Aim:To identify airway fibroblast miRNAs that contribute to interleukin-13 (IL-13)-induced airway remodeling in asthma.
Methods: Mild asthmatic (n = 13) and non-asthmatic human subjects (n = 16) underwent bronchoscopy with endobronchial biopsy. Cultured airway fibroblasts were treated with or without dexamethasone (dex) or IL-13 for 24 h. Total RNA was isolated and expression of miRNAs and elastin mRNA was measured by quantitative real time polymerase chain reaction.
Results: Three miRNAs were differentially expressed in unstimulated fibroblasts from asthmatic vs. non-asthmatic subjects (miR-21 and miR-125b were elevated, while miR-146a was decreased in asthma). Levels of miR-21 and miR-125b were inversely related to measures of lung function in asthmatic subjects and were repressed by dex. IL-13 affected the expression of 9 miRNAs in asthmatic airway fibroblasts (up-regulated: miRs-422, -98, -570-3p, -346, -1291, -937, and -338-3p; down-regulated: Let-7d and miR-106a), but had no effect on non-asthmatic control fibroblasts. Levels of miRs-937, -346, and -422 post-IL-13 stimulation were significantly associated with elastin mRNA levels in airway fibroblasts. Transforming growth factor-beta signaling was predicted to be a top target of the differentially regulated miRNAs.
Conclusion: Airway fibroblast miRNAs may play roles in IL-13-induced airway remodeling, thereby serving as potential therapeutic targets and novel biomarkers in asthma.
Keywords: MicroRNA, differential expression, asthma, allergy, interleukin-13, airway remodeling
Asthma is a common disease that is characterized by chronic inflammation that produces reversible narrowing and can lead to permanent obstruction of the airways. One of the most concerning features of asthmatic inflammation is the potential for airway remodeling, which refers to the structural changes that include the loss of epithelial integrity, thickening of the basement membrane, sub-epithelial fibrosis, and increased smooth muscle mass. These factors contribute to reduction in lung function, changes in airway hyper-responsiveness, and over time may lead to irreversible airway obstruction. The factors that underlie these changes are not well elucidated, but type 2 cytokines, interleukin-13 (IL-13) in particular, may play an important role in this process.
IL-13 is produced by T-helper 2 cells and innate lymphoid type 2 cells, and has been well established as a promoter of smooth muscle hypertrophy,[1-3] bronchial mucus secretion, and sub-epithelial fibrosis. IL-13 binds to a dimeric receptor (IL-13Rα1/IL-4Rα) that is present on airway fibroblasts, smooth muscle, and epithelial cells. Signaling through the IL-13 receptor is mediated by JAK/STAT6, and leads to induction of IL-13-dependent gene transcription.[1-4] IL-13 stimulates the production as well as the activation of preformed transforming growth factor-beta (TGF-β), which is thought to be a crucial determinant of airway remodeling. TGF-β has been shown to induce sub-epithelial fibrosis, airway smooth muscle proliferation, and mucus hypersecretion in asthma.[6,7]
MiRNAs are non-coding RNAs that are emerging as crucial regulators of inflammation. These small (~20 nucleotide long) RNAs are transcribed from their own genes or from introns of other genes, processed in the nucleus to pre-miRNA hairpins, and exported to the cytoplasm where they associate with the RNA-induced silencing complex (RISC). Binding of the miRNA/RISC to mRNA [usually in the 3’ untranslated region (UTR)] leads to inhibition or induction of translation of mRNA. A number of miRNAs have been found to be dysregulated in blood and lung fluid of asthmatics compared to non-asthmatic subjects.[9,10]
We previously identified miR-570-3p as a pro-inflammatory miRNA, which we found to be differentially expressed in the blood and airways of asthmatics compared to non-asthmatic controls, and was an effective inducer of cytokines and chemokines in airway epithelial cells. Recently, miR-21 was found to be up-regulated in a mouse model of infectious asthma, and found to induce steroid insensitivity through a phosphoinositide 3-kinase pathway. A number of anti-inflammatory miRNAs have been discovered as well. MiR-146a, a negative regulator of nuclear factor-kappa B (NF-κB) signaling, has been shown to be de-regulated in asthma and a number of inflammatory processes.[13-15] In addition, Kumar et al. demonstrated that miRNAs of the let-7 family repress IL-13, and we found that this miRNA was down-regulated in the lung fluid of asthmatics compared to healthy subjects.
