MiR-107 and Its Association with House Dust Mite Sensitization: Implications for Asthma

Abstract

Introduction

MicroRNAs (miRNAs) have been linked to allergic diseases but their effects on sensitization to allergens in individuals with asthma are unknown. We aimed to identify miRNAs associated with house dust mite (HDM) sensitization in childhood asthma.

Methods

Serum samples from 1,126 children with asthma who participated in the Genetics of Asthma in Costa Rica Study (GACRS) were profiled for 304 miRNAs. We first divided according to HDM sensitization, and then tested whether miRNAs were differentially expressed (DE) between the two groups. Gene enrichment analysis for target genes of the DE miRNAs was then performed to identify potential causal pathways. A replication analysis was performed in the Childhood Asthma Management Program (CAMP), in which expression data of 258 miRNAs in 491 children were available. A mediation analysis was conducted to discern relationships between miRNA and phenotype differences according to HDM sensitization in GACRS cohort.

Results

There were 906 (80.5%) and 220 (19.5%) subjects in the GACRS HDM+ and HDM- groups. Compared with HDM- participants, those in the HDM+ group were more likely to be severe in variables including pulmonary function, oral corticosteroids use, and blood tests. A total of 17 miRNAs were differentially expressed (p < 0.05) between the two groups. with miR-642a-3p, let-7c-5p, and miR-107 most significantly associated with HDM sensitization. In CAMP, there were 39 DE miRNAs and increased expression of miR-107 in HDM+ children were replicated in this cohort. In both GACRS and CAMP, the cadherin-binding pathway was enriched in an analysis of target genes for DE miRNA. In a mediation analysis, miR-107 showed significant indirect effects on eosinophil count and total IgE that were mediated by HDM sensitization.

Conclusion

In children with asthma, miR-107 is associated with HDM sensitization. Further, miR-107 was indirectly associated with total IgE and eosinophil count through HDM sensitization.

Graphical Abstract

In children with asthma, miR-107 is associated with HDM sensitization and MiR-107 was indirectly associated with total IgE and eosinophil count through HDM sensitization. MiR-107 may affect worsening of asthma outcomes with two key intermediate phenotypes of asthma.

Introduction

Asthma is a chronic respiratory condition characterized by airflow obstruction, airway hyper-responsiveness and airway inflammation (1). Asthma is a heterogeneous disease comprising several phenotypes, with allergic asthma being most frequent and often associated with other allergic diseases during childhood.

In children with allergic asthma, exposure to common allergens such as those in house dust mite (HDM) can trigger innate and adaptive immune responses that ultimately lead to increased airway inflammation, worse asthma symptoms, asthma attacks and possible development of asthma (2). The rate of sensitization to HDM allergens varies by geographic region, but it is generally known to be around 50–85% in patients with asthma (3).

MicroRNAs (miRNA) are a class of non-coding RNAs that act as important post-transcriptional regulators and, therefore, are promising biomarkers for human diseases. MiRNA also play important roles in many pathologic processes including regulation of allergic inflammation. Several miRNAs have been linked to lung function, airway hyperresponsiveness, and asthma exacerbations (4–6). Also, it has shown its impact of childhood with previous studies. Several miRNA showed its relationship with pulmonary function, airway hyperresponsiveness, and asthma exacerbation in Childhood Asthma Management Program (CAMP) study (5–7). However, it is not known whether miRNAs affect sensitization to allergens such as HDM in children with asthma.

The purpose of this study was to identify miRNAs associated with HDM sensitization in children with asthma and to further examine whether such miRNAs are in turn linked to asthma and asthma-related outcomes.

