isolated from transfected hPFB cells. These data demonstrated that cells transfected with miR-155 did not significantly decrease hAT 1 R steady state mRNA levels (Fig. 3B). To determine whether the reduction in hAT 1 R density also resulted in decreased Ang II-induced signal transduction, hPFBs were transfected with miRNA-124a or -155 and activated with 0.1 M Ang II for 5 min and phospho-ERK1/2 levels were determined. These results demonstrated that only hPFB cells transfected with miR-155 exhibited decreased phospho-ERK1/2 levels (52.4% Ϯ 5.3%, p Ͻ 0.001) compared with controls (Fig. 3, C and D). Taken together, these experiments suggest that miR-155 markedly decreased hAT 1 R expression by inhibiting the translation process and not by targeting hAT 1 R mRNA for degradation. Importantly, decreased hAT 1 R expression also resulted in a significant decrease in Ang II-induced signaling events.
MiR-155 Is Expressed in Human Fibroblasts at Physiologically Important Levels-The hAT 1 R is expressed in a wide variety of human tissues (1,33). Therefore, to begin to investigate the hypothesis that miR-155 may play a physiological role in regulating the expression of the hAT 1 R, we determined whether hPFB cells and human tissues expressed the miR-155 gene. Real-time PCR experiments demonstrated that miR-155 was expressed in hPFB cells and in all human tissues studied although at varying levels (Fig. 4). The TaqMan probes used to generate the data presented in Fig. 4 will fluoresce only in the presence of amplicon; therefore the data represent true miR-155 expression and not background. The data were classified as tissues with high (lung, spleen, and thymus), intermediate (colon, esophagus, ovary, small intestine, and trachea), low (adipose, bladder, brain, cervix, heart, kidney, prostate, skeletal muscle, and thyroid), and very low (liver, pancreas, placenta, and testes) miR-155 expression. The miR-155 expression in hPFB cells fell into the intermediate category (Fig. 4). The expression of miR-155 precursor paralleled that of mature miR-155 in both the tissues and hPFB cells (data not shown).
To investigate whether the level of miR-155 expression in hPFBs is physiologically relevant, these cells were transfected with antisense RNA oligonucleotides (i.e. anti-miR-Control, anti-miR-124a, anti-miR-155, and anti-miR-365) complementary to each miRNA utilized in this study, and the cells were then subjected to radioreceptor binding assays. Importantly, these experiments demonstrated that only hPFBs transfected with anti-miR-155 showed an increase in hAT 1 R levels (32.5% Ϯ 3.9%, p Ͻ 0.01, Fig. 5A). To investigate whether the antisense RNAs also modulated Ang II-induced signaling events, phospho-ERK1/2 levels were determined (Fig. 5, B and C). These experiments demonstrated that cotransfection of anti-miR-155 not only increased hAT 1 R expression but also enhanced Ang II-induced signaling via the hAT 1 R (31.2% Ϯ 5.1%, p Ͻ 0.01, Fig. 5, B and C). Taken together, these results suggest that although the miR-155 gene is expressed at relatively low levels in hPFBs, the inhibition of the endogenously expressed miR-155 by anti-miR-155 resulted in enhanced levels of hAT 1 R, indicating that miR-155 plays a physiological role in regulating the expression of hAT 1 Rs in human fibroblasts.
