Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) is altered in individuals with the ΔF508 CFTR mutation. We previously reported differential expression of microRNA (miRNA) in CF airway epithelium; however, the role of miRNA in regulation of CFTR expression here remains unexplored. In this study, we investigated the role of upregulated miRNAs in CFTR regulation in vivo in bronchial brushings from individuals homozygous or heterozygous for ΔF508 CFTR, validated our observations in vitro, and assessed the impact of defective chloride ion conductance, genotype, and colonization status on miRNA expression. miRNA target prediction was performed in silico, and expression of miRNA and target genes were measured by quantitative real-time PCR and/or Western blotting. Overexpression and inhibition studies were performed with pre-miRs or antimiRs, respectively, and a luciferase reporter gene was used to elucidate direct miRNA–target interactions. miR-145, miR-223, and miR-494 were upregulated in CF versus non-CF bronchial brushings and cell lines; in ΔF508 CFTR homozygotes versus heterozygotes; in subjects positive for P. aeruginosa; and in cells treated with a CFTR inhibitor or IL-1β. Reciprocal downregulation or upregulation of CFTR gene and/or protein expression was observed after miRNA manipulation and direct miRNA–target relationships demonstrated via a reporter system containing a wild type or mutated full-length CFTR 3′ untranslated region. Increased expression of miR-145, miR-223, and miR-494 in vivo in bronchial epithelium of individuals carrying the ΔF508 CFTR mutation correlates with decreased CFTR expression. Defective CFTR function, Pseudomonas colonization, and inflammation may affect miRNA expression and contribute to the regulation of ΔF508 CFTR.
Cystic fibrosis (CF) is a lethal genetic disease principally affecting white individuals. It has a high incidence in Europe and North America, and a carrier rate of ∼4% in the general population. The primary cause of CF is defective chloride ion transport due to mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein (1). CFTR is an ATP-regulated chloride channel present within the apical surface of epithelial cells. More than 1800 CFTR mutations have been identified (CFTR mutation database: www.genet.sickkids.on.ca) and these can be subdivided into six classes (2). The most common CFTR mutants are those in class II, which occur because of defective protein processing; ΔF508 CFTR, which belongs to this class, is the most prevalent mutation worldwide (3).
Expression of CFTR is high in epithelial cells, where it is localized to the apical plasma membrane (4). Although CFTR expression is more abundant in intestinal, pancreatic, or kidney epithelial cells, it is expressed in nasal and bronchial biopsy tissue (5), and is expressed and functional in ciliated cells within the epithelium (6). Much is known about the spatial and developmental transcriptional regulation of CFTR (7). Other than alternative splicing (8, 9), there is little known regarding the posttranscriptional regulation of CFTR expression. However, a recent study has reported microRNA (miRNA) involvement in CFTR regulation in vitro (10).
miRNAs are short, noncoding RNAs that hybridize to miRNA recognition elements in the 3′-untranslated region (3′-UTR) of mRNA transcripts and predominantly block translation (11–13). We have previously reported differential expression of miRNA in CF bronchial brushings (14). Of the 255 miRNA that are predicted to target CFTR (TargetScan 6.2), few have been examined and none in the context of CF in vivo. In this article, we describe the increased expression of specific miRNAs predicted to target CFTR in vivo and in vitro. We investigate the relationship between miRNAs and CFTR expression in individuals homozygous or heterozygous for the ΔF508 CFTR mutation and examine the effect of inhibition of the CFTR chloride ion channel on miRNA expression. We also determine the effects of miRNA manipulation on CFTR expression and function in vitro, and examine direct miRNA/CFTR 3′-UTR interactions. In addition, we explore how colonization with Pseudomonas aeruginosa, Staphylococcus aureus, or Aspergillus species relate to miRNA and CFTR expression, and examine the effects of Pseudomonas-conditioned media (PCM), LPS, and IL-1β on miRNA levels.
