Zmniejszona ekspresja microRNA-16 i microRNA-451a w przeroście migdałka gardłowego związana jest z alergią

Introduction

Adenoid hypertrophy (AH) is a common childhood pathology, which causes the obstruction of upper airways that may eventually lead to sleep apnea, craniofacial abnormal growth, or conductive hearing loss and cognitive impairment [1]. Several studies underline higher incidence of AH among children with allergy, but underlying molecular mechanisms are still not fully understood [2]. MiRNAs are small, non-coding molecules that serve as gene regulators of many biological processes, such as cell differentiation, proliferation, apoptosis or angiogenesis [3]. Their role in allergic type of inflammation has recently been described in some studies [4]. Allergic type of inflammation is based on Th cell imbalance, where the cytokines secreted in Th2 type of inflammation dominate over cytokines produced in Th1 type [5]. Recent studies showed that particular miRNAs or their clusters regulate differentiation of Th cells into Th2 type. Several murine models demonstrated that either enhancing or silencing those miRNAs might rebuild Th cell balance [6].
Our assumption that specific miRNAs might take part in adenoid enlargement pathogenesis in allergic patients comes from the concept of the united airways disease (UAD). According to the latest evidence, the upper and lower respiratory tract mucosae in allergic patients have a similar potential to trigger the Th2 immune response and secrete cytokines stimulating the allergic type of inflammation in each of its compartments. IgE-mediated allergic inflammation is based on immunoglobulin class switching (from IgM to IgE) after antigen sensitization and secretion of specific cytokines, such as interleukin 4 (IL)-4 or IL-13, which are responsible for maintenance of the allergic response. This results in vasodilatation, bronchoconstriction and increased mucus production [7]. Immunoglobulin class switching is present in respiratory mucosa of patients with AR and asthma but also has been observed in the gastrointestinal tract in patients with food allergy [8]. Nguyen et al. compared several levels of cytokines in adenoids, middle ear fluid and torus tubarius biopsies, and found that the eosinophils, T lymphocytes and IL-4 mRNA levels were significantly higher in the allergic group. This proved the assumption that all three compartments share the allergic pattern of inflammation [9].

Aim

Therefore, we hypothesized that nasopharynx, with its adenoid tissue, is a part of the united airways and that the mechanism of AH formation in allergic patients might depend on a specific regulatory mechanism that is absent in non-allergic children. Taking into account the high regulatory potential of miRNAs and their involvement in allergic inflammatory processes, we sought to determine whether miRNA expression differs between patients with and without allergy, suggesting that the pathomechanism of tonsil enlargement in these two groups of patients might be distinct.

Material and methods

The study was approved by the Bioethics Committee of Poznan University of Medical Sciences. Patients were recruited from inpatients at the Department of Pediatric Otolaryngology, Poznan University of Medical Sciences. The study was approved by the Poznan University of Medical Sciences Bioethics Committee. Subjects were from the Wielkopolska region of Poland, which is considered ethnically homogeneous. Written informed consent was obtained from a parent or legal guardian. The study group consisted of children diagnosed with adenoid hypertrophy. Exclusion criteria included: craniofacial abnormalities, cleft palate, genetic syndromes, immune deficiencies, cystic fibrosis, immotile cilia syndrome, steroids, and antihistamine or leukotriene drug intake 2 weeks prior to the surgical procedure.
Full otolaryngologic examination was performed at the admission. Each child was carefully investigated for a history of allergic diseases using a detailed questionnaire. Children with positive skin prick tests or blood tests for food or inhalant allergens or with allergic rhinitis diagnosis based on ARIA [10] criteria or diagnosed allergic asthma according to GINA [11] criteria were assigned to the allergy group and compared against children without allergic history. The allergy group consisted of 16 patients whereas the non-allergic group had 21 patients. Samples were taken during the adenoidectomy procedure, before the tonsil removal. Nasal respiratory mucosa swabs were also taken. The material was then transferred to the laboratory and frozen at –80°C for further experiments. Total RNA was isolated with miRCURY miRNA Isolation Kit – Cell and Plant (Exiqon), according to the manufacturer’s instructions and transcribed to cDNA with TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific). MiRNA expression was analyzed with TaqMan MicroRNA Assays and TaqMan Universal Master Mix II, no UNG (Thermo Fisher Scientific), according to the manufacturer’s protocol. MiRNA expression datasets were analyzed with DataAssist software v.3.01 after global normalization. Pathway enrichment analysis was performed for miRNAs that had significant changes in expression. We selected validated target genes (via reporter assay) from miRTarBase (available at http://mirtarbase.mbc.nctu.edu.tw/php/index.php). To identify KEGG pathways, the list of validated targets was analyzed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) v.6.7. Results

Patient characteristics

Samples were taken from 37 subjects (10 girls and 27 boys). Mean age was 6.22 ±2.4 years. Adenoid hypertrophy was more common in boys independently of allergy status (75% in allergic group and 71% in non-allergic group). Mean total IgE serum level was higher among allergic children (392.8 ±125.1 and 90 ±574.5 in non-allergic patients). Mean eosinophil count was also higher in the allergy group (6.2 ±2.3% in allergy group and 3.5 ±3.3% in non-allergic group). These results correspond to other studies underlining that IgE serum level as well as eosinophils is elevated in allergic patients. 69% of allergic children and 67% of non-allergic children attended kindergarten. 18.7% of allergic children and 14.2% of non-allergic children were under chronic exposure to tobacco smoke at home, which is a separate allergy factor (Table 1).