Methods

Study populations

The Genetic Epidemiology of Asthma in Costa Rica study (GACRS) was a cross-sectional study of 1,165 children with asthma. Subject recruitment and the study protocol for the GACRS were previously described (8). In brief, children aged 6–14 years who had asthma (defined as physician-diagnosed asthma and either ≥2 asthma symptoms or at least one asthma attack in the previous year) and a high likelihood that at least six of their great-grandparents were born in the Central Valley of Costa Rica were recruited between February 2001 and July 2011. Study participants completed a protocol including questionnaires, spirometry, collection of blood samples, and allergy skin testing. Skin testing was performed according to the protocol of the International Study of Asthma and Allergies in Childhood, and HDM testing was considered positive if the maximum diameter of the wheal to either Dermatophagoides farinae or Dermatophagoides pteronyssinus was ≥3 mm after subtraction of the maximum diameter of the negative control. Serum total IgE was measured using the UniCAP 250 system (Pharmacia & Upjohn, Kalamazoo, Mich), and total IgE levels were transformed to a log10 scale for data analysis. Peripheral blood eosinophil count was determined using Coulter-Counter techniques. Spirometry was conducted with a Survey Tach Spirometer (Warren E. Collins, Braintree, Mass) following American Thoracic Society recommendations for children. After completing baseline spirometry, study participants were given 200 mg (2 puffs) of an albuterol metered-dose inhaler using a spacer, and spirometry was repeated after 15 minutes. For data analysis, bronchodilator responsiveness was calculated as: ([post-bronchodilator FEV1 – baseline FEV1]/baseline FEV1) x 100. Oral corticosteroid (OCS) use was defined as OCS use more than three days because of asthma.

Written parental consent and the child’s assent consent were obtained for all participants. The study was approved by the Institutional Review Boards of the Hospital Nacional de Ninos (San Jose, Costa Rica) and Brigham and Women’s Hospital (Boston, Mass, USA)

Replication analyses were conducted in the CAMP study. The CAMP study was a multi-center, randomized, placebo-controlled trial of budesonide, nedocromil, or placebo in 1,041 children over mean follow-up of 4.3 years (9). Children aged 5–12 years who had chronic asthma symptoms for at least 6 months in the prior year were enrolled. Asthma was defined by having 2 or more symptoms per week, using an inhaled bronchodilator at least twice weekly or asthma medication daily, and airway responsiveness to methacholine < 12.5 mg/ml (4). House dust mite sensitization was defined in the same way as in the GACRS. The CAMP Genetics Ancillary Study was approved by the Brigham and Women’s Hospital Internal Review Board, protocol #2015P001622/BWH. Informed consent and assent were obtained from parents and participants respectively.

MiRNA profiling

Serum samples from 1,159 participants in the GACRS and 492 CAMP participants were profiled for 1,901 and 1,696 miRNAs, respectively. All samples were collected during a stable asthma condition: at patient enrollment for the GACRS cohort regardless of treatment and two weeks prior to treatment randomization for the CAMP cohort. MiRNA extracted from samples stored properly in appropriate facilities is very stable (10). Therefore, small RNA-seq libraries were prepared using the Small RNA Library Prep Kit (Norgen Biotek Corp., Thorold, ON Canada) and sequenced on the NextSeq 500 platform (Illumina, San Diego, CA). The exceRpt pipeline was employed for the QC of the RNA-seq data (11). miRNAs with mapped read counts <5 in ≥50% of all subjects were excluded from the analyses. Read counts were normalized using the DESeq2 R package and then log transformed.

Statistical analysis

Subjects were divided into HDM-positive (HDM+) and HDM-negative (HDM-) groups according to the results of skin prick tests to HDM. When comparing clinical and demographic characteristics of the two groups, Student’s T-test was used for continuous and χ² test was used for categorical variables.

We identified miRNAs that were differentially expressed (DE) between children with asthma who were and were not sensitized to HDM using the DESeq2 package in R (version 4.1.2). DESeq2 is a package that is used to estimate variance-mean dependence in count data from high-throughput sequencing assays and to test for differential expression based on a model using the negative binomial distribution (12) Gene targets for the identified DE miRNAs were identified by multiMiR package in R with “Functional MTI (miRNA-target interactions)” support type, i.e., functionally validated targets. multiMiR package is a package that finds target genes of multiple miRNAs using a collection of miRNAs/targets from external resources, including validated miRNA-target databases (miRecords, miRTarBase and TarBase) and predicted miRNA-target databases (DIANA-microT, ElMMo, MicroCosm, miRanda, miRDB, PicTar, PITA and TargetScan) (13). Pathway enrichment analysis with the gene ontology (GO) was performed with the enrichr package in R. enrichr package analyzes gene sets and returns any enrichment of common annotated biological features (14) We replicated the DE miRNAs and pathway analyses in the CAMP cohort. P values lower than 0.05 were considered statistically significant.