Materials and Methods
Study populations, bronchial brush sampling, and miRNA profiling
Following informed consent under a protocol approved by our Institutional Review Board, bronchial brushings were sampled from 23 individuals and RNA was isolated as previously described (14); CF (n = 14; 27.2 ± 2.7 y; male/female ratio, 8:6; confirmed by sweat testing and/or genotyping; Table I) and non-CF controls (n = 9; 50.8 ± 5.4 y; male/female ratio, 4:5). miRNA expression profiling was previously described for CF (n = 5; non-CF: n = 5) (14). Only CF samples from individuals with at least 1 ΔF508 CFTR allele were included in this study, which consisted of 4 samples from the original miRNA expression profiling screen and 10 additional brushings.
|Patient No. .||Sex .||CFTR Genotype .||P. aeruginosa .||S. aureus .||Aspergillus Sp. .|
|1||M||ΔF508/R506T > K||Positive||Positive||Negative|
|8||M||ΔF508/621+1G > T||Positive||Negative||Positive|
|11||F||ΔF508/621+1G > T||Negative||Negative||Positive|
|Patient No. .||Sex .||CFTR Genotype .||P. aeruginosa .||S. aureus .||Aspergillus Sp. .|
|1||M||ΔF508/R506T > K||Positive||Positive||Negative|
|8||M||ΔF508/621+1G > T||Positive||Negative||Positive|
|11||F||ΔF508/621+1G > T||Negative||Negative||Positive|
Cell culture and treatments
All experiments were repeated in triplicate on at least three separate occasions. Human bronchial epithelial 16HBE14o− (15) and ΔF508 homozygous CFBE41o− cell lines were obtained as a gift from D. Gruenert (California Pacific Medical Center Research Institute, San Francisco, CA). HEK293 cells (human embryonic kidney cell line) were obtained from the European Collection of Cell Cultures (Salisbury, U.K.). Cell lines were routinely grown to 60–80% confluency in MEM (MEM+GlutaMax; Life Technologies) supplemented with 10% FCS (Life Technologies) and 1% penicillin-streptomycin (Life Technologies) in 75-cm2 flasks and maintained in a 37°C humidified incubator containing 5% CO2.
Before agonist treatment and after 24-h serum starvation, cells were washed with serum-free media and then placed in serum-free medium or IL-1β (10 ng/ml) or media containing 1% FCS for P. aeruginosa LPS (10 μg/ml; Sigma-Aldrich, St. Louis, MO) and Pseudomonas-conditioned media stimulation experiments. P. aeruginosa strain 01 was a gift from S. Smith (Trinity College Dublin, Ireland). PCM 1% (v/v) was prepared by filter sterilizing supernatants from 72-h P. aeruginosa strain 01 trypticase soy broth cultures. PCM contains multiple TLR agonists including lipopeptides, LPS, and uCpG DNA (16). For CFTR-inhibitor studies, 16HBE14o− were treated with 10 μM CFTR inhibitor-172 (CFTRinh172; Sigma-Aldrich) in serum-free medium for 24 h. Total RNA was isolated for miRNA analysis by quantitative real-time PCR (qRT-PCR) analysis.
miRNA qRT-PCR and target prediction
miRNA expression in brushings and cell lines was measured using TaqMan miRNA assays (Applied Biosystems, Foster City, CA) on the Roche LC480 LightCycler. Expression of miRNA relative to miR-218 was determined using the 2(−ΔΔCt) method (17). miR-218 was chosen for normalization because of a high degree of similarity in its expression levels between CF and non-CF brushings. All qRT-PCR experiments in cell lines were performed in triplicate, a minimum of three times, and included no-template controls. In silico target prediction analysis was performed using TargetScan 6.2 (release June 2012), MicroRNA.org, PITA, miRDB v4.0 (release Jan 2012), and Microcosm v5.0.
Target gene expression analysis
Total RNA was extracted using TriReagent, and equal quantities were reverse transcribed into cDNA using Quantitect Reverse Transcription Kit (Qiagen, Valencia, CA) following the manufacturer’s protocol. qRT-PCR was performed on the Roche LC480 LightCycler. CFTR gene expression was assessed in CF versus non-CF brushings and in cell lines. Primers for CFTR (forward 5′-CAAGGAGGAACGCTCTATCG-3′, reverse 5′-AGAACACGGCTTGACAGCTT-3′) and β-actin (forward 5′-GGACTTCGAGCAAGAGATGG-3′, reverse 5′-AGGAAGGAAGGCTGGAAGAG-3′) were obtained from MWG Eurofins Genetics (Ebersberg, Germany). Expression of CFTR relative to β-actin (data presented as fold differences) was determined using the 2(−ΔΔCt) method.