The goals of our study were to identify candidate miRNAs that may play roles in airway remodeling in allergic asthma and serve as potential non-invasive biomarkers to identify populations of asthmatics that are at risk for airway remodeling. We found that a subset of miRNAs was differentially regulated in fibroblasts cultured from the airways of asthmatic subjects compared to non-asthmatic controls. In addition, IL-13 induced changes in expression of multiple miRNAs in asthmatic, but not control subject fibroblasts. The implications of these findings are discussed herein.
Twenty-nine subjects were recruited from the Denver, Colorado and Durham, North Carolina communities. The asthmatics fulfilled criteria for asthma exhibiting a provocative concentration of methacholine resulting in a 20% fall in the forced expiratory volume in 1 s (PC20) of < 8 mg/mL and reversibility, as demonstrated by at least a 12% and 200 mL increase in the forced expiratory volume in 1 s (FEV1) or the forced vital capacity (FVC) with inhaled albuterol. All subjects provided consent in these Duke University and National Jewish Health Institutional Review Board-approved studies.
Bronchoscopy and airway fibroblast culture
Subjects underwent bronchoscopy with endobronchial biopsy as previously described.[19,20] Endobronchial tissue was placed on collagen-coated plates and airway fibroblasts were cultured as previously described. As numerous passages can induce cell senescence and phenotypic changes, only cells from the first three passages were used for experiments. Non-asthma and asthmatic airway fibroblasts were cultured and passaged under identical conditions.
Airway fibroblasts were plated at 40,000 cells/well onto 12-well plates as previously described and incubated until 7 days past confluence. At baseline, these cells express alpha-smooth muscle actin and secrete collagen type 1, indicating a myofibroblast phenotype. Following 24 h in serum-free media, mediators were added and cells incubated for 24 h with IL-13 (IL-13; ProSpec, Rehovot, Israel; 50 ng/mL) or dexamethasone (dex) (Sigma-Aldrich, St. Louis, MO; 10-7 mol/L) or serum-free media (no treatment) as control. Total RNA from airway fibroblasts was extracted using TRIzol reagent (Gibco BRL, Rockville, MD) according to manufacturer’s protocol. Samples were stored at -80 °C until they were used for analysis.
A NanoDrop Lite was used to measure mRNA concentration and the A260/A280 ratio. A 3’ UTR adapter system was used to convert miRNA to complimentary deoxyribonucleic acid (cDNA) using the qScript miRNA cDNA Synthesis Kit (Quanta) per manufacturer instructions. Briefly, a poly A tail was added by mixing 300 ng of RNA with poly A polymerase and the Poly A tailing buffer and incubating for 60 min at 37 °C and then 5 min at 70 °C to stop the reaction. A 3’ adapter was added by annealing an oligo dT-linked to a universal adapter sequence (proprietary to Quanta), and this was used as a primer for reverse transcription using the qScript reverse transcriptase kit (reaction mix was incubated for 20 min at 42 °C followed by 5 min at 85 °C). For quantification via quantitative real time polymerase chain reaction (qPCR), a Bio-Rad CF1000 Touch Thermal Cycler was used by combining 1 µL of cDNA (diluted 1:10), 1 µL of Quanta 3’ universal reverse primer (2 µmol/L), 1 µL dd H2O, 5 µL PerfeCTa IQ SYBR Green Supermix, and 2 µL of primers specific to the miRNAs of interest (1 µmol/L). The thermocycle program was as follows: 95 °C for 3 min followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. Expression of a panel of 30 miRNA candidates previously found to be de-regulated in asthma was analyzed.[10,17] The qPCR data was normalized to a geometric mean of internal standards (SNORD44 and SNORD68). A stand curve was generated using known concentrations of miRNAs (synthesized in vitro by Integrated DNA Technologies), reverse transcribed with the qScript miRNA cDNA Synthesis Kit, and analyzed by qPCR using serial dilutions of cDNA. The standard curve was used to convert the normalized threshold cycle (CT) values from subject samples to copy number in the reaction mix.