Finally, we conducted a mediation analysis in the GACRS using structural equation modeling to discern the relationship among miR-107, house dust mite sensitization (HDM), and various asthma-related outcomes, specifically baseline percent predicted FEV1, baseline percent predicted FVC, bronchodilator response, peripheral blood eosinophil count, total IgE level, oral corticosteroid use, and the number of ER visits. The analysis was carried out with 1000 bootstraps using the Lavaan version 0.6–15 R package (15).

Results

Of the 1,159 GACRS participants with serum samples, 1,126 had adequate miRNA profiling. The main characteristics of these 1,126 participants are shown in Table 1, according to whether they did (n=906 or 80.4%) or did not (n=320 or 19.6%) had HDM sensitization. Compared with participants without house dust mite sensitization (HDM- group), those in the HDM+ group were significantly more likely to have used oral corticosteroids in the previous six months (730/906 or 80.6% vs. 154/22 or 70%, p < 0.001) and had significantly lower FEV1 (98.2 ± 17.5% of predicted value vs. 102.0 ± 16.0% of predicted value, p = 0.002) and FEV1/FVC ratio (83.7 ± 8.1% vs. 86.0 ± 6.5%, p < 0.001), but higher bronchodilator responsiveness (6.2 ± 10.8% vs. 3.2 ± 6.6, p < 0.001), peripheral blood eosinophil count (621.7 ± 415.5 vs. 258.9 ± 230.0, p < 0.001) and total IgE level. (862.9 ± 967.3/μl vs. 211.9 ± 471.0/μl, p < 0.001). In addition, use of inhaled corticosteroids, short acting beta-2 agonist was more frequent in HDM+ group and use of leukotriene inhibitor was more frequent in HDM- group (52.1% vs. 44.1%, p = 0.04, 83.9% vs. 77.7%, p = 0.04, and 4.6% vs. 6.4%, p = 0.04, respectively). In contrast, there were no significant differences between the two groups in terms of asthma duration, medication usage rates.

Table 1.

Clinical characteristics of the house dust mite sensitized (HDM+) and non-sensitized (HDM-) groups from the Genetic Epidemiology of Asthma in Costa Rica study

	HDM+ group (N = 906)	HDM- group (N = 220)	p value
Age (yr)	9.3±1.9	8.9±1.8	0.006
Male no. (%)	532 (58.7%)	130 (59.1%)	0.92
Onset of asthma (yr)	2.4±2.3	1.7±2.0	<0.001
Duration of asthma (yr)	6.88±2.8	7.24±2.6	0.07
FEV1 (% of predicted value)	98.2±17.5	102.0±16.0	0.002
FVC (% of predicted value)	104.5±16.8	105.8±15.5	0.27
FEV1/FVC (%)	83.7±8.1	86.0±6.5	<0.001
Bronchodilator response (%)	6.2±10.8	3.2±6.6	<0.001
Eosinophil (/μl)	621.7±415.5	258.9±230.0	<0.001
Total IgE (IU/ml)	862.9±967.3	211.9±471.0	<0.001
Oral corticosteroid uses within past 6 m	730 (80.6%)	154 (70%)	<0.001
ER visit or hospitalization within past 1 yr	51 (5.6%)	7 (3.2%)	0.19
Taking medication (%)	881 (97.2%)	209 (95.0%)	0.13
Inhaled corticosteroids (%)	472 (52.1%)	97 (44.1%)	0.04
Leukotriene inhibitor (%)	42 (4.6%)	14 (6.4%)	0.04
Long acting beta-2 agonist (%)	17 (2.3%)	5 (1.9%)	0.91
Short acting beta-2 agonist (%)	760 (83.9%)	171 (77.7%)	0.04

Abbreviation; FEV₁, forced expiratory volume in 1 second; FVC, forced vital capacity; ER, emergency room.