Luciferase reporter plasmid transfection
HEK293 cells (1 × 105 in triplicate) were transiently transfected with 250 ng of a luciferase reporter vector containing the full-length wild type (WT) 1.5-kb CFTR 3′-UTR (WT-CFTR-3′-UTR) or a negative control reporter containing full-length 1.5-kb CFTR 3′-UTR with mutations in the predicted binding sites (underlined) for miR-145 (WT: AATGATAACTGGAAACTTC, mutant: AATGATAACCGGTAACTTC), miR-223 (WT: ACTCCAAACTGACTCTTA, mutant: ACTCCAAATTAACTCTTA), and miR-494 (WT: TTAATAATGTTTCAAACATA, mutant: TTAATAATGCATCAAACATA) called Mut-CFTR-3′-UTR (Origene). Endogenous red fluorescent protein expressed within both the WT and MUT-CFTR 3′-UTR reporters was used to monitor transfection efficiency. Cells were cotransfected with a total of 30 nM synthetic pre-miR mimics (PMs) for miR-145, miR-221, miR-223, and miR-494 or a scrambled control (Applied Biosystems). PMs were transfected at 30 nM individually, all three combined at 10 nM each if three used, or 15 nM if just two used. Transfections were performed using Genejuice (Novagen, Madison, WI) for plasmid DNA and Ribojuice (Novagen) for miRNA in OptiMEM reduced serum media (Life Technologies) per the recommended conditions. Lysates were prepared 24 h after transfection, and measurement of firefly luciferase was performed using the Luciferase assay system (Promega, Madison, WI).
16HBE14o− (1 × 105 for RNA or 3 × 105 for protein) or CFBE14o− (8 × 104 for RNA) cells were transfected with a total of 30 nM of a scrambled control and either synthetic PMs or antimiRs (AMs) for miR-145, miR-223, and miR-494 (Applied Biosystems), respectively, using Ribojuice transfection reagent (Novagen). As an additional control, a PM and AM for miR-221, an miRNA also increased in CF in the expression profiling screen but not predicted to target CFTR was included. Cells were cotransfected with a fluorescently labeled nontargeting miRIDIAN miRNA mimic (Dharmacon) to monitor transfection efficiency. Total RNA was extracted from 16HBE14o− and CFBE14o− cells 48 h after transfection, and qRT-PCR was performed to measure CFTR mRNA and miRNA expression. Transfection efficiency was assessed by visualization of epifluorescence of the miRIDIAN negative control labeled with Thermo Scientific Pierce Dy547. For Western blotting, equal volumes of whole-cell lysates from 16HBE14o− cells transfected for 48 h were separated on 4-12% Bis-Tris acrylamide gels (Invitrogen), transferred to polyvinylidene difluoride membranes (Roche), and probed with mouse anti-CFTR (R&D Systems catalog no. MAB25031) or rabbit anti-actin Abs (Millipore catalog no. MAB1501). Signals were detected using appropriate HRP-conjugated secondary Abs (Cell Signaling Technologies) and visualized by chemiluminescence (Millipore) on the Syngene G:Box chemi XL gel documentation system. Membranes were analyzed by densitometry using GeneTools software on the same system.
Measurement of intracellular chloride
16HBE14o− cells grown in a monolayer in 24-well plates were loaded with 1 mM fluorescent membrane permeable chloride indicator N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) in phenol red–free DMEM at 37°C, 5% CO2 as previously described (18–20). After 6 h, the cells were washed and maintained in MQAE-free, phenol red–free DMEM. MQAE fluorescence was measured using a Wallac Victor2 plate reader (Perkin Elmer) at excitation and emission wavelengths of 355 and 460 nm, respectively. A decrease in MQAE fluorescence reflects an increase in intracellular chloride concentration ([Cl−]i). Fluorescence was measured in 0.1-s intervals for 1 min to determine the baseline of each experiment. Changes in [Cl−]i upon cell stimulation were determined by expressing alterations in MQAE fluorescence in response to respective treatments relative to DMSO controls. After treatment with DMSO (vehicle control, 0.1%), CFTRinh172 (10 μM), or forskolin (FSK; 10 μM), fluorescence was measured for a further 10 min in 1-s intervals. Both CF and non-CF epithelial cell lines were transfected with a scrambled control and either a combination of AM145, -223, and -494 or PM145, -223, and -494 (100 nM in total), respectively. Cells were loaded with 1 mM MQAE 24 h posttransfection, and intracellular chloride levels were determined as described earlier. Transfection efficiency was estimated using a fluorescently labeled nontargeting miRIDIAN miRNA mimic to monitor transfection efficiency where uniform transfection was observed across all wells.