Elastin mRNA analysis
First-strand cDNA was prepared using 1 µg of total RNA and random hexamers in a 50 µL reaction according to the manufacturer’s protocol (Applied Biosystems, Foster City, CA). Expression of elastin (ELN) (Genbank, accession # NM-000501; Applied Biosystems, Hs0355783-mL) and the housekeeping gene GAPDH (Applied Biosystems, Hs9999995-mL) was evaluated using the following 50 µL quantitative polymerase chain reaction (PCR) reaction: 50 ng cDNA, 100 nmol/L fluorogenic probe and 200 nmol/L primers and other components contributed from Applied Biosystems 2X reverse transcription polymerase chain reaction (RT-PCR) Master Mix. The specificity of PCR was verified by lack of a signal in no-template controls or RT (-) RNA samples. The CT was recorded for each sample, in each condition. Fold changes in ELN mRNA expression were calculated by normalization of all expression levels to GAPDH mRNA levels and then compared to those of untreated cells by the ∆∆CT method.
All analyses were performed using JMP (SAS, Cary, NC) and Prism (GraphPad, La Jolla, CA) statistical software. Expression differences, as depicted by copy numbers, between miRNAs in asthma vs. non-asthma was determined by two tailed t-tests with false discovery rate correction of 5% to adjust for multiple hypothesis testing. Changes in expression with either dex or IL-13 were calculated using two-tailed, paired t-tests comparing each condition to untreated cells with a significance cutoff of P < 0.05. Single linear regression analyses were performed to demonstrate the associations between miRNA copy numbers and the provocative PC20, the pre-bronchodilator FEV1 (L), or IL-13-induced mean percentage fold change in ELN mRNA expression data for each subject to determine the Pearson’s correlation coefficient.
Samples were collected from 29 subjects (n = 13 asthmatics, n = 16 non-asthmatic controls). The asthmatic subjects were predominantly male, while age, ethnicity, and body mass index were similar between the groups. Diagnosis of asthma and airway hyper-responsiveness was confirmed by methacholine testing (asthmatics PC20 of 0.65 ± 0.23 mg/mL vs. > 16 in the non-asthmatic group) and response to bronchodilator [Table 1]. As expected, the asthmatics were characterized by increased airway obstruction [significantly reduced FEV1%-predicted (78 ± 4% vs. 97 ± 2%, P < 0.01) and post-bronchodilator FEV1/FVC ratio: 73 ± 2% vs. 83 ± 2%, P < 0.01]. The asthmatic subjects used albuterol as needed, and no controller medications. The non-asthma control subjects took no chronic medications.
Table 1: Subject demographicsClick here to view
MiRNA differences in airway fibroblasts between asthmatic and non-asthma controls
The expression of 30 miRNAs that we previously found to be differentially expressed in blood and lung fluid of asthmatic vs. non-asthmatic subjects was profiled in airway fibroblasts.[10,17] Quantitative real time PCR analysis revealed three miRNAs that were differentially expressed in airway fibroblasts of asthmatic and non-asthmatic groups. In unstimulated fibroblasts from asthmatic vs. non-asthmatic subjects, expression of miR-21 and miR-125b were significantly elevated, while expression miR-146a was reduced [Figure 1A].
Figure 1: miRNAs differentially expressed in untreated airway fibroblasts of asthmatics vs. non-asthmatics. (A) miRNA differences between asthmatic and non-asthma control airway fibroblasts without stimulation and with dex stimulation; (B) baseline expression of miR-21 in airway fibroblasts is significantly and inversely related to FEV1 in asthmatics, but not non-asthma control subjects; (C) baseline expression of miR-125b is significantly and inversely related to methacholine PC20 values. dex: dexamethasone; FEV1: forced expiratory volume in 1 s; PC20: concentration of methacholine that decreases FEV1 by 20%. *: P < 0.05; NS: not significantClick here to view
We next sought to determine whether miRNA expression in asthmatic airway fibroblasts was associated with any clinical features of asthma. Baseline expression of miR-21 was significantly and inversely related with FEV1 in asthmatic subjects only, suggesting that higher miRNA expression correlates with increased airway obstruction [Figure 1B]. In addition, baseline levels of miR-125b were significantly and inversely associated with methacholine PC20 values, demonstrating a link between miRNA expression and airway hyper-reactivity [Figure 1C]. This relationship was not assessed in the non-asthmatic group as they did not exhibit a significant drop in lung function with methacholine.