After filtering and normalization in the GACRS, there were 304 miRNAs which passed our filtering criteria for differential analysis between participants with and without HDM sensitization (Figure 1). Of these, 17 were differentially expressed between those with and without HDM sensitization, with 11 up-regulated miRNAs and 6 down-regulated miRNAs (Figure 2 and Supplementary table S1). In the CAMP, there were 491 subjects with 258 miRNAs after filtering and normalization. Of these, 39 miRNAs were differentially expressed between the HDM+ and HDM- groups, with 19 up-regulated miRNAs and 20 down-regulated miRNAs (Figure 3 and Supplementary table S2). Notably, miR-107 was up-regulated in HDM+ participants in both cohorts.

Figure 1.

Illustration of miRNA analysis

Figure 2.

Differentially expressed microRNAs between house dust mite sensitized vs. non-sensitized asthmatics in the GACRS cohort

Figure 3.

Differentially expressed microRNAs between house dust mite sensitized vs. non-sensitized asthmatics in the CAMP cohort

Validated target genes for 17 DE miRNAs from GARCS were searched using the multiMiR package in R, yielding 268 genes. Similarly, we found 674 validated target genes based on the 39 DE miRNAs in CAMP. For those target genes, we performed pathway enrichment analysis in both cohorts using the enrichr package in R. Among the enriched pathways in HDM+ groups from both cohorts, the cadherin binding pathway was identified in GACRS and CAMP. (Table 2, 3).

Table 2.

Enriched pathways in HDM+ group from GARCS cohort using GO analysis.

Term	Overlap	P-value	Adjusted P-value	Odds ratio	Combined score
RNA binding (GO:0003723)	1243/1387	6.59×10−75	7.58×10−72	4.021	686.811
cadherin binding (GO:0045296)	284/313	1.51×10−19	8.67×10−17	4.324	187.399
ubiquitin-like protein ligase binding (GO:0044389)	270/297	6.04×10−19	2.32×10−16	4.412	185.096
ubiquitin protein ligase binding (GO:0031625)	258/284	4.68×10−18	1.35×10−15	4.375	174.584
protein kinase binding (GO:0019901)	425/495	1.85×10−17	4.26×10−15	2.690	103.651

Table 3.

Enriched pathways in HDM+ group from CAMP cohort using GO analysis.

Term	Overlap	P-value	Adjusted P-value	Odds ratio	Combined score
RNA binding (GO:0003723)	1022/1387	1.51×10−76	1.72×10−73	2.984	520.894
cadherin binding (GO:0045296)	240/313	2.67×10−22	1.52×10−19	3.322	164.994
ubiquitin-like protein ligase activity (GO:0061659)	153/186	5.91×10−20	2.25×10−17	4.662	206.394
ubiquitin-protein transferase activity (GO:0004842)	299/417	1.30×10−19	3.69×10−17	2.564	111.499
ubiquitin protein ligase activity (GO:0061630)	156/192	2.73×10−19	6.23×10−17	4.357	186.235

We also examined the relation between miR-107 and other variables. MiR-107 was not significantly associated with asthma-related outcomes such as lung function measures or use of corticosteroids in in HDM+ participants in either GACRS or CAMP. Other clinical characteristics including sensitization to other allergens did not show any significant relationships with miR-107 except sex and use of inhaled corticosteroid in GACRS cohort (Supplementary table S4 and S5). In a mediation analysis, however, miR-107 showed significant indirect effects on peripheral blood eosinophil count (estimate = 0.026 and p = 0.026) and total serum IgE (estimate = 0.019, and p = 0.043), which were each mediated by sensitization to house dust mite allergen (HDM).