All analyses were performed using GraphPad PRISM 4.0 (San Diego, CA). Results are expressed as the mean ± SEM and were compared by Student t test or ANOVA as appropriate. Differences were considered significant at p ≤ 0.05.
miR-145, -223, and -494 are increased in CF bronchial epithelium in vivo and in vitro, and target CFTR
We have previously reported differential expression of miRNA in CF versus non-CF bronchial brushings (14). It is possible that miRNA that are increased in vivo in CF bronchial epithelium might regulate expression of CFTR. Of the 255 miRNA predicted to regulate CFTR (TargetScan 6.2), 7 were differentially expressed in CF versus non-CF airway epithelium, 4 were upregulated (miR-101, -145, -223, and -494), and 3 were downregulated (miR-31, miR-331-3p, and miR-362-5p). For miR-101, only two samples passed the relative expression cutoff; therefore, we did not analyze it further. miR-145, miR-223, and miR-494 were modestly increased 2.16 ± 0.85, 5.7 ± 1.8, and 3.22 ± 0.84-fold, respectively. Three miRNA target prediction databases (TargetScan 6.2, microRNA.org, and PITA) predict these three miRNA to regulate CFTR. Fig. 1A depicts the full-length human CFTR 3′-UTR with locations of predicted binding sites for miR-145, miR-223, and miR-494, and Fig. 1B shows the locations and base-pair matches of predicted binding sites adapted from TargetScan 6.2.
Quantification of CFTR mRNA expression in the same bronchial brushings and also in CFBE41o− versus 16HBE14o− cell lines demonstrated that CFTR expression was significantly decreased in the CF versus non-CF samples both in vivo and in vitro (p = 0.0004 and 0.004, respectively; Supplemental Fig. 1A, 1B). Only individuals carrying at least one ΔF508 CFTR allele were included in this study; genotypes are listed in Table I.
Next, the expression of miR-145, miR-223, and miR-494 from the miRNA expression profiling study (14) were independently verified by qRT-PCR using TaqMan miRNA assays. This was performed using the original 4 ΔF508 CFTR samples and 10 additional CF brushings, and also in the original and additional controls (total CF = 14; non-CF = 9). miR-145, miR-223, and miR-494 were significantly increased in the CF samples by 8.4-, 32.8- and 3.6-fold, respectively (p = 0.0017, 0.0054, and 0.0236, respectively; Fig. 2A). These higher fold changes are likely to be a truer reflection of the in vivo situation having been determined using miR-specific assays in 14 rather than the original 4 samples used for profiling. Expression of miR-145, miR-223, and miR-494 were also found to be significantly increased in vitro in ΔF508/ΔF508 CF versus non-CF bronchial epithelial cell lines (CFBE41o− versus 16HBE14o−, p = 0.02, 0.0011, and 0.0014, respectively; Fig. 2B).
miR-145, miR-223, and miR-494 mediate repression of luciferase in WT CFTR 3′-UTR reporter
HEK293 cells were transiently transfected with a luciferase reporter vector containing the full-length WT 1.5-kb CFTR 3′-UTR (WT-CFTR-3′-UTR) or a negative control reporter with mutations in the predicted binding sites for these miRNAs (Mut-CFTR-3′-UTR) to determine whether CFTR is a true molecular target of miR-145, miR-223, and miR-494. Cotransfection with PM145, PM223, and PM494, alone or in various combinations, resulted in significant decreases in luciferase gene expression from WT-CFTR-3′-UTR compared with Mut-CFTR-3′-UTR demonstrating direct miRNA–target interaction (p ≤ 0.05; Fig. 3). The magnitude of miRNA-mediated luciferase repression was in the order of PM145+PM494 > PM145 > PM145+PM223 > all 3 > PM223+PM494 > PM223. Additive effects were not evident; however, this may be due to the fact that PMs were used individually at 30 nM and for the combination, only 10 nM of each was used (or 15 nM each when two were combined), so that the total concentration did not exceed 30 nM. Differences in luciferase expression between WT and mutant reporters after transfection with PM221 (included as an additional control) were not significant. Delivery of reporter constructs, and hence transfection efficiency, was monitored by visualization of endogenous red fluorescent protein within luciferase reporter vectors (Supplemental Fig. 2) where representative uniform transfection (estimated to be ∼50%) was observed across all wells.