TGF-␤ 1 Inversely Regulates hAT 1 R and miR-155 Expression in Human Fibroblasts-To determine whether physiological stimuli could modulate miR-155 expression, we utilized TGF-␤ 1 (4 ng/ml for 4 h) since it has been previously demonstrated that hAT 1 R protein expres- A, schematic representation of the f-luc reporter constructs utilized. The pMIR/883 plasmids contain the full-length 3Ј-UTR of the hAT 1 R in either the 5Ј33Ј or 3Ј35Ј orientation with respect to the f-luc open reading frame and were designated pMIR/883/5Ј33Ј and pMIR/883/3Ј35Ј, respectively. The firefly luciferase start and stop codons are also shown. B, CHO cells were cotransfected with pMIR/883/5Ј33Ј or pMIR/883/3Ј35Ј, pRL-CMV, and a 50 nM concentration of a given RNA oligonucleotide. Forty-eight hours after transfection luciferase activities were measured. f-luc activity was normalized to r-luc expression and the mean activities Ϯ S.E. from five independent experiments are shown (*, p Ͻ 0.01 versus mocktransfected). C, CHO cells were cotransfected with pMIR/883/5Ј33Ј, pRL-CMV, and either miR-Control or miR-155 at the concentrations indicated, and luciferase activities were calculated as described above. The mean activities Ϯ S.E. from five independent transfection experiments are shown (*, p Ͻ 0.01 versus miR-Control at each concentration shown). D, CHO cells were cotransfected with pMIR/883/5Ј33Ј/⌬miR-155, pRL-CMV, and a 50 nM concentration of the appropriate miRNA as shown. Forty-eight hours after transfection luciferase activities were calculated as described above. The mean activities Ϯ S.E. from five independent transfection experiments are shown.
sion levels were enhanced in TGF-␤ 1 -treated fibroblasts (37). Thus, hPFBs were incubated with TGF-␤ 1 for the times indicated, and PCR and radioreceptor binding assays were performed. Importantly, TGF-␤ 1 treatment significantly decreased miR-155 expression levels (Fig. 6A). Although TGF-␤ 1 also up-regulated the expression of the hAT 1 R gene (Fig. 6B), the time course for the increased production of hAT 1 R mRNA did not correlate well with the time course for the enhanced synthesis of hAT 1 R protein levels since these values remained higher than expected at the 8 and 12 h time points (Fig. 6, C versus B). Therefore, the sustained increase in hAT 1 R protein levels cannot be accounted for by only a transcriptional mechanism and suggests that post-transcriptional mechanisms must also be involved.

DISCUSSION
Computational studies have suggested that ϳ10% of human genes (38) are targets of the 326 known human miRNAs (39). To date, however, relatively few target sites have been experimentally identified (Tar-Base; Ref. 40). An on-line search of the miRBase Target data base (38) suggested that all components of the renin-angiotensin system may be regulated by miRNAs (data not shown). A specific search of the AT 1 R gene demonstrated that 9 -16 putative miRNA target binding sequences are present in the 3Ј-UTR of these mRNAs, depending upon which species are analyzed. The aim of this study was to begin to determine whether the identified miRNAs actually regulate the expression of the hAT 1 R. Therefore, three specific miRNAs (i.e. miR-124a, miR-155, and miR-365) were chosen and utilized to investigate whether they could mediate the translational repression of a luciferase/hAT 1 R 3Ј-UTR reporter construct and endogenous hAT 1 R expression. Interestingly, only miR-155 could efficiently reduce luciferase activity and hAT 1 R density (Figs. 2 and 3) suggesting that miR-155 could interact specifically with the 3Ј-UTR of the hAT 1 R mRNA and inhibit translation. At present it is not clear why miR-124a and -365 do not interact with hAT 1 R mRNAs. Current computational search strategies apply different assumptions about how to best identify functional target sites; therefore these approaches may only capture subsets of real targets and/or may include a number of background matches (41). It has recently been demonstrated that sites with as few as 7 bp of complementarity (designated as the seed sequence) to the miRNA 5Ј end are suffi- . miR-155 inhibits hAT 1 R expression and Ang II-mediated signaling in fibroblasts. hPFBs were either mock-transfected or transfected with the miRNAs 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 represent specific binding. The values are shown as percent of maximal specific binding of mock-transfected hPFBs and represent the mean Ϯ S.E. from four independent experiments (*, p Ͻ 0.001 versus mock-transfected cells). B, real-time PCR experiments were performed as described previously under "Experimental Procedures" utilizing total RNA isolated from transfected cells. The relative gene expression of hAT 1 R mRNA was normalized to 18S rRNA expression and is