Biomedicine & Pharmacotherapy Emerging role of non-coding RNAs in allergic disorders

RNA transcripts that not undergo translation into polypeptides recently came into focus of research. Long non- coding RNAs (lncRNAs), microRNAs (miRNAs) and circular RNAs (circRNAs) comprise the most important groups of these transcripts. LncRNAs have a length over 200 nucleotides and like mRNAs, have regulated transcription in a tissue speci ﬁ c manner. Biogenesis and function of lncRNAs is related to cell di ﬀ erentiation, response to stimuli and regulation of immune responses. LncRNAs can interact with both miRNAs and mRNAs. MiRNAs are characterized by a length of 22 – 24 nucleotides. MiRNAs regulate expression of genes at the post-transcriptional level. LncRNAs together with miRNAs are considered as regulators of the immune system. Alterations in their biogenesis is an important mechanism in the development immune related disorders. CircRNAs are products of aberrant maturation of protein-coding transcripts in a process of back-splicing, in which a single strand RNA molecule attains a closed circle shape. Despite a low expression, some circRNA were found to titrate miRNAs and interfere with maturation of legitimate protein-coding transcripts. We summarize the current knowledge on the role of non-coding transcripts in allergic disorders: asthma, atopic dermatitis, allergic rhinitis and urticaria. The reviewed data suggest lncRNA and miRNAs as therapeutic targets and bio- markers of allergic disorders.