Overexpression of pre–miR-145, -223, and -494 decreases CFTR mRNA and protein expression in 16HBE14o− cells
Next, the effect of miR-145, miR-223, and miR-494 overexpression on CFTR mRNA and protein was assessed. Non-CF 16HBE14o− cells were transfected with synthetic pre-miRs for each of these miRNA individually and in combination (all three). A scrambled PM and PM221 were included as controls. Cells were cotransfected with a fluorescently labeled nontargeting miRIDIAN miRNA mimic to monitor transfection efficiency. Supplemental Fig. 3 shows the transfection efficiency of nontransfected (NT) and transfected cells, where >80–90% of cells are transfected as monitored by epifluorescence, and miRNA expression was increased after pre-miR transfection. Although levels appear greatly increased compared with controls, it has previously been determined that ectopic overexpression of miRNA at physiological levels using an expression plasmid has similar biological effects to the mature pre-miR introduced at supraphysiological levels (21).
Fig. 4A shows that pre-miR overexpression (PM145, PM223, PM494, and all three) significantly decreases CFTR mRNA expression by an average of 36.5% in 16HBE14o− cells; PM221 has no effect. Overexpression of any single pre-miR induced as potent an effect on CFTR mRNA as a combination of all three. CFTR protein expression examined by Western blot was also decreased after transfection of PM145, PM223, or PM494, and to a greater extent with the combination of all three miRNAs (Fig. 4B). In SDS-PAGE, WT CFTR migrates with a characteristic banding pattern: band A (130 kDa), the core glycosylated band B (135 kDa), and the diffusely migrating mature glycosylated band C (150–170 kDa). The densitometry in Fig. 4C shows the fold change of bands A+B+C normalized to actin and confirms that CFTR protein is decreased by PM145, PM223, and PM494 by ∼33 and 52% (p = 0.0471) by all three combined.
Knockdown of miR-145, miR-223, and miR-494 enhances ΔF508 CFTR expression in CFBE41o− cells
AMs are chemically modified siRNA-like molecules designed to knock down miRNA expression. CFBE41o− cells were transfected with AM145, AM223, and AM494 alone and in combination, and ΔF508 CFTR mRNA expression was assessed. Both nontransfected (NT) and transfected cells are shown in Supplemental Fig. 4 where transfection efficiency is estimated qualitatively to be >80–90%, and miRNA expression after AM transfection is ∼35–40% decreased. Fig. 5 shows that knockdown of miR-145, miR-223, or miR-494 leads to a reciprocal increase in ΔF508 CFTR mRNA expression. When all three miRNAs are knocked down in combination, ΔF508 CFTR expression is increased, but not in a synergistic fashion. Control AM221 did not increase ΔF508 CFTR mRNA expression.
Functional inhibition of CFTR in vitro and the ΔF508 genotype causes increased expression of miR-145, -223, and -494
To examine whether altered miR-145, miR-223, and miR-494 expression is a consequence of a lack of CFTR function, we treated 16HBE14o− cells with CFTRinh172, a thiazolidine channel inhibitor highly selective for CFTR (22). After 24-h treatment with CFTRinh172, expression of all three miRNAs was significantly enhanced (p ≤ 0.05; Fig. 6A). Expression of miR-218 (the normalization control), not predicted to target CFTR, was not altered in response to CFTRinh172 (Fig. 6B). These results suggest that miR-145, miR-223, and miR-494 are constitutively upregulated in CF bronchial epithelium because of a lack of CFTR function. To assess the impact of ΔF508 CFTR on this finding, miRNA expression was quantified in bronchial brushings from ΔF508 CFTR homozygotes or heterozygotes versus non-CF controls (Fig. 6C–E). Expression of miR-145 and miR-223 was significantly higher in both groups compared with the non-CF controls (miR-145: p = 0.0035 and 0.0071; miR-223: p = 0.021 and 0.0122 for homozygotes and heterozygotes, respectively). miR-494 was significantly increased in the homozygote group compared with non-CF controls (p = 0.0415). There were no significant differences in miRNA expression between ΔF508 CFTR heterozygotes versus homozygotes.
