Rationale: Chronic obstructive pulmonary disease (COPD) is characterized by progressive inflammation in the airways and lungs combined with disturbed homeostatic functions of pulmonary cells. MicroRNAs (miRNAs) have the ability to regulate these processes by interfering with gene transcription and translation.
Objectives: We aimed to identify miRNA expression in induced sputum and examined whether the expression of miRNAs differed between patients with COPD and subjects without airflow limitation.
Methods: Expression of 627 miRNAs was evaluated in induced sputum supernatant of 32 subjects by stem–loop reverse transcription-quantitative polymerase chain reaction. Differentially expressed miRNAs were validated in an independent replication cohort of 41 subjects. Enrichment of miRNA target genes was identified by in silico analysis. Protein expression of target genes was determined by ELISA.
Measurements and Main Results: Thirty-four miRNAs were differentially expressed between never-smokers and current smokers without airflow limitation in the screening cohort. Eight miRNAs were expressed at a significantly lower level in current-smoking patients with COPD compared with never-smokers without airflow limitation. Reduced expression of let-7c and miR-125b in patients with COPD compared with healthy subjects was confirmed in the validation cohort. Target genes of let-7c were significantly enriched in the sputum of patients with severe COPD. The concentration of tumor necrosis factor receptor type II (TNFR-II, implicated in COPD pathogenesis and a predicted target gene of let-7c) was inversely correlated with the sputum levels of let-7c .
Conclusions: let-7c is significantly reduced in the sputum of currently smoking patients with COPD and is associated with increased expression of TNFR-II.
MicroRNAs (miRNAs) are small regulatory noncoding RNAs that control important biological processes, including cellular differentiation, apoptosis, and proliferation. At present, their role in chronic obstructive pulmonary disease (COPD) pathogenesis is unknown.
miRNAs are measurable in induced sputum supernatant. Cigarette smoking has a significant influence on miRNA expression. Several miRNAs are associated with the severity of airway obstruction in COPD, independent of smoking.
COPD combines two major features: narrowing of the small airways (mainly because of chronic bronchiolitis) and destruction of the alveolar walls (emphysema) (4). A chronic inflammatory process that persists even after smoking cessation is thought to be responsible for the pathological features of COPD. Impaired antimicrobial responses with subsequent low-grade infections or microbial colonization could maintain the inflammatory process (5, 6). In the more severe cases of COPD, defective regulation of tolerogenic immune mechanisms could lead to autoimmune responses, which in turn contribute to the damaging inflammation (7, 8). In addition, dysfunction of pulmonary homeostatic processes, increased apoptosis, and accelerated aging have been demonstrated in the lungs of patients with COPD (9, 10). Genetic and epigenetic changes could contribute to these altered immune responses and homeostatic mechanisms in the lungs of smokers susceptible to COPD (11, 12).
MicroRNAs (miRNAs) are a class of small noncoding RNAs with a regulatory function on gene expression. They are approximately 19- to 25-nucleotide, single-stranded and highly conserved RNAs. miRNAs bind the 3′ untranslated region (3′ UTR) of target messenger RNAs, leading to direct inhibition of protein translation or degradation of messenger RNA (13, 14). In addition, miRNAs can alter gene expression by targeting transcription factors and DNA methyltransferases. In this way, miRNAs can interact with hundreds of genes simultaneously and regulate several biological processes such as cellular proliferation, differentiation, and apoptosis. miRNAs have been implicated in the pathogenesis of various malignancies (15) and cardiovascular (16), endocrine (17), and neurological diseases (18). Studies have described a role for certain miRNAs in asthma and lung fibrosis (19–22). The role of miRNAs in the pathogenesis of smoking-induced nonmalignant respiratory diseases such as COPD has not yet been elucidated.
Taking into account the deregulated mechanisms in immune responses and cellular homeostasis in COPD and the regulatory role of miRNAs, we hypothesized that miRNA expression is altered in the lungs of smokers and patients with COPD. Therefore, we optimized a method to detect miRNAs in induced sputum supernatant, which is a minimally invasive assessment of the biological processes in lower airways. We found significantly differential expression of miRNAs in patients with COPD and current smokers compared with never-smokers. Moreover, we found a significant association between several miRNAs and FEV1. In silico analysis demonstrated that these miRNAs target a network of several key molecules in COPD pathogenesis. Some of the results of this study have been previously reported in the form of an abstract (23).