To gain insight into potential targets of these miRNAs, we performed a pathway analysis using the online tools Targetscan, DIANA-miRPath v3.0, and Reactome. The predicted mRNA targets generated from the Targetscan analysis were used to classify the main predicted functions with the Reactome tool [Supplemental Figure S1]. The majority of predicted targets were immune-related genes and regulators of signal transduction [Supplemental Figure S1, yellow lines], which are consistent with the expected roles of inflammatory mediators in asthmatic airway fibroblasts. Kyoto encyclopedia of genes and genomes (KEGG)-pathway analyses indicated that the three miRNAs regulate mediators of Toll-like receptor signaling and tumor necrosis factor-alpha signaling, including cytokines (IL-1B, IL-6, IL-8, CCL20, CXCL10), TGF-β signaling (TGFB1, TGFB2, TGFBR1, TGFBR2) signal transduction molecules (PIK3-Akt signaling, AP-1, NF-kB signaling) and other inflammatory molecules (MMP9, VEGF, ICAM1) [Table 2].
Table 2: Predicted inflammatory genes regulated by miR-21, miR-125b, and miR-146aClick here to view
Effects of dexamethasone and IL-13 on asthmatic and non-asthma control airway fibroblasts
We next sought to determine whether glucocorticoids affected miRNA expression, as this class of medication is the main anti-inflammatory treatment for asthma and may prevent airway remodeling. Therefore, miRNAs altered by glucocorticoids may represent potential therapeutic targets. Treatment of the asthmatic airway fibroblasts with dex resulted in normalization of expression of miR-21 and miR-125b to levels of the non-asthma controls. Expression of miR-146a did not significantly change with dex, but there was a trend towards increased expression. Glucocorticoids did not have any effect on expression of other miRNAs in airway fibroblasts of asthmatics, and had no effect on any miRNAs in non-asthma control subjects. In contrast, IL-13 significantly affected the expression of nine miRNAs in asthmatic airway fibroblasts (up-regulated: miRs-422, -98, -570-3p, -346, -1291, -937, and -338-3p; down-regulated: Let-7d and miR-106a) [Figure 2A]. IL-13 did not significantly alter the expression of any miRNAs in the non-asthma subjects, suggesting that the asthmatic airway fibroblasts are uniquely susceptible to the effects of this type 2 cytokine.
Figure 2: Effects of interleukin-13 (IL-13) on miRNA expression in airway fibroblasts. (A) Differences between asthma and non-asthma, IL-13 stimulation; (B-D) associations between miRs-937, -346, -422 and elastin (ELN) mRNA expression levels in post-IL-13-stimulated asthmatic and non-asthmatic airway fibroblasts. *: P < 0.05; NS: not significantClick here to view
Next, we sought to determine whether IL-13-induced miRNAs were associated with production of the extracellular matrix protein, elastin, a marker of fibrosis. We found that post-IL-13-stimulated levels of miRs-937, -346, and -422 were significantly and positively associated with IL-13-induced elastin mRNA levels in airway fibroblasts from asthmatics [Figure 2B-D]. In non-asthma control subject fibroblasts, miR-937 expression was inversely related to elastin mRNA levels, while there was no significant association found between miRs-346 and -422 and elastin mRNA expression [Figure 2B-D].
We then performed a similar pathway analysis as described above to gain insight into the functions of the miRNAs whose expression was altered by IL-13. The top KEGG-pathway that was predicted to be regulated by the subset of miRNAs was TGF-β signaling [Figure 3 and Table 3]. Multiple genes, including TGFBR2, TFGBR1, and TGFB2 were targets of multiple IL-13-regulated miRNAs. Other signaling mediators, such as SMADs (SMAD2, SMAD3, SMAD4, SMAD5) and mitogen-activated protein kinases were also targets.
Figure 3: Kyoto encyclopedia of genes and genomes pathway analysis of interleukin-13 (IL-13)-regulated miRNAs from airway fibroblasts. IL-13 regulated miRNAs in airway fibroblasts are predicted to regulate multiple components of transforming growth factor-beta signalingClick here to view
Table 3: Interleukin-13 altered miRNAs in asthmatics that are predicted to regulatetransforming growth factor-beta signalingClick here to view
MiRNAs are emerging as crucial regulators of inflammation and immunity. However, their roles in asthma, their function in lung cells, and contributions to specific features of asthma pathobiology such as airway remodeling are not well understood. Our findings that expression of miRNAs is modulated at baseline in airway fibroblasts in asthmatics raise the possibility that they may regulate asthmatic inflammation and could be either therapeutic targets, and/or potential biomarkers to assess airway remodeling. In addition, IL-13 is known to contribute to airway remodeling through stimulation of airway fibroblast differentiation, proliferation, invasion and matrix production[19,22,23,27] though the exact mechanisms and mediators that drive these actions on airway fibroblasts have not been elucidated. MiRNAs may convey the effects of IL-13 and could shed light onto how this cytokine mediates profibrotic processes. The implications of these findings will be discussed herein.