MiRNA expression analysis

We found that expression of analyzed miRNAs in nasal mucosa did not differ significantly between allergic and non-allergic patients with AH (Figure 1). In adenoid tissue, we observed significantly decreased expression of 2 miRNAs, miR-16 (p = 0.048) and miR-451a (p = 0.019), in allergic patients as compared to non-allergic children (Figure 2).

Target analysis

A list of validated target genes regulated by miR-16-5p or miR-451a was analyzed with the DAVID annotation tool to identify the most enriched pathways possibly regulated by this miRNA. Several pathways were significantly enriched for both miRNAs (Tables 2 and 3).

Discussion

While several studies have debated whether allergy is a separate risk factor of AH, there is still insufficient molecular evidence supporting this hypothesis [12]. Nguyen et al. reported that IL-4 levels and eosinophil infiltration were increased in adenoids and middle ear effusions from allergic patients compared to controls [9]. Huo et al. linked adenotonsillar regrowth with the allergy status. They established levels of GATA3+ cells (Th2-type cells) and found that they were increased in the adenoids from the allergic subgroup [13]. To our knowledge, our study is the first to analyze miRNA expression in adenoid tissue in the pediatric population and whether it depends on allergy status.
Expression of 5 analyzed miRNAs (miR-320e, miR-16-5p, miR-451a, miR-223-3p, miR-25-3p) was not significantly different in nasal mucosa between allergic and non-allergic children, so these miRNAs are not likely to play a role in allergic inflammation in the nose. Interestingly, we found that miR-16 and miR-451a were significantly decreased in the adenoid tissue of allergic patients, suggesting that they may favor the expression of genes participating in adenoid hypertrophy in allergic patients. MiR-16 takes part in the allergic inflammation. Pangiban et al. demonstrated that several miRNAs, including miR-16, had different expression in patients with asthma and allergic rhinitis (AR) as compared to the controls and underlined their potential as future noninvasive biomarkers of allergic diseases [14]. Another study in asthmatic patients revealed the possible role of miR-16 in asthma exacerbation by regulating Th2 cytokine expression and favoring airway inflammation [15]. Yu et al. also demonstrated that miR-16 has the potential to serve as an asthma biomarker. Moreover, miR-16 regulates mRNA expression of adrenoreceptor β2, which is an agonist receptor for bronchodilators; thus miR-16 may affect their efficacy [16].
Pathway analysis of predicted target genes for miR-16 and miR-451a have identified several potential regulatory mechanisms that might be involved in allergic inflammation. For example, the PI3K-Akt pathway, regulated by both the miRNAs, modulates airway inflammation and airway hyper-responsiveness [17]. MAPK signaling pathway contributes to the expression of proinflammatory genes [18]. Moreover, the neurotrophin signaling pathway modulates biological effects of infiltrated eosinophils in the allergic airways [19]. MiR-451 plays a role in pathogenesis of various cancers, but its exact role in allergic inflammation is still not fully understood. Macrophages activated by reactive oxygen species have altered miR-451 expression, suggesting its important role in macrophage maturation [20]. Chung et al. investigated the role of miR-451 in a mouse model of allergic asthma and found that its levels are significantly decreased, affecting macrophage activation in lungs. Macrophages isolated from mice’s lungs had increased levels of CCL17 and sirtuin-2, indicating miR-451 function in regulating the allergic response [21]. Identifying the altered expression of particular miRNAs in allergic patients could help to understand the pathogenesis of AH formation, linking it to allergic inflammation. Further miRNAs studies would enable the mechanisms underlying morbidities to be explained and could possibly lead to the development of biomarkers or even therapeutic options in cases where current therapy is unfortunately insufficient. There are two major limitations to this study that are going to be addressed in future research. The primary limitation is the small sample size. Another limitation is the lack of target gene verification addressed at altered miRNAs, which will be dealt with in a future study.

Conclusions

We have documented that miRNAs are expressed in adenoid tissue in children and that the expression of two of them, miR-16 and miR-451a, differs between allergic and non-allergic patients. These miRNAs may be involved in adenoid hypertrophy formation in allergic patients. Further studies are needed to better understand their exact role in the induction of allergic-type inflammation.

Acknowledgments

This work was supported by the Poznan University of Medical Sciences (Grant number: 502-01-01105122-04348). The data that support the findings of this study are available from the corresponding author, upon request.

Conflict of interest

The authors declare no conflict of interest.