Inhibition or overexpression of miR-145, miR-223, and miR-494 alters intracellular chloride levels in CF and non-CF cell lines in vitro
The chloride-sensitive, quinoline-based dye MQAE, which can measure [Cl−]i, was used to establish whether inhibition or overexpression of miR-145, miR-223, and miR-494 affects CFTR function (23). In control experiments, non-CF 16HBE14o− cells treated with CFTRinh172 (10 μM, 10 min) had significantly reduced MQAE fluorescence when compared with DMSO controls corresponding to an increase in [Cl−]i content (p = 0.0152; Fig. 7). FSK treatment (10 μM, 10 min) induced the opposite effect, leading to a decrease in [Cl−]i content (p = 0.0243; Fig. 7). After transfection of 16HBE14o− cells with a combination of PM145, PM223, and PM494, MQAE fluorescence was significantly reduced, similar to that observed with CFTRinh172 (p ≤ 0.0001; Fig 7). Transfection of CFBE41o− cells with AM145, AM223, and AM494 in combination led to an increase in MQAE fluorescence (p ≤ 0.0001; Fig 7). Together, these data suggest that manipulation of these miRNAs can influence chloride ion conductance, possibly via altered CFTR levels.
Increased expression of miR-145, miR-223, and miR-494 may also be attributed to Pseudomonas colonization within the CF lung
Next, the effect of colonization status on miRNA and CFTR mRNA expression was assessed (Fig. 8, Table I). Of the 14 CF samples, 9 tested positive for P. aeruginosa, 7 for S. aureus, and 6 for Aspergillus species at the time of sampling. miR-145 expression was increased in all CF subgroups compared with the non-CF group (Fig. 8A). All subgroups, with the exception of P. aeruginosa negative, were associated with increased miR-223 expression; levels of this miRNA were significantly increased in P. aeruginosa–positive versus P. aeruginosa–negative samples (Fig. 8B). miR-494 expression was most significantly elevated in P. aeruginosa–positive versus non-CF samples, compared with any other subgroup (Fig. 8C). The P. aeruginosa–positive subgroup was also associated with the most significant decrease in CFTR mRNA expression (Fig. 8D).
Overall, P. aeruginosa colonization appeared to be associated with the most significant increases in expression of all three miRNAs and the most significant decrease in CFTR mRNA expression. Also, of the three organisms evaluated, only P. aeruginosa showed a significant difference in CFTR (and miR-223) expression in positive versus negative samples. The mean forced expiratory volume in 1 s percentage predicted was 49.67 ± 8.3 for P. aeruginosa–positive and 85.8 ± 4.3 for P. aeruginosa–negative individuals.
Pseudomonas-conditioned media, but not LPS, leads to increased expression of miR-145, -223, and -494 in vitro
Finally, to assess whether P. aeruginosa affected miRNA expression in vitro, CFBE41o− cells were stimulated for 6 h with Pseudomonas-conditioned media, and the effects were compared with cells treated with P. aeruginosa LPS or IL-1β (Fig. 9). To confirm that the agonists were inducing a proinflammatory effect, IL-8 qRT-PCR and ELISA were performed; all stimuli significantly increased IL-8 mRNA and protein expression (data not shown). miRNA expression was measured by qRT-PCR. Fig. 9A shows that PCM increases expression of miR-145, miR-223, and miR-494; however, only the miR-145 response is significant (p = 0.0206). Conversely, treatment with LPS decreased expression of all three miRNA (miR-223 and -494; p = 0.0206 and p = 0.0465, respectively; Fig. 9B), indicating that LPS is not the factor present in PCM responsible for increased miRNA expression. IL-1β, which was used as a proinflammatory control, enhanced expression of both miR-145 (p = 0.0130) and miR-223 (p = 0.0022; Fig. 9C).