In total, 73 subjects participated to this study. First, 32 subjects participated in the initial screening cohort. Participants were classified into three groups: never-smokers without airway obstruction and current smokers without airway obstruction (recruited by an advertising campaign), and currently smoking patients with COPD (recruited from our outpatient clinic). Second, to validate the results obtained in the screening cohort, 41 additional subjects were recruited for an independent validation cohort. Current smokers were defined as smokers who still smoked at the moment of participation in the study or had quit smoking for less than 1 year before their participation in the study. Subjects were classified in the COPD group if they had a postbronchodilator FEV1/FVC ratio of less than 0.70 (1). All patients with COPD had stable disease: patients with symptoms or clinical signs of a COPD exacerbation in the 2 months before the study were excluded. For safety reasons, patients suffering from severe and very severe COPD (GOLD [Global Initiative for Chronic Obstructive Lung Disease] stages III and IV) were excluded. Other exclusion criteria included a diagnosis of asthma, bronchiectasis, lung cancer, or an upper or lower respiratory tract infection in the preceding 4 weeks. Written informed consent was obtained from all subjects according to protocols approved by the medical ethics committee of Ghent University Hospital (Ghent, Belgium).
Sputum induction and processing were performed as previously described (24). A summary of the protocol is available in the online supplement. One hundred microliters of sputum supernatant was processed with an RNeasy micro kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.
miRNA expression profiling was performed as described previously (25). miRNA expression data were normalized on the basis of the mean miRNA expression per sample, as described previously (26). More details and the miRNA expression data sets are available in the online supplement.
Sputum thin-layer cell preparations (Cytospins) (Shandon Cytospin 4; Thermo Fisher Scientific, Waltham, MA) were incubated with locked nucleic acid probes for miRNA detection (Exiqon, Vedbæk, Denmark) as described in the online supplement.
The concentration of soluble tumor necrosis factor receptor II (sTNFR-II) (official name: TNF superfamily member 1B, 75 kD) was measured in sputum supernatant with a human sTNFR-II ELISA kit (R&D Systems Inc, Minneapolis, MN).
Network core analysis of predicted miRNA target genes was performed with the Ingenuity Pathway Analysis platform (Ingenuity Systems Inc, Redwood City, CA). Gene set enrichment analysis (GSEA) (27) and evaluation of enrichment of miRNA binding sites in the 3′ UTR of selected genes are described in the online supplement.
Demographic and clinical characteristics of the study population are expressed as means and standard deviations. Categorical variables were analyzed by means of the Fisher exact test. Continuous variables were analyzed by the Kruskal-Wallis test, using SPSS 16.0 software (SPSS Inc, Chicago, IL).
Statistical analysis on reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed with R Bioconductor software (version 2.9). Differential expression of an miRNA between any of the two different study groups was assessed by Mann-Whitney U test. Correlations between FEV1 and miRNA expression data were analyzed by the Spearman rank test. In the screening cohort, significant P values were determined after correction for multiple testing by the Benjamini-Hochberg algorithm. The association between FEV1 or FEV1/FVC and miRNA expression was further analyzed by linear regression analysis, using the enter method. P values less than 0.05 were considered significant.
Ten never-smokers, 10 current smokers without airway obstruction, and 12 subjects with stable COPD (no exacerbation in the preceding 2 mo) were included in the initial screening cohort. Ten patients with COPD were classified as stage II according to the GOLD criteria, whereas two patients were GOLD stage I. All patients with COPD were current smokers. The characteristics of the different study groups are summarized in Table 1. Total and differential sputum cell counts are shown in Table E1 in the online supplement.