Discussion

In the GACRS, HDM sensitization was very common and associated with greater asthma severity, as participants in the HDM+ group were more likely to have used oral corticosteroids for asthma and demonstrated lower FEV1 and FEV1/FVC and higher bronchodilator responsiveness, peripheral blood eosinophil count, and total serum IgE than the HDM- group. Also, use of asthma controller and short acting beta-2 agonist was more frequent in HDM+ group. Of the 17 miRNAs that were differentially expressed by HDM sensitization in the GACRS, miR-107 was replicated in the CAMP in the same direction of association as in the GACRS. Further, a pathway enrichment analysis using target genes of differentially expressed miRNAs in both cohorts identified the cadherin signaling pathway signaling pathway in both GACRS and CAMP. Moreover, we showed an indirect effect of miR-107 on eosinophil count and total IgE in the GACRS that was mediated by HDM sensitization.

HDM can thrive in many sites throughout the house and is difficult to eradicate (1). HDM is one of the most important sources of indoor allergens and has powerful antigenic capabilities. Exposure to HDM is associated with development of asthma, worse asthma control and severe disease exacerbations in children with asthma (16), through innate immunity and IgE-dependent, adaptive immunologic responses (3, 17). Although the exact pathways are diverse depending on effector molecules, HDM allergen inhalation leads to release of innate pro-Th2 cytokines (3) and causing Th2 immune responses and allergic airway inflammation (18) Further, HDM allergen can also damage the airway epithelium through proteases and proteolytic activity (19, 20). In addition, HDM exposure can activate both TLR2 and TLR4 in sensitized individuals, which in turn activates calpains, Ca2+-dependent protease, and activated calpains degrading transmembrane junctional proteins including occluding and E-cadherin (21), thus facilitating penetration of the airway epithelium by HDM allergens.

There are several lines of evidence that show the relationship between asthma phenotype and circulating miRNAs. In the CAMP cohort, miR-296–5p and miR-16–5p were associated with airway hyperresponsiveness (6), several miRNAs including miR-15b-5p and miR-27b-3p were associated with FEV1 and FEV1/FVC (5), and miR-146b, miR-206 and miR-720 were linked to asthma exacerbations (4). MiR-451b, miR-7–5p, miR-532–3p, miR-296–5p, and miR-766–3p showed association with exacerbation of childhood asthma (7). In addition, miRNAs also affect the immune responses in the airways of subjects with asthma (22). MiR-155 showed an association with innate lymphoid cells and IL-33 level with allergen-exposed knockout mice thus influencing innate immunity (23). MiR-19b targets TSLP and is observed to be downregulated in allergy and treatment with miR-19b showed suppression of allergic inflammation (24). Inhibition of miR-126 suppressed the inflammatory response upon allergen contacts via TLR signaling (25). Regarding adaptive immunity, let-7 miRNAs has shown the potential to regulate Th2 inflammation by inhibiting IL-13 (26). Regarding HDM sensitization, A study showed 13 miRNAs were increased in HDM-sensitized asthma patients (18). In addition, several miRNAs were differentially expressed after HDM exposure (27).

We found 17 miRNAs and 39 miRNAs which were differentially expressed according to HDM sensitization in GARCS and CAMP cohort, respectively. Of these, miR-107 was up-regulated among children with asthma and HDM sensitization in GARCS and in CAMP. Within the literature, miR-107 seems to have various and complicated roles in the pathogenesis of several diseases (28–33). On the contrary, the role of miR-107 in the pathogenesis of allergic diseases has not been formally defined. In one study, miR-107 showed a positive correlation with peripheral blood eosinophil count and an association with asthma (28), with another study of the same dataset showing that miR-107 was related to migration of airway smooth muscle cells, and also, this paper provides functional evidence with mouse model that miR-107 significantly affects the migration of airway smooth muscle cells. (29).