expressed in arbitrary units. The mean of hAT 1 R steady state mRNA levels from four independent transfection experiments are shown. C, 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. D, the 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 mock-transfected cells and represent the mean Ϯ S.E. from four independent transfection experiments (*, p Ͻ 0.001). . Mature miR-155 is expressed in human tissues. miRNA certified human total RNAs were reverse transcribed utilizing miR-155-and 18 S rRNA-specific primers, and PCR experiments were performed as described under "Experimental Procedures." Alternatively, total RNA was isolated from non-treated hPFBs and utilized as described above. The relative expression of the mature miR-155 gene was normalized to 18 S rRNA. The values shown were obtained from a single PCR experiment. cient to confer regulation in vivo and are used in biologically relevant targets (30,42). Additionally, it has been demonstrated that G:U wobble pairing in the seed sequence is highly detrimental to miRNA function despite its favorable contribution to RNA:RNA duplexes (30). Finally, it has been established that miRNAs that have weak 5Ј pairing need substantial 3Ј pairing to function (42). Therefore, based on these derived criteria, it might be expected that of the three miRNAs utilized in this study, only miR-155 should regulate hAT 1 R expression since this miRNA harbors a seed sequence of 7 nucleotides and does not contain a G:U base pair in this complementary region (Fig. 1).
While the luciferase reporter gene and miRNA transfection experiments were useful in confirming that miR-155 could regulate the expression of hAT 1 Rs, it is important to demonstrate that endogenous miR-155 can regulate the translatability of endogenously expressed hAT 1 R mRNA. Therefore, to investigate whether or not the expression levels of miR-155 in hPFB (Fig. 4) were physiologically relevant, these cells were transfected with antisense miRNA inhibitors (Fig. 5, A-C). For the first time, we have demonstrated that the inhibition of endogenously expressed miR-155, by anti-miR-155, markedly increased hAT 1 R expression (Fig. 5A). Importantly, the increased hAT 1 R expression levels also lead to enhanced Ang II-induced phospho-ERK1/2 activation (Fig.  5C). Taken together, these data clearly suggest that miR-155 regulates the in vivo expression of hAT 1 R in fibroblasts. Furthermore, our PCR data demonstrated that miR-155 was expressed at varying levels in a number of human tissues (Fig. 4) implying that miR-155 could regulate the expression of the hAT 1 R in any tissue or cell type where both RNAs are coexpressed. Since the vasoconstrictor response of Ang II is mediated via the AT 1 R expressed on vascular smooth muscle cells, we also investigated whether these cells expressed miR-155. 3 Interestingly, human vascular smooth muscle cells expressed 5-6-fold more miR-155 versus fibroblasts. Therefore, this observation may 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.
To further demonstrate the physiological relevance of miR-155 in regulating hAT 1 R expression levels, we investigated whether TGF-␤ 1 stimulation also modulated miR-155 expression since it was previously demonstrated that this cytokine could regulate hAT 1 R synthesis in fibroblasts (37). Importantly, TGF-␤ 1 treatment markedly decreased miR-155 expression levels (Fig. 6A) suggesting that a miRNA mechanism was involved in the enhanced hAT 1 R protein levels (Fig. 6C). While it was clear from these data that hAT 1 R protein levels were also up-regulated by a TGF-␤ 1 -mediated transcriptional mechanism (Fig. 6B), it was apparent that other mechanisms must be involved since the hAT 1 R protein levels remained high even though hAT 1 R mRNA levels returned to basal values (see the 12-h TGF-␤ 1 time point, Fig. 6, C versus B). Additional support for the involvement of a non-transcriptional mechanism in regulating hAT 1 R expression is based on a recently published study, which demonstrated that TGF-␤ 1 -treated hPFBs predominantly synthesized a hAT 1 R mRNA splice variant (i.e. hAT 1 R-B, a mRNA comprised of exons 1, 2, and 4, which was up-regulated 6-fold) that harbored multiple upstream open reading frames (33). Importantly, the inclusion of these upstream open reading frames decreased hAT 1 R protein levels. Therefore, the hAT 1 R levels measured in TGF-␤ 1 -treated hPFBs were much higher than expected (Fig. 6C). Since miR-155 inversely regulates hAT 1 R expression (Figs. 3A and 5A), the TGF-␤ 1 -mediated down-regulation of miR-155 could explain, in part, the high hAT 1 R protein levels observed in the data shown in Fig. 6C. Taken together, our studies suggest that miR-155 can translationally repress the expression of hAT 1 R in vivo.