Current Smokers with COPD
|Age, yr||53.9 (6.1)||53.3 (6.4)||63.2 (8.9)||0.01|
|Pack-years||0||44.6 (28.6)||62.2 (25.1)||<0.001|
|FEV1 (% pred)||105.9 (18.6)||98.3 (12.9)||71.9 (11.2)||<0.001|
|FEV1/FVC, %||79.9 (4.8)||78.8 (3.9)||57.8 (9.3)||<0.001|
Of the 627 miRNAs tested, 212 individual miRNAs were successfully detected in induced sputum. Given that only two small RNA controls were expressed in all sputum samples we used the mean expression of all expressed miRNAs per sample as a normalization factor. To validate the stability of the mean expression value we first selected a set of miRNAs, the expression profile of which is similar to that of the mean expression value, by calculating the geNorm pair-wise variation V value (28). The 10 miRNAs with the lowest V value were used for a geNorm analysis together with the mean expression value as described previously (26). geNorm analysis of sputum miRNA revealed that the mean expression value ranked among the most stable normalizers, suggesting it serves as a suitable normalization factor in our data set (Figure 1).
We first compared the expression of 627 individual miRNAs between never-smokers and current smokers without airflow limitation (Table 2, column A). This revealed a set of 34 miRNAs that were differentially expressed between these two study groups. The majority of these differential miRNAs were decreased in current smokers (27 of 34 miRNAs). Correlations between the expression levels of these miRNAs and sputum cell counts are displayed in Table E2. Differentially expressed miRNAs correlated mainly with macrophage and lymphocyte cell counts, but not with neutrophils or eosinophils.
A: Current Smokers without COPD vs. Never-Smokers
B: Current Smokers with COPD vs. Never-Smokers
|MicroRNA ID||Fold Change (median)||P Value||Fold Change (median)||P Value|
Binding sites of these differentially expressed miRNAs were analyzed for enrichment in gene sets of interest. Genome-wide association studies discovered genes that were associated with reduced lung function (29, 30). Binding sites of miR-150 were significantly enriched genes that were associated with impaired lung function (Table E3).
In patients with COPD, the expression of eight miRNAs was significantly lower compared with never-smokers (Table 2, panel B). There was no significant difference in sputum miRNA expression between current smokers and patients with COPD. However, significant associations between FEV1 (% predicted) and eight individual sputum miRNAs were identified by univariate analysis (Table E4).
The individual miRNAs that correlated with FEV1 were further analyzed by linear regression to adjust for possible confounders including age, sex, smoking (number of pack-years), and treatment with inhaled corticosteroids. The expression of six individual miRNAs remained significantly associated with FEV1 (% predicted), even after adjustment for these confounders (Table 3). The relationship between each of these miRNAs and FEV1 is plotted in Figure 2. In silico network core analysis of the predicted target genes of these miRNAs is shown in Figure E1.
|hsa-let-7c||0.49||FEV1 (% pred)||0.493||0.011|
|hsa-miR-34b||0.43||FEV1 (% pred)||0.460||0.024|
|hsa-miR-34c||0.51||FEV1 (% pred)||0.442||0.019|
|hsa-miR-125a-5p||0.49||FEV1 (% pred)||0.379||0.047|
|hsa-miR-30a-3p||0.57||FEV1 (% pred)||0.493||0.013|
|hsa-miR-30e-3p||0.52||FEV1 (% pred)||0.433||0.021|
Characteristics of the 41 study subjects of the validation cohort are shown in Table 4. Eight currently smoking patients with COPD and nine ex-smoking patients with COPD were classified as GOLD stage II. All other patients with COPD were GOLD stage I. Sputum differential cell counts are shown in Table E5. Table 5 shows the differentially expressed miRNAs from the screening study that were also differentially expressed between study groups in the validation cohort. let-7c and miR-125b levels were significantly lower in the sputum of currently smoking patients with COPD compared with never-smokers without airflow limitation (Figure 3). Moreover, let-7c and miR-125b levels were also significantly lower in currently smoking patients with COPD compared with current smokers without airflow limitation. The sputum levels of let-7c and miR-125b were not different between ex-smoking patients with COPD and subjects without airflow limitation. When focusing on the subgroup of current smokers with or without COPD, there was an independent association between let-7c and miR-125b expression and the FEV1/FVC ratio, which remained significant even after adjusting for confounders including time since the subject had quit smoking and the number of pack-years (Table 6). Seven miRNAs (let-7c, miR-150, miR-146a, miR-449, miR-203, miR-340, and miR-222) from the initial screening study that were down-regulated by current smoking were confirmed in the validation cohort, especially in the comparison between current and ex-smoking patients with COPD.