Introduction
Some sequences of a genome are transcribed but not translated into polypeptides. These non-coding RNA (ncRNA) can be detected in the cytoplasm or in extracellular fluids. Deep RNA sequencing and bioinformatics tools allowed to study (ncRNAs). Participation of ncRNA in RNA splicing, transposon reassembly or genes rearrangements [1] was known for long time. However, regulatory ncRNAs constitute a large proportion of ncRNAs. The most recently described circular RNAs are produced from protein-coding transcripts by abnormal splicing and have a closed circle shape. Some circRNA were found to trap miRNAs and interfere with maturation mRNA [2]. Other ncRNA are named long ncRNAs (lncRNAs, length > 200 nucleotides) or microRNAs (miRNAs, length 22-24 nucleotides) [2,3]. MiRNAs regulate translation of polypeptides at post-transcriptional level [4] contributing to epigenetic regulation. All ncRNAs are encoded by their respective genes and regulate many biological processes in an evolutionary conserved manner [5]. Both lncRNAs and miRNAs participate in the regulation of immunity by the preservation of hematopoietic stems cells, differentiation and apoptosis of myeloid cell and the stimulation of monocytes, macrophages and dendritic cells (DCs) [6]. Moreover, several lncRNAs regulate proliferation, differentiation and induction of immune cells from inactive state. During innate or adaptive immune responses monocytes, macrophages, DCs, neutrophils, T and B lymphocytes change their expression of ncRNAs [7]. Assessment of expression of lncRNAs in CD8+ and CD4 + T cells led to identification of numerous RNAs species with a phase-or tissue-specific signatures [8,9]. Similarly, miRNAs were showed to alter cell development, differentiation and release of inflammatory cytokines [10]. The role of miRNAs in regulation of innate immune responses, especially macrophages and granulocytes, was well established [11]. MiRNAs also participate in a control adaptive immune responses and abnormal expression of miRNAs was found in autoimmune disorders [11]. We summarize the available data about the role of these ncRNA in asthma, atopic dermatitis (AD), allergic rhinitis (AR), and urticaria.
A histopathological component of asthma is hyperplasia and hypertrophy of airway smooth muscle cells (ASMCs). Epigenetic mechanisms have prominent roles in the regulation of these cells [13]. Austin et al. reported on differential expression of lncRNAs comparing ASMCs from patients with non-severe and those with severe asthma. Also, a lower expression of PVT1 lncRNA was found in patients with corticosteroidsensitive non-severe asthma, whereas over-expression of this lncRNA was present in asthmatics with corticosteroid-insensitive severe phenotype. Functional studies showed the role of PVT1 in the regulation of cell proliferation and IL-6 production of ASMCs [14]. Zhu et al. assessed expression of lncRNAs in peripheral blood samples of patients with eosinophilic asthma, neutrophilic asthma and healthy controls using RNA-sequencing. Several differentially expressed lncRNAs were identified. Over-expression of LNC_000127 in eosinophilic asthma was replicated in Jurkat immortalized human T lymphocytes and isolated human CD4 + T cells following stimulation with phorbol ester or anti-CD28. The function of this lncRNA in Th2 inflammation pathway was thus verified [15]. Ye et al. examined expression of ANRIL lncRNA in patients with asthma during exacerbation (BA-E), patients at remission (BA-R) and healthy controls. Of interest, a higher expression of ANRIL/ miR-125a axis was observed in BA-E patients compared with BA-R or control groups. Expression of this regulatory axis negatively correlated with respiratory functional tests results in the study participants. Another correlation was found between ANRIL/miR-125a axis expression and severity of asthma, particularly in exacerbations [16]. In addition, receiver operating characteristic curve showed that the axis expression could discriminate disease status with a performance comparable to the measurements of TNF-α, IL-1β, IL-6 and IL-17 cytokines [16]. Presented results are consistent with the role of ANRIL/miR-125a axis in regulation of immune response and suggest its involvement into the pathogenesis of asthma [17,18]. Some other studies also reported aberrant expression of a number of lncRNAs in asthma. Fig. 1A shows the role of TIMMDC1 and lncTCF7 in the pathophysiology of asthma. Fig. 1B illustrates TUG1 lncRNA in the control of miR-590-5p regulated bronchial remodeling. The profile of expression and a tentative function of lncRNAs in asthma are summarized in the Table 1.
Several studies assessed the role of miRNAs in asthma. A group of miRNAs which regulate the balance between Th1 and Th2 cells are particular candidates for immunoregulation of the disease. Qui et al. evaluated expression of miRNAs that targeted the transcription factor Runx3 contributing in differentiation of T helper cells. They showed an imbalance of Th1/Th2 cells in the asthmatic patients they studied. Moreover, a lower expression of Runx3 and higher expression of a several miRNAs targeting this transcription factor in the CD4 + T cells was detected in asthmatic patients. Experimentally, it was verified that miR-371, miR-138, miR-544, miR-145, and miR-214 could directly bind to the 3'-UTR of Runx3. These miRNAs could contribute to the Th1/Th2 imbalance in asthma by regulating Runx3 [26].
Several other miRNAs participate in suppression of inflammation in airways, thus down-regulation of these miRNAs contribute to the pathogenesis of asthma. MiR-21 role in regulation of allergic asthma model in mouse is illustrated by the Fig. 2. Ma et al. established a mouse model of asthma by sensitizing and challenging the mice with ovalbumin. In this animal model, they showed that miR-20b mimic reduced both the number of the total leukocytes, neutrophils and eosinophils in the bronchoalveolar lavage fluid (BALF) and mucus production in the airway. Moreover, this treatment decreased VEGF levels in BALF [53]. The role of some other miRNAs in the pathogenesis of asthma was evaluated in human subjects. Zhang et al. found decreased abundance of miR-192 in asthmatic children as compared with controls. In vitro experiments documented that miR-192 inhibited activation pathway of T follicular helper cells by targeting CXCR5 [43]. Table 2 shows the list Knock-down of lncTCF7 has led to down-regulation of TIMMDC1 at transcript and protein levels. Up-regulation of TIMMDC1 increases phosphorylation and activation of AKT. Activated AKT increases expression of β-catenin through inhibition of GSK-3β leading to modulation of expression of genes which are involved in the airway remodeling [19]. (B) TUG1 acts as a competing endogenous RNA to decrease miR-590-5p levels. miR-590-5p has a role in suppression of FGF1 expression through binding with its 3' UTR region, thus inhibition of this miRNA by TUG1 increases expression of FGF1. FGF1 is a pro-angiogenic growth factor and vascular endothelial growth factor, therefore it is involved in the airway remodeling [20]. Table 1 Expression and function of lncRNAs in asthma.