Although the miR-155 target site harbored in the 3Ј-UTR of AT 1 R mRNAs is highly conserved across species (Fig. 1B), the mRNA sequence, which is complementary to the miR-155 seed sequence, is not perfectly conserved in the mouse and rat AT 1 R genes. Therefore, we speculate that miR-155-mediated translational repression of the AT 1 R will only occur in humans, chimpanzees, and dogs.
As mentioned earlier, bioinformatics analysis suggested that hAT 1 R levels may be regulated by at least nine distinct miRNAs (data not shown). Importantly, all six of the miRNAs, not experimentally tested in this study, have the appropriate seed complementarity with distinct target sites in the hAT 1 R 3Ј-UTR, suggesting that they may also regulate hAT 1 R expression levels. This observation raises the possibility that the translatability of hAT 1 R mRNAs may be subject to combinatorial regulation by multiple miRNAs. In support of this hypothesis, Doench and Sharp (30) demonstrated that multiple miR-NAs can simultaneously repress a given target mRNA, and these investigators demonstrated a cooperative regulation between coexpressed miRNAs in translationally repressing their target mRNA (30). Therefore, future studies will investigate whether multiple miRNAs can simultaneously regulate hAT 1 R expression.
In conclusion, AT 1 R expression can be regulated by a number of distinct transcriptional and post-transcriptional mechanisms (1,7). Alternative splicing provides an additional mechanism by which the expression and function of the AT 1 R can be "fine-tuned" (14,15,43). Our study demonstrates, for the first time, that translational repression by miR-155 provides yet another mechanism by which AT 1 R expression can be modulated. Since AT 1 R activation initiates a cascade of pathological events, including altered vascular tone, endothelial dysfunction, structural remodeling, and vascular inflammation, all of which may significantly contribute to the development of cardiovascular and kidney disease (2)(3)(4)(5)(6), it has been speculated that the overproduction of the AT 1 R could lead to these health disorders. As a result of our current study, one possible scenario that would lead to abnormally high levels of AT 1 R would be if miR-155 levels were atypically low. Recently, it has been demonstrated that miRNAs are involved in the pathogenesis of solid tumors and support their function in either dominant or recessive fashion, by controlling the expression of protein-coding tumor suppres-FIGURE 6. TGF-␤ 1 inversely regulates hAT 1 R and miR-155 expression in fibroblasts. hPFBs were grown to confluence, serum-starved for 24 h, and incubated with or without 4 ng/ml of TGF-␤ for the times indicated. A, total RNA was isolated, and relative miR-155 expression was determined as described under "Experimental Procedures." The values shown represent the mean Ϯ S.E. from at least three independent transfection experiments (*, p Ͻ 0.001) and are expressed as fold over mocktransfected values, which was set to "1." B, total RNA was isolated and relative hAT 1 R expression was determined as described under "Experimental Procedures." The values shown represent the mean Ϯ S.E. from at least three independent transfection experiments (*, p Ͻ 0.001). C, AT 1 R radioreceptor binding assays were performed as described above. Values are expressed as fold increase over non-treated hPFBs and represent the mean Ϯ S.E. from four independent transfection experiments (*, p Ͻ 0.001 versus non-treated cells).
sors and oncogenes (44). Therefore, future studies will begin to investigate the potential role of miRNAs in mediating cardiovascular disease.