Current Smokers with COPD
Ex-Smokers with COPD
|Age, yr||42.3 (15)||41.8 (12)||58.4 (7)||73.5 (5)||<0.01|
|Pack-years||0||17.4 (13)||53 (24)||33.0 (15)||<0.01|
|FEV1 (% pred)||104.7 (15)||107.2 (18)||72.2 (11)||63.8 (11)||<0.01|
|FEV1/FVC, %||81.8 (7)||81.7 (6)||57.6 (12)||55.0 (9)||<0.01|
A. Current Smokers without COPD vs. Never-Smokers
B. Current Smokers with COPD vs. Never-Smokers
C. Current Smokers with COPD vs. Current Smokers without COPD
D. Current Smokers with COPD vs. Ex-Smokers with COPD
|MicroRNA ID||Fold Change||P Value||Fold Change||P Value||Fold Change||P Value||Fold Change||P Value|
|Quit smoking, yr||−0.06||0.792|
|Quit smoking, yr||−0.12||0.568|
Gene set enrichment analysis (GSEA) was performed for target genes of the miRNAs that were confirmed to be differentially expressed between subjects without airflow limitation and patients with COPD. GSEA was computed on the basis of a publicly available data set (Gene Expression Omnibus GSE22148). This data set contains sputum messenger RNA expression profiles of patients with COPD. GSEA showed significant enrichment for target genes of let-7c in patients with severe and very severe COPD compared with patients with moderate COPD (P = 0.04) (Table E6). Target genes of let-7c enriched in these sputum samples are shown in Table E7. TNFR-II (TNF superfamily member IB) is a target gene of let-7c that is enriched in the sputum of patients with severe and very severe COPD.
Levels of soluble (s) TNFR-II were measured in the available sputum supernatant of a subgroup of the validation cohort (n = 27). The study group for this experiment consisted of never-smokers (n = 7), current smokers without airflow limitation (n = 4), patients with COPD that were current smokers (n = 6), and COPD ex-smokers (n = 10). Results are shown in Figure 4. The sputum concentration of sTNFR-II was significantly higher in patients with COPD compared with never-smokers (P = 0.04). The protein level of sTNFR-II in sputum was inversely correlated with the level of let-7c (r = −0.43; P = 0.03).
let-7c expression was detected in sputum cells on Cytospins by in situ hybridization (Figure 5). let-7c expression was located mainly in sputum macrophages and bronchial airway epithelial cells. We could not detect expression of let-7c in granulocytes and lymphocytes.
This study extends the work of Schembri and colleagues, who were the first to assess miRNA levels in airway epithelium of nonsmokers and smokers without airflow limitation (31). Our study identified and validated miRNAs in induced sputum that were significantly reduced by current smoking. Importantly, two miRNAs (let-7c and miR-125b) were significantly lower in currently smoking patients with COPD compared with healthy subjects.
This study focused on induced sputum supernatant. Sputum induction is a safe and minimally invasive assessment of airway biological processes (32). Previously, the presence of miRNAs has been reported in whole induced sputum, serum, and some other body fluids such as saliva (33–35).
Screening for differential miRNA expression in induced sputum supernatant of current smokers versus never-smokers revealed a set of 34 individual miRNAs. Most of these miRNAs are decreased in smokers, indicating that the overall suppressive effect on gene transcription and translation through miRNAs is reduced by smoking. Seven miRNAs that were differentially expressed between never-smokers and current smokers without airflow limitation were also down-regulated in a rat model of short-term cigarette smoke exposure (miR-34c, let-7c, miR-125b, miR-26a, miR-34b, miR-191, and miR-222). In addition, three of these miRNAs were also down-regulated in cigarette smoke–exposed mice (miR-125b, miR-26a, and miR-34b) (36, 37). Interestingly, of all 34 miRNAs that are influenced by cigarette smoking, miR-150 could be of importance in the initiation of smoke-induced decline of lung function, as genes that were associated with lung function impairment in genome-wide association studies are significantly enriched in binding sites for this miRNA.