LncRNA
Expression pattern

Numbers of clinical samples Targets/ Regulators
Signaling Pathways Function Ref

Atopic dermatitis (AD)
Wang et al. established an fluorescein isothiocyanate-induced animal model of AD. Next, they assessed expression of ncRNAs and mRNAs in this model using microarray technique. They showed dysregulation of 5766 lncRNAs, 4025 mRNAs, and 202 miRNAs after provocation of the AD recurrence. Most notably, expression of 419 lncRNAs, 349 mRNAs and 23 miRNAs remained altered in the remission stage [56]. In silico prediction steps led to identification of seven lncRNAs that were subjected to expression assay in the ear tissue of animals by qRT-PCR. They reported upregulation of lincRNA0016+, uc008thl.1, uc029qxr.1, and AK077345, and down-regulation of uc029ycn.1, ENSMUST00000164311, and ENSMUST00000149791. Finally, they selected five lncRNAs (AK077345, uc008thl.1, uc029ycn.1, ENSMUST00000164311, and ENSMUST00000149791) with the highest expression change to identify their potential mRNA targets [56]. These lncRNAs have been suggested as novel targets for modulation of AD recurrence in mice [56].
Several studies assessed the role of miRNAs in the development of AD. Expression profiling of miRNAs in AD facilitated identification of pathogenetic pathways and helped in differentiation of this allergic condition from other disorders like cutaneous T-cell lymphoma (CTCL) or mycosis fungoides (MF). Ralfkiaer et al. showed differential expression of 38 miRNAs between early MF vs. AD. While miR-155, miR-146a, 146b-5p, miR-342-3p and let-7i* were down-regulated in AD, miR-203 and miR-205 had the opposite trend [57]. It is worth mentioning, that up-regulation of a certain miRNA in AD tissues does not necessary imply the pathogenic role of this miRNA in the development of this disorder. Rebane et al. described higher expression of miR-146a in keratinocytes and chronic lesions of the skin in patients with AD. However, the role of this miRNA was confirmed as suppression several proinflammatory transcripts such as IFN-γ-inducible and AD-associated chemokines CCL5, CCL8, and ubiquitin D (UBD) in vitro. Moreover, in vivo experiments confirmed the presence of more robust inflammatory responses in miR-146a-deficient mice. Functional studies revealed that this miRNA inhibits the nuclear factor kappa-B signal transducers [58]. The function of up-regulated and down-regulated miRNAs in AD was summarized in Table 3.

Allergic rhinitis (AR)
AR is characterized by induction of the inflammatory response in the nasal mucosa after exposure to an allergen. The inflammatory responses comprise an immediate IgE-mediated mast cell degranulation followed by recruitment of eosinophils, basophils, and T cells. Cells that contribute in the late phase produce Th2 cytokines such as IL-4 and Il-5 which facilitate IgE synthesis and expansion of eosinophils [64]. Based on the role of lncRNAs and miRNAs in the regulation of Th2 responses, it is not surprising that altered expression of these transcript contributes to the pathogenesis of AR. Ma et al. assessed lncRNA signature in nasal mucosa of AR patients to predict possible function of these transcripts in the pathogenesis of AR Their microarray investigation demonstrated differential expression of a total of 2 259 lncRNAs (1,033 up-regulated and 1,226 down-regulated) in the nasal mucosa of AR patients compared with healthy controls. Analysis of lncRNA-mRNA co-expression showed their enrichment in cellular signaling pathways associated with AR development such as positive regulation of IL-13 production, Fcepsilon receptor-1 and NF-kappa B signaling pathways [65]. Another microarray-based analysis reported differential expression several lncRNAs including 110 upregulated and 48 downregulated lncRNAs in Fig. 2. (A) miR-21 has been shown to be increased in allergic asthmatic mice, while IL-12 and STAT4 are decreased. These factors are involved in regulation of Th1/ Th2 balance. Dysregulated expression of these factors in asthma leads to Th2 dominance [29]. (B) IFN-γ is one of the Th1 cytokines which inhibits Th2 differentiation. IL-4, -5 and -13 are Th2 cytokines which enhance production of eosinophils in the bone marrow and their transport to the lungs. While production of eosinophils is increased by IL-5, IL-4 and -13 enhance expression of VCAM-1 on endothelial cells. Binding of VLA-4 molecules on eosinophils with VCAM-1 molecules leads to extravasation of eosinophils. Then, IL-5 acts as an exotoxin to enhance transport of these cells to the lungs. Th2 cytokines increase survival of eosinophils as well [54,55].