The miRNAs that we observed as differentially expressed at baseline in airway fibroblasts of asthmatics, miR-21, miR-125b, and miR-146a, are known candidate biomarkers and suspected regulators of inflammation in asthma. The significant associations between airway fibroblast miR-21 expression and FEV1, and miR-125b expression and methacholine PC20 in asthma subjects indicate that these miRNAs are associated with features of worsening asthma severity and airway hyper-reactivity. These findings are in line with prior studies that have demonstrated that miR-21 promotes eosinophilia, mucus production, smooth muscle hypertrophy and airway hyper-responsiveness, which are hallmarks of asthmatic inflammation. Recently, a mouse model of infection and allergy demonstrated that miR-21 was elevated in the setting of corticosteroid-insensitive airway disease. This effect appeared to be due to disruption of the Akt-PI3K pathway, which was identified as a target for miRNA regulation in our in silico studies. Furthermore, the authors found that inhibition of miR-21 attenuated airway inflammation and restored corticosteroid sensitivity, suggesting that miR-21 might be an important therapeutic target in asthma.
Similarly, miR-125b may act to promote inflammation in the airway. An association was shown in the upper airway between miR-125b expression in epithelial cells from nasal mucosa and chronic eosinophilic rhinosinusitis with polyps. A subsequent study found that miR-125b was part of panel of six miRNAs that was able to discern whether a person had allergic rhinitis or asthma. These results were similar to our previous findings that this miRNA was dysregulated in asthma, and expression miR-125b was increased in allergic and asthmatic inflammation in all three studies, in line with our findings in this study.[10,28,29]
In contrast to miR-21 and miR-125b, miR-146a may serve as an anti-inflammatory mediator of transcription. MiR-146a is part of the negative feedback loop between T-cell receptors and NF-κB. When miR-146a is absent from T-cells there is a release of the repression of NF-κB, one of the central pro-inflammatory transcription factors in asthma.[13-15] In addition, our lab has demonstrated that miR-146a exerts anti-inflammatory effects in airway epithelial cells, and over-expression enhances the anti-inflammatory effect of the glucocorticoids (Ishmael lab, unpublished data). Thus, the reduced expression of miR-146a coupled with elevated expression of miRs-21 and 125b in asthmatic airway fibroblasts would be expected to promote inflammation and fibrosis in the airway. Moreover, the ability of glucocorticoids to normalize miR-21 and miR-125b levels to the levels of non-asthma control subjects raises the possibility that at least some of the effects of steroids are mediated by altering miRNA expression levels. Further research will be needed to determine whether the effects of steroids on these miRNAs is lost in severe or steroid-resistant asthma, and whether targeting these miRNAs could be tapped for novel therapeutic approaches.
The pattern of miRNA response to IL-13 may shed some light on how these miRNAs interact to regulate a pathogenic state in allergic asthma. It is interesting to note that none of the miRNAs that were differentially expressed in asthmatic vs. non-asthmatic fibroblasts (miRs-21, -125b, -146a) were altered by IL-13. These findings suggest that these miRNAs may be de-regulated in asthma via IL-13 independent mechanisms, and that IL-13 may exert its effects via other miRNAs. One of the IL-13 down-regulated miRNAs, Let-7d, has been shown to regulate IL-13 expression, and administration of exogenous Let-7 in mice reduced IL-13 levels and led to a decrease in airway inflammation, airway hyper-responsiveness, mucus production, and subepithelial fibrosis in an asthma model. Further evidence of the importance of Let-7 in airway fibroblasts (and possibly airway remodeling) was demonstrated by its anti-inflammatory role in idiopathic pulmonary fibrosis. Thus, reduction of Let-7a, as we observed with IL-13, may enhance the pro-fibrotic responses of fibroblasts.
The roles of the other miRNAs that exhibited IL-13-dependent expression changes have not been as well characterized in asthma. The results of our gene ontology and pathway analyses shed light on how these miRNAs may interact to regulate airway inflammation and remodeling. A KEGG-pathway analysis found that TGF-β signaling was the top target when all nine of the IL-13-regulated miRNAs were combined into the in silico analysis. Predicted targets included TGF-β, its receptors, SMADs, and other signaling mediators. The exact mechanisms of how these miRNAs function in regulation of TFG-β signaling is not clear. As miRNAs most often repress gene expression, it would be expected that the miRNAs that directly target TGF-β, its receptor, or signal transduction mediators would inhibit this pathway. In this setting, it makes sense than down-regulation of Let-7d and miR-106a could promote TGF-β signaling.