In this article, we present the first report, to our knowledge, on a direct correlation between miRNA and CFTR expression in vivo in the bronchial epithelium of individuals with CF carrying a ΔF508 CFTR mutation. The data show that miR-145, miR-223, and miR-494 directly regulate CFTR expression via inhibition of luciferase expression from a WT-CFTR-3′-UTR reporter, and that modulation of these miRNAs can reciprocally regulate CFTR gene expression. Alterations in miRNA based on genotype and colonization status were observed, with the highest increases in miR-145, miR-223, and miR-494 expression occurring in ΔF508 CFTR homozygotes, and those positive for P. aeruginosa. Levels of CFTR expression in vivo based on P. aeruginosa status were also assessed where those positive for P. aeruginosa were found to have marked reduction in CFTR expression compared with P. aeruginosa–negative subjects. In vitro PCM and IL-1β amplified miRNA expression in a CF bronchial epithelial cell line, whereas LPS had the opposite effect, suggesting the involvement of other P. aeruginosa components. Chemical inhibition of CFTR increased expression of all three miRNAs, and overexpression or inhibition of the three miRNAs together led to alterations in intracellular chloride levels, implying functional effects on CFTR. The data suggest that regulation of CFTR by miRNAs represents a previously unidentified mechanism controlling CFTR levels, at least in those carrying a ΔF508 mutation. Many of the in vivo observations could be replicated in cell culture in the absence of patient-specific stimuli, indicating an intrinsic effect.
This study validates CFTR as a target of miR-223, and complements and extends the findings of recent reports documenting miRNA influences over CFTR expression (10, 24, 25). Gillen et al. (10) reported varying degrees of miRNA/CFTR interaction in pancreatic and colonic cell lines, and of 13 miRNAs examined found 4 that are predicted to target CFTR and are expressed in primary bronchial and tracheal cells (miR-101, -145, -331-3p, and -494). Consistent with this, our expression profiling screen detected the same four miRNAs in CF and non-CF bronchial epithelial cells in vivo. miR-101, -145, and -494 were upregulated in CF (together with miR-223), whereas miR-331-3p was downregulated. For miR-101, only two of five samples passed the relative expression cutoff; therefore, it was not analyzed further here. Although several of the 255 miRNAs predicted to regulate CFTR by TargetScan 6.2 may be important regulators of CFTR in different cells types, we found only 7 to be differentially expressed in CF versus non-CF airway epithelium. However, this does not account for those not tested in our arrays, which may also be of significance in the CF lung or miRNAs targeting CFTR by indirect means. For example, Ramachandran et al. (25) reported indirect regulation of CFTR by miR-138, an miRNA also increased in CF in our profiling study but not predicted to target CFTR, and therefore not pursued in this study.
There have been several reports on miR-145, miR-223, and miR-494. miR-145 is decreased in a variety of malignancies including lung cancer and is now considered a tumor suppressor (26). miR-223 and miR-494 have been described as oncomirs targeting the tumor suppressors F-box and WD-40 domain protein 7 (27), and phosphatase and tensin homolog (28), respectively. A role in allergic airway disease has also been proposed for miR-145 (29). Decreased expression of miR-145 and miR-223 was reported in cigarette smoke–exposed rat lungs and similarly, miR-223 was downregulated in primary human bronchial epithelial cells exposed to cigarette smoke (30, 31). Conversely, miR-223 was one of the most highly expressed miRNAs in chronic obstructive pulmonary disease smokers versus normal smokers in a study by Ezzie et al. (32). miR-223 expression can be increased in response to LPS in murine bronchial epithelial cells and alveolar macrophages of mild asthmatics (33, 34). In CF airway epithelial cells, we found LPS decreased miR-223 expression. In this article, we show that CFTR is a target of miR-145, miR-223, and miR-494, and that their expression is increased in vivo in the bronchial epithelium of ΔF508 CFTR carriers.