SOCS1
-miR-155 drives differentiation of Th17 cells, directly inhibits SOCS1 in AD enhancing function of Th17 cells [59] skin biopsy from and patients with AD (n = 18) and healthy subjects (n = 29) CTLA-4 -miR-155suppresses CTLA-4 and by enhancing T-cell proliferation is involved in the regulation of T-cell responses [60] miR-151a blood leukocytes from AD patients (n = 117) and healthy subjects (n = 166)
the CD4 + T cells in an AR murine model. Differentially expressed genes were enriched in some pathways such as regulation of calcium ion transport, B cell activation and chemokine-signaling [66]. In a human study, Qian et al. compared expression of ANRIL in nasal mucosa samples between AR patients and non-atopic obstructive snoring patients. Notably, up-regulation of ANRIL characterized AR patients. Moreover, expression of this lncRNA positivly correlated with levels of TNF-α, IL-4, IL-6, IL-13, and IL-17, whereas, it negatively correlated with IL-10 and IFN-γ levels [67]. ANRIL expression also positively correlated with: increased AR risk, severity and inflammation indices, showing some diagnostic properties of this transcript in AR (Sensitivity = 81.3 % and specificity = 56.3 %) [67]. Table 4 summarize the results of studies which reported up-regulation and down-regulation of lncRNAs in AR. Function of miRNAs was also investigated in the pathophysiology of AR. Jia et al. assessed miRNA signature in nasal mucosa of AR patients and non-atopic subjects using microarray technique and qRT-PCR. Upregulation of miR-126-5p, miR-19a-5p and miR-26a-5p was reported in AR patients as compared to healthy subjects [69]. Hou et al. assessed expression of miRNAs in AR mice before and after treatment with ipratropium bromide (IB) effective in the control of AR symptoms. Differential expression of 87 miRNAs in IB group was found by comparison with the placebo group. Notably, mmu-miR-124-3p/5p, -133b-5p, -133a-3p/5p, -384-3p, -181a-5p, -378a-5p and -3071-5p were among the most up-regulated miRNAs. Based on these observations, authors suggested that IB treatment regulated expression of immune-associated miRNAs in the nasal mucosa of allergic mice and corresponded with amelioration of the nasal allergic symptoms [70]. Table 5 summarizes the results of studies which reported up-regulation or down-regulation of miRNAs in AR.
One of the most practical aspects of miRNA profiling in human disorders is application of differentially expressed molecules as predictive disease biomarkers. However, only a few studies assessed diagnostic performance of miRNA in diagnosis of AR. Table 6 summarizes the results of these studies.

Urticaria
Urticaria is an acute or chronic dermal edema which is caused by dilatation of vessels and leakage of fluid into the skin. Chronic spontaneous urticaria (CSU), also called chronic idiopathic urticaria (CIU), is characterized by spontaneous development of wheals and/or angioedema for more than 6 weeks without obvious triggering factors [93]. Mast cells granules mediators have essential roles in the pathogenesis of this disorder. CSU/CIU is an autoimmune disease [94]. A few studies evaluated ncRNAs in the pathophysiology of this disorder. Lin et al. reported on identification of miRNAs in CIU. By assessment of miRNAs in plasma, profiles of these molecules were obtained in groups comprising active hives or no hives and presence or absence of CIU. Differentially expressed 16 miRNAs were found in patients with active hives. MiR-2355-3p, miR-4264, miR-2355-5p, miR-29c-5p and miR-361-3p were over-expressed in exacerbated CIU patients. Target prediction of these miRNAs showed their enrichment in regulatory pathways such as TGF-β, glucocorticoid receptor, and p53 signaling. Some other enriched terms were p21-activated kinase, phosphoinositide-3 kinase, protein kinase B and neuroactive ligand-receptor interaction [95]. Zhang et al. explored the role of miRNAs in the CIU. Up-regulation of miR-125a-5p and CCL17 was found in patients sera. Although serum levels of miR-125a-5p were even higher in refractory CSU patients, its levels were down-regulated in patients who experienced remission [96]. Table 7 summarizes the results of studies that reported alterations of expression of miRNAs in urticaria.