In the validation cohort, only one differentially expressed miRNA was confirmed when comparing never-smokers and current smokers without airflow limitation. This is probably due to the smaller number of pack-years smoked in the current smoker group of the validation cohort compared with the current smokers of the screening cohort. Interestingly, of the 34 individual miRNAs that were significantly different when comparing never-smokers and current smokers without COPD in the screening cohort, 7 miRNAs were also differentially expressed between current smokers with COPD versus ex-smoking patients with COPD in the validation cohort, confirming the reduction of the expression of these miRNAs by smoking as such.
When comparing the sputum of patients with COPD with that of never-smokers, a smaller set of eight differential miRNAs was found in our initial screening cohort. All these miRNAs were decreased both in patients with COPD and in current smokers without airflow limitation compared with never-smokers (except for miR-125a-5p). There were no significant differences in miRNA expression between the patients with COPD and the current smokers without airflow limitation in our initial screening cohort. It is possible that the initial screening study is somewhat underpowered to detect differences in miRNA expression between smokers without airflow limitation and patients with COPD. Large intragroup variation of miRNA expression levels found in the COPD group, reflecting the heterogeneity of the disease and the use of different medications, such as inhaled corticosteroids, could contribute to the lack of differences in miRNA expression between current smokers and patients with COPD.
To address the issues of various, possibly confounding factors, we performed a linear regression analysis, investigating the association between miRNA expression in induced sputum and FEV1 (% predicted). By this approach, we identified a set of six miRNAs that were significantly associated with FEV1, even after adjustment for age, sex, number of pack-years smoked, and treatment with inhaled corticosteroids.
Target prediction and pathway analysis revealed that these six miRNAs work as brakes on the translation of intracellular signaling molecules such as mitogen-activated protein kinase-14 and STAT-3 (signal transducer and activator of transcription-3). These signaling molecules are located downstream of TNF-α, a well-known key molecule in the pathogenesis of COPD. Reduced expression of these miRNAs in smokers susceptible to COPD could increase the expression of these signaling molecules, enhancing the proinflammatory effect of TNF-α. Evidence that the expression of STAT-3 is increased in COPD supports this concept (38).
miRNAs that were differential between never-smokers and patients with COPD in the screening cohort were analyzed in the independent validation cohort. Our validation study confirmed the significant decrease in expression of let-7c and miR-125b in currently smoking patients with COPD compared with never-smokers. More importantly, these miRNAs were also significantly lower in current smokers with COPD compared with current smokers without COPD. This indicates that both miRNAs were not only affected by current smoking, but that their expression was even further reduced in currently smoking patients with COPD. This was already suggested by the independent correlation of let-7c with FEV1 in our initial screening cohort and is confirmed by the association between FEV1 or FEV1/FVC and let-7c in our validation cohort.
Importantly, sputum levels of let-7c and miR-125b were not altered in ex-smoking patients with COPD compared with healthy control subjects. This suggests that these miRNAs are involved mainly in the development of COPD due to smoking, but not in the persistence of airway inflammation despite smoking cessation.
To elucidate the biological relevance of the detected miRNAs in the pathogenesis of COPD, we performed gene set enrichment analysis. We found significant enrichment of predicted target genes of let-7c in the sputum of patients with severe and very severe COPD. In addition, sputum let-7c levels were significantly inversely correlated with sputum protein levels of sTNFR-II, which is a primary target of let-7c. These findings are compatible with the functional role of let-7c in inhibiting the translation of its target genes.