We have shown that IL-13 stimulates significantly increased airway fibroblast invasion and type I collagen production in asthma through mechanisms that require TGF-β signaling.[23,27] Furthermore, we have demonstrated that airway fibroblasts derived from allergic asthmatics exhibit significantly reduced cell surface expression of IL-13Rα2, a possible negative regulator of IL-13-signaling, as compared to cells from non-asthma control subjects. Thus, airway fibroblasts in allergic asthma are intrinsically primed to respond to IL-13, resulting in IL-13-induced pro-fibrotic responses.
For the miRNAs that were upregulated by IL-13, such as miR-1291, it is possible that these are induced by inflammatory stimuli as a form of feedback inhibition. Thus elevation of these miRNAs may not be pathogenic, but rather a secondary change as the result of an inflammatory stimuli. This “chicken or the egg” scenario is one of the limitations this and other miRNA studies. This scenario may be the case for miRs-937, -346, and -422, which were induced by IL-13 and significantly associated with elastin mRNA expression. We previously demonstrated that IL-13 reduces elastin levels in the airway of asthmatics, which may result in enhanced airway collapsibility and loss of elastic recoil, thereby increasing airway obstruction. As IL-13 induces expression of these miRNAs, this could be a counter-regulatory mechanism to limit the effects of IL-13 on elastin levels. For miR-937 specifically, we found opposing associations with elastin levels in asthmatic vs. non-asthmatic fibroblasts after IL-13 stimulation. Clearly, IL-13 has different effects on the miRNA in these cells, but it remains to be seen whether miR-937 mediates pathogenic effects, could alternatively have a protective effect, or could just be a disease marker. Further work will need to be performed to determine whether these miRNAs have functional effects in this process. However, it is clear that IL-13 is a central contributor to airway remodeling, and our findings implicate miRNAs as potential mediators of transcriptional regulation of airway fibrosis.
It is important to point out that a key limitation in our study was a sex disparity in the asthmatic and non-asthmatic groups. Our findings that dex restored levels of miR-21 and miR-125b in asthmatic fibroblasts to that of non-asthmatic fibroblasts, suggests that the elevation of these miRNAs was due to the inflammatory state rather than sex. Furthermore, our prior work did not find sex-specific differences in miRNA expression in asthmatic subjects vs. non-asthmatic controls.[10,17] However, this point will need to be further studied, both for miRNAs differentially expressed in untreated, and IL-13 simulated asthmatic vs. non-asthmatic fibroblasts.
In summary, our findings that miRNAs are dysregulated in asthmatic airway fibroblasts and are altered by IL-13, have significant biomarker and therapeutic implications. For instance, measurement of a miRNA profile in lung fluid (such as sputum, exhaled breath condensate, or bronchoalveolar lavage fluid) could have predictive value to identify subjects who are at risk for airway remodeling. As some of the miRNAs returned to normal levels with glucocorticoids, maximizing inhaled glucocorticoid treatment could be envisioned as a treatment approach in these patients. In addition, as it is possible to deliver aerosolized miRNAs or inhibitors to the lungs, these findings may be targeted for novel therapeutics. For example, delivering miRNAs such as miR-146a or Let-7d to the lungs could have anti-inflammatory effects, and a similar approach could be used with antagomirs to inhibit pro-inflammatory miRNAs like miR-21 and miR-570-3p. Taking this a step further, miRNA therapies could be uniquely tailored to a specific patient, to replace or inhibit miRNAs based on their unique miRNA profile in the lungs. As such, these results may represent the next step to personalize asthma diagnosis, prognosis, and management.
Designing and performing study: F.T. Ishmael, J.L. Ingram, M. Kraft
Data collection/analysis and writing of the manuscript: R. Vender, K. Trank
Financial support and sponsorship
This work was supported by NIH/NHLBI 2R01 HL064619-071A (J.L.I., M.K.), Doris Duke Charitable Foundation CSDA grant (F.T.I.), and NSF ATE 1204209 (K.T., F.T.I).
Conflicts of interest
There are no conflicts of interest.
Written consent was provided by the patient for the present study.
The study has been approved by the Duke University Institutional Review Board.
Supplemental Figure S1: Reactome based on predicted functions of miRs-21, -125b, and -146a identify targets in immune-related genes and regulators of signal transductionClick here to view
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