Discussion
The current research on lncRNAs and miRNAs in allergic disorders is summarized, however, no circRNA studies were conducted so far. These data could be used for design of novel therapeutic strategies. Integrative assessment of lncRNAs, miRNAs, circRNA with mRNA-encoded proteins production is proposed as competing endogenous (ce) network. This approach was successfully applied in AD [56]. A similar analysis in AR identified mRNA-lncRNA network and suggested possible therapeutic targets [65]. More recently, comprehensive assessment of lncRNA-miRNA and mRNA-miRNA interaction data led to a construction of lncRNA-miRNA-mRNA ceRNA network in asthma. These analyses showed the importance of lncRNAs in the disease. Moreover, results suggested tentative novel targets for treatment via drug repositioning techniques [97]. Assessment of miRNAs/lncRNAs expression in paired tissue and serum samples to compare patients vs. healthy subjects facilitates identification of disease biomarkers and explores the correlation between their expressions in two tissues. The latter can elucidate source of alterations in peripheral blood. Such comparative analyses seems highly recommended in allergic diseases.
An imbalance between Th1 and Th2 responses is a common finding in allergic conditions. Assessment of the specific roles of miRNAs and lncRNAs in the regulation of these cells and identification of the ceRNA network would facilitate recognition of biomarkers for these disorders. This approach can be applied using the available expression profiles from high-throughput studies. Moreover, variable expression of ncRNAs in different stages of a disorder, e.g. remission or exacerbation, is necessary to fully address the function of these transcripts. Similarly, a response of ncRNA levels to a treatment evaluated by appropriate clinical scores would confirm importance of the observed alterations. For example, alterations in the expressions of miRNAs were reported in nasal mucosa after specific immunotherapy for AR in mice, thus inflicting miRNA in specific immunotherapy [98].  lower expression of miR-21 and higher expression of TGFBR2 in.CB associated with antenatal IgE production and development of AR. [84] miR-let-7e nasal mucosa from 23 patients with AR and 18 control patients SOCS4, JAK1/STAT3 miR-let-7e through the SOCS4/JAK1/STAT3 signaling pathway regulates progression and development of AR. [85] miR-21 -PTEN traditional chinese medicine yupingfeng upregulates PTEN-induced miRNA-21 while improving imbalance in theTh1/Th2 ratio in allergic rhinitis [86] miR-155, miR-181a 25 AR children and 20 healthy children IL-10 decreased regulatory cells (Tregs)-derived miR-155 and miR-181a correlated with reduced number and function of Tregs in AR children. [87] let-7e 159 young adult subjects subdivided into control (n = 34), AR (n = 50), AR + asthma (n = 36), and non-allergic rhinitis (NAR, n = 39) let-7e importance in allergic inflammation of nasal mucosa. [77] miR-106b -Egr-2 miR-106b by targeting Egr-2 regulates pro-allergic properties of dendritic cells and Th2 polarization in vitro [88] miR-143 nasal mucosal from AR (n = 23) and non-AR sibjects (n = 18). IL13Ra1 miR-143 by targeting IL13Ra1 inhibits IL-13-induced inflammatory cytokine and mucus production in nasal epithelial cells of AR patients [89] miR30a-5p 20 AR and 20 control subjects SOCS3 miR30a-5p/SOCS3 involved in the pathogenesis of AR [90] miR-181a, miR-155 20 AR and 20 healthy subjects SOCS1, SIRT1, IL-10, TGF-β, PI3K/ Akt miR-155 and miR-181a closely correlated with the proliferation and function of Tregs in AR [91] miR-15a-5p nasal mucosa of 20 patients with long-term AR and 20 non-AR subjects ADRB2 in AR stimulated by IL-13, ADRB2 inhibits inflammatory response of NECs, miR-15a-5p has a negative regulatory effect on ADRB2 [92] miR-375 -JAK2, STAT3 miR-375 via inhibiting JAK2/STAT3 pathway ameliorates AR and prevents nasal mucosa cells from apoptosis in mice mode. [73] S. Ghafouri-Fard, et al.  Upregulated 12 patients stratified to: normal chronic urticaria index and no active hives; positive disease index no active hives; active hives with a negative disease index; active hives and positive disease index modulation of inflammation-related pathways [95] miR-125a-5p Upregulated 20 active CIU patients and 20 healthy controls CCL17 miR-125a-5p could serve as potential serum biomarkers for CIU. [96] S. Ghafouri-Fard, et al.
The presented data appeals for the role of both lncRNAs and miRNAs, and possibly circRNAs in the pathogenesis of asthma, AR, AD and urticaria. Identified miRNAs/lncRNAs alterations awaits replication studies.

Declaration of Competing Interest
The authors declare they have no conflict of interest.