In the past, our group has shown that TNFR-II is implicated in COPD pathogenesis. In a mouse model of COPD, TNFR-II knockout mice were protected against cigarette smoke–induced inflammation and emphysema (39). In addition, other groups have shown that TNFR-II is increased in the sputum of patients with COPD (40, 41). Our data suggest that the cigarette smoke–induced decrease in let-7c expression could be of importance in COPD pathogenesis through the impaired control of the translation of TNFR-II, resulting in higher expression of TNFR-II in the airways of patients with COPD. Importantly, we demonstrated expression of let-7c in macrophages and bronchial epithelial cells. These cells are known key players in COPD pathogenesis and express TNFR-II (42).
Many of the differentially expressed microRNAs in current smokers and patients with COPD are not only involved in regulating inflammatory genes, but are also implicated in oncogenesis. Previously, let-7c expression has already been associated with lung cancer. Hence, deregulated miRNA expression can form the bridge between sustained airway inflammation, impaired cellular homeostasis, and oncogenesis, contributing to the increased risk of lung cancer in patients with COPD. This phenomenon is known as the COPD–lung cancer nexus (43, 44).
There are several aspects that contribute to the strengths of this study. First of all, this is the first study to assess the expression of miRNAs in the sputum of patients with COPD and current smokers without airflow limitation compared with never-smokers. Second, our results were validated in a second, independent cohort. Third, data were obtained by RT-qPCR and normalized on the basis of the global mean expression level, currently considered the “gold standard” for high-throughput miRNA expression analysis and normalization, respectively. Fourth, sputum miRNA expression was associated with spirometric values and clinical parameters, linking airflow limitation to the regulatory role of miRNAs. Last, in silico analysis identified relevant target genes of differentially expressed miRNAs, which are known to be implicated in COPD pathogenesis.
There are also some limitations to this study that should be addressed. First, miRNAs in induced sputum supernatant are essentially located in the extracellular compartment, where their exact cellular origin and function are unclear. Some studies suggest that miRNAs could be actively released by various cell types in exosomes as a part of a biological signaling process and intercellular communication (45, 46). Alternatively, the miRNAs in induced sputum could be a result of cell death with rupture of the cell membrane, which could be part of the airway pathology or part of an artifact due to processing of the sputum sample. The latter is less likely to induce differences in miRNA expression between study groups, as no difference in cellular viability was present between study groups. Importantly, the majority of the differentially expressed miRNAs correlated with sputum macrophage and lymphocyte counts, but not with the other sputum cells, thereby suggesting a possible specific cellular origin of these miRNAs. In the case of let-7c, we demonstrated expression of this miRNA in bronchial epithelial cells and sputum macrophages. As miRNA expression patterns are largely cell and compartment specific, it is difficult to draw definitive conclusions as to the miRNAs involved in the pathogenesis of COPD on the basis of this single study. A better understanding of the role for miRNA in COPD will likely require careful integration of larger cohort studies from whole lung, airway, and sputum as well as allied in vitro studies to validate functional targets and effects on cellular phenotype.
Second, a subset of patients with COPD used inhaled corticosteroids, which could have influenced miRNA expression. However, associations between FEV1 and individual miRNAs were analyzed by linear regression analysis, adjusting for this possible confounder. Last, the subjects with COPD in both cohorts are older than the healthy subjects. This age difference could influence miRNA expression. However, age was incorporated into all our linear regression models, taking into account its possible confounding effect.
miRNA expression can be detected in induced sputum supernatant. Current smoking significantly alters the miRNA expression profile in induced sputum. let-7c and miR-125b are significantly decreased in currently smoking patients with COPD. The decrease in let-7c is associated with the increase in TNFR-II in the airways of patients with COPD.
The authors thank Mrs. Ann Neesen, Mrs. Eliane Castrique, Mrs. Indra Deborle, Mr. Sven Verschraegen, and Mrs. Kim De Rijck (Laboratory for Translational Research in Obstructive Pulmonary Diseases, Department of Respiratory Medicine, Ghent University Hospital, Belgium) and Mrs. Gaëlle Van Severen (Center for Medical Genetics, Ghent University Hospital, Belgium) for technical contributions to this work. The authors also thank Mrs. Hermine Middendorp, Mrs. Frauke Vandewalle, and Mrs. Tasja Verstraete (Department of Respiratory Medicine, Ghent University Hospital, Belgium) for their support on data management.
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