**2. The Diagnostic Role of miRNAs**

Due to diagnostic difficulties, delays in initiating appropriate treatment and the need to deal with complications of untreated diseases, asthma is a resource-consuming condition in the healthcare sector. Finding and implementing into clinical practice noninvasive, fast, and sensitive diagnostic methods that can undoubtedly be based on miRNA molecules would largely solve this growing problem. Research has been conducted on these biomolecules that could play a role in this process. Increased expression of miRNA-221 and miRNA-485-3p has been shown in the pediatric asthma population. The first molecule is associated with increased adhesion and migration of mast cells and cytokine production after the body comes into contact with the antigen. In addition, these biomolecules reduce the concentration of the Spred-2 protein (Sprouty-related EVH1 domain-containing 2), contributing to increased cell proliferation and the eosinophilic inflammatory response in the airways through IL-5 [7,11]. MiRNA-21 levels are higher in children with confirmed asthma and positively correlated with disease severity and serum and sputum eosinophilia in these patients. This molecule acts on the Smad-7 gene and inhibits the production of a protein that is an inhibitor of the TGFβ1/Smad pathway. The consequence of this is the excessive synthesis of collagen, α-smooth muscle actin, proliferation and differentiation of fibroblasts, and deposition of the extracellular matrix. This results in remodeling, fibrosis of the airways, and increased airway obstruction [12]. In a study conducted by M. Atashbasteh et al. among patients with severe asthma, increased expression of miRNA-125 was demonstrated, as well as decreased expression of miR-124, miR-130a, and miR-133b. The level of the first of these was correlated with the concentration of CRP and IgE in the serum. These molecules are involved in the pathway of phosphorylation of sphingosine to sphingosine-1 phosphate (S1P) by sphingosine kinase type 1 (SphK1). The effect of S1P on cells is possible by affecting receptors, e.g., S1PR and S2PR. The former is present in large amounts on the surface of lymphocytes and is involved in the process of their maturation (in particular, release from lymphatic organs). In addition to affecting the immune system cell population, S1P also participates in the activation of molecules that regulate inflammation, proliferation, and cell death: NF-kB (nuclear factor kappa B) and STAT3

(signal transducer and activator of transcription 3). For this reason, the significant role of this pathway in the development of autoimmune diseases is emphasized. The increased concentration of this compound in the airways of patients with asthma is responsible for the increased hyperreactivity of the smooth muscle cells of the bronchial tree and the increased inflammatory process. MiRNA-124 is associated with sphingosine kinase 1 (SphK1), which is responsible for the formation of S1P, miRNA-130a with an effect on the S1PR2 receptor (it was proven that the reduction in its expression is associated with an increase in the concentration of inflammatory mediators, including TNF-α and increased expression of inflammatory genes). MiRNA-133b, on the other hand, plays a significant role in controlling the level of sphingosine-1-phosphate receptor protein [13].

An increased blood concentration of some miRNAs in the pediatric population suffering from asthma, e.g., miRNA-3162-3p, miRNA-1260a, miRNA-let-7c-5p, and miRNA-494, has become the subject of further research interest in understanding their role in the pathogenesis of allergic diseases. Particular attention was paid to the miRNA-3162-3p increase in serum in a study of an allergic asthma model conducted in albumin-sensitized mice. Its relationship with β-catenin, which is responsible for the reconstruction of the respiratory tract, and its inhibitory effect on the expression of the gene encoding this protein was proven. The experimental use of antagomir (anti-miRNA) resulted in a reduction in airway hyperreactivity, inflammation, and an increase in B-catenin levels. Thus, signaling involving this molecule seems to be crucial in models of asthma provoked by allergens [14,15].

In childhood the model of allergic asthma dominates, with an increased Th2 cell response and eosinophilic inflammation of the airways. Eosinophils play a key role in the development and maintenance of the inflammatory process. It was shown that by their effect on smooth muscle cells (ASMC) in laboratory conditions, they promote proliferation and hypertrophy. This only occurs in the environment of eosinophilic cells obtained from the blood of sick people. These processes include, for example, TGF-β1 and WNT-5 genes [16]. Eosinophils are also a source of miRNAs which can be transferred between the cells of the body through exosomes, becoming a potential regulator of gene expression [17].

In a study evaluating differences in the expression of miRNAs in eosinophils in patients with the allergic march and healthy subjects, 18 biomarkers were isolated that were associated with the expression of genes involved in cellular regulation, immune response, angiogenesis, and smooth muscle cell proliferation. This highlights the role of miRNAs, which may play a role in the atopic march and the occurrence of many allergic diseases in one patient. Attention was paid to miRNA-590, which was then also shown to be down-expressed in previous studies. Its target is the CITED-2 gene, which is responsible for the regulation of the TGF-beta pathway and the proliferation of airway smooth muscle cells [18,19]. Another study demonstrated a higher concentration of miRNA-144-3p in severe disease and its positive correlation with blood eosinophilia. This biomolecule affects genes responsible for the inflammatory process and remodeling of the airways, e.g., GATA3, STAT6, SOCS5, RHOA, NR3C1, and PTEN, which makes it a potential diagnostic marker for severe asthma [20]. In non-allergic asthma, miRNA-629-3p, miRNA-223-3p, and miRNA-142-3p were significantly elevated in patients with severe symptoms and associated with increased inflammation mediated by neutrophils. In particular, the miRNA-629-3p molecule was responsible for the increased production of IL-8, which stimulates the migration of neutrophils, monocytes, and T cells and the adhesion of neutrophils to the endothelium [21]. Individual miRNAs also affect the expression of interleukins. Involvement in inflammatory processes, e.g., miRNA-1248 (increased concentration in asthmatics) stimulates the synthesis of IL-5, responsible for survival, growth, differentiation, and recruitment of eosinophils. MiRNA-181b-5p, on the other hand, showing reduced expression in people with asthma, is characterized by targeting the SPP1 molecule, i.e., phosphoprotein 1, otherwise known as osteopontin. It is a component of the extracellular matrix that is involved in the migration of eosinophils to the respiratory tract and the intensification of IL-13-induced expression of IL-1 and CCL 11 (eosinophil chemotactic protein) in bronchial epithelial cells [7,22]. MiR-146a and miR-106b, in turn, are upregulated in pediatric asthma patients

and lead to increased production of IL-5 and IL-13, which stimulate inflammatory cell recruitment, epithelial cell hyperplasia, smooth muscle hyperplasia, goblet cell metaplasia, and extracellular matrix deposition in the respiratory tract [23].