To find the pathway that explains the relationship between miR-107 and asthma, we performed pathway enrichment analysis with target genes of differentially expressed miRNAs in two cohort. Among the enriched pathways in HDM+ groups from both cohorts, the cadherin binding pathway was in common. E-cadherin plays crucial role in intercellular junctions forming the structural adhesiveness in airway epithelium (34). In situations like cigarette smoking, functional alteration of E-cadherin in the intercellular junction appears to be a pivotal step in epithelial-mesenchymal transition and loss of barrier function. Like cigarette smoking, protease activity of molecules in HDM such as Der p 1 also cause destruction of E-cadherin mediated intercellular contact (20). This process can also lead to an increase in antigen permeability, creating a vicious cycle where barrier alteration occurs again due to the influence of infiltrated antigens. In addition, although it is not a findings of airway disease, there are several evidence that miR-107 itself might affect to intercellular junction. In one study, miR-107 was related to alteration of blood-brain barrier endothelial junction (35). Also, miR-107 expression was associated with TLR4 activation in other metabolic diseases (30). HDM can also weaken barrier function by activating TLR, which could partly explain the association between HDM sensitization and worse asthma severity and increased expression of miR-107 in the HDM+ group in the current study.

Mediation analysis is a statistical method used to understand the relationship between variables and how a mediator variable influences or explains the relationship between that. Indirect effect in mediation analysis shows the mediator has the impact of the independent varable on the dependent variable. In our study, we observed a significant indirect influence of miR-107 on peripheral blood eosinophil and total serum IgE levels, with the effect being mediated via HDM sensitization. Additionally, a positive association between miR-107 and HDM sensitization was identified. Eosinophils and total IgE play a significant role in asthma among children with HDM sensitization (18). This suggests that miR-107 may contribute to the elevation of HDM sensitization, subsequently leading to an increase in blood eosinophil count and IgE levels. Consequently, the combined influence of miR-107 and HDM sensitization may have a greater impact on asthma severity.

This study has several strengths. Firstly, it is the first paper to elucidate the relationship between miR-107 and HDM sensitization related to asthma. This paper not only explores this relationship but also explains its relevance to HDM sensitization through two cohorts. Secondly, GACRS and CAMP are clinically well-characterized cohorts with standardized methodologies, providing validated large sample sizes. The use of such cohorts with large sample size in genetic expression studies, including miRNA, is a significant advantage. Thirdly, through the result of cadherin binding pathway with enrichment analysis, we proposed two hypotheses that can explain the results and with mediation analysis we showed the effect of miR-107 to asthma via HDM sensitization.

Despite its strengths, our study has limitations. First, the lack of direct functional validation through experimental studies is a limitation. Additional experiments are needed to confirm the role of miR-107 and validate our findings functionally. Second, although we focused on nominal p-values for association in the individual cohorts, the combined p-value for the association of miR-107 with HDM in GACRS and CAMP using Fisher’s method was 0.0002 with a Bonferroni-corrected p-value of 0.049. It is noteworthy that the p-value remained statistically significant even after undergoing the highly stringent Bonferroni correction, which is particularly rigorous in controlling for Type I errors. This outcome strongly suggests that the relationship between miR-107 and HDM sensitization demonstrated in our study is well-supported and carries substantial validity.

In summary, we report a novel association between miR-107 expression and HDM sensitization among children with asthma in two cohorts. Our findings further suggest that miR-107 may affect worsening of asthma outcomes with two key intermediate phenotypes of asthma (peripheral blood eosinophils and total IgE) through HDM sensitization. Through further research into the mechanisms underlying the association between miR-107 and HDM sensitization, we can enhance our understanding of asthma. This promising direction may also reveal potential targets for diagnosis or treatment related to miR-107.

Key messages.

• In children with asthma, miR-107 is associated with HDM sensitization

• MiR-107 was indirectly associated with total IgE and eosinophil count through HDM sensitization.

• MiR-107 may affect worsening of asthma outcomes with two key intermediate phenotypes of asthma.

Abbreviations:

CAMP

Childhood Asthma Management Program

DE

differentially expressed

FEV₁

forced expiratory volume in 1 sec

FVC

forced vital capacity

GACRS

Genetics of Asthma in Costa Rica Study

HDM

house dust mite

HDM+

HDM-positive

HDM-

HDM-negative

miRNA

mcroRNA