The ΔF508 CFTR mutation encodes a misfolded protein that is rapidly degraded (35). More than 95% of ΔF508 CFTR is retained in the endoplasmic reticulum and subsequently degraded by the proteosome; consequently, less protein reaches the apical membrane and chloride ion transport is impaired. ΔF508 CFTR accounts for up to 70% of CFTR mutations worldwide, and its frequency is even higher in the Irish population, with 90% of CF individuals carrying one or two alleles (36). Whether the altered miRNA pattern evident here is unique to ΔF508 individuals or is more broadly representative of other CFTR mutations remains to be determined.
A number of possibilities exist regarding the relationship between increased miR-145, miR-223, and miR-494 expression and decreased CFTR. It may be an intrinsic event directly linked to ΔF508 CFTR, although this is unlikely given that miRNA modulation in 16HBE14o− cells expressing WT CFTR and in ΔF508-expressing CFBE41o− cells generated comparable data. Alternatively, the observation that CFTR dysfunction impacts on the expression of these specific miRNAs may point toward an autoregulatory loop and the fact that the miRNAs are regulated by defective chloride ion transport. In support of this, we found that miR-218 was not regulated in response to CFTRinh172, indicating a specific effect on these miRNAs. In addition, we demonstrated that manipulation of these miRNAs can influence CFTR function based on alterations in intracellular chloride levels in CF and non-CF cell lines after inhibition and overexpression studies. Thus, regulation by miRNAs may be a previously unidentified mechanism regulating CFTR. Interestingly, miR-494 also targets SLC12A2, an Na-K-Cl transporter (10). Therefore, the decrease in MQAE fluorescence caused by overexpression of the combined miRNAs may be due, in part, to altered function of SLC12A2 or other ion channels, not only CFTR; conversely, the upregulation of chloride conductance after inhibition of miRNAs may be caused by stimulation of alternative channels to CFTR.
Although the data presented in this study support the concept that P. aeruginosa–positive CF bronchial brush samples express higher levels of miR-145, miR-223, and miR-494, the levels of these miRNAs in P. aeruginosa–negative samples were also increased, most likely as a result of altered chloride ion conductance. Low numbers of samples in the P. aeruginosa–negative population is, in part, responsible for this variation in the data. Another factor potentially confounding these data is the possibility that the change in miRNA levels related to P. aeruginosa–positive sputum also may be caused by contributions from neutrophils harvested with the epithelial cells from the bronchial brushings of these patients. Nonetheless, P. aeruginosa can contribute to increased miRNA expression given that the expression of these miRNAs is increased in bronchial epithelial cell lines in vitro in response to stimulation with PCM, where there are no neutrophils present.
With respect to other organisms in the CF lung that could contribute to altered miRNA expression, although Aspergillus and S. aureus colonization were somewhat associated with increases in miR-145, miR-223, and/or miR-494, the patterns were less clear than for P. aeruginosa. Further in-depth studies examining the contribution of these organisms and their components to altered miRNA expression in CF bronchial epithelium are warranted given their importance as CF lung pathogens.
In conclusion, the observations that miR-145, miR-223, and miR-494 regulate CFTR expression and that their increased expression in vivo correlates with decreased ΔF508 CFTR expression are novel. We show that defective CFTR, IL-1β, and Pseudomonas colonization not due to LPS upregulate miR-145, miR-223, and miR-494 expression in CF bronchial epithelium in vitro and/or in vivo. These data implicate intrinsic, inflammatory, and infective factors as influences on miRNA regulation of CFTR. This knowledge may influence future therapies for CF and other diseases where modulation of CFTR expression might be important.
16HBE14o− and CFBE41o− cell lines were obtained as a gift from D. Gruenert (Pacific Medical Center Research Institute, San Francisco, CA). We thank Drs. S. Keely and M. Mroz (Molecular Medicine, Royal College of Surgeons in Ireland) for advice on MQAE experiments.
This work was supported by the Science Foundation Ireland (12/TIDA/B2265) and the Medical Research Charities Group and Health Research Board.
The online version of this article contains supplemental material.
Abbreviations used in this article:
cystic fibrosis transmembrane conductance regulator
cystic fibrosis transmembrane conductance regulator inhibitor-172
intracellular chloride concentration
human embryonic kidney
quantitative real-time PCR
The authors have no financial conflicts of interest.