Structure and functional impact of seed region variant in MIR-499 gene family in bronchial asthma

Bronchial asthma is a chronic heterogeneous respiratory disease that is characterized by airway inflammation, recurring bronchial obstruction, and airway hyper-responsiveness [1, 2]. Most common histopathological features are inflammatory cell infiltration, sub-basement fibrosis, smooth muscle hypertrophy, mucus hypersecretion, injury to epithelial cells, and angiogenesis [3, 4]. Treatment with anti-inflammatory drugs or bronchodilators usually improves some of these features. Nevertheless, therapeutic response relies on the interplay between environmental exposure and genetic background [2]. Despite several advances over the past decades in understanding the underlying mechanisms involved in the disease, there are no current satisfactory strategies for the cure or prevention of long-term decline in pulmonary function [5]. Given the significant morbidity and burden of childhood asthma worldwide, better therapeutic modalities are mandatory to counteract the development and progression of the disease. In prior studies, a sizeable proportion of genetic influence existed, ranging from 35 to 95% for asthma and 30 to 66% for bronchial hyper-responsiveness [6]. Genome-wide association studies provide evidence for multiple novel loci associated with the disease. However, the exact maestro fine tuning these putative genes is still uncovered.

In early 90’s, the presence of small non-coding RNAs (ncRNAs) was discovered in the mammalian genome [7]. These microRNAs are transcriped via specific cellular machinery to form short single-stranded mature RNAs of 19–24 nucleotides long. They function by complementary base pairing with mRNA targets, leading to its degradation or translational repression [8]. They are estimated to modulate gene expression of 60% of protein-coding genes, and to regulate many cellular processes; including proliferation, apoptosis, immunomodulation, stress response, and angiogenesis [9]. As a result the focus of human genome studies has witnessed a shift from mRNAs to ncRNAs as major key players in human disorders. Currently, there are emerging opportunities for targeting these disruptions of ncRNAs using novel therapeutic approaches. Some strategies aimed to increase the levels of abnormally down-regulated microRNAs via epigenetic drugs as DNA demethylating agents and histone deacetylase (HDAC) inhibitors or by replacement of miRNAs using virus delivery systems [10]. On the other hand, over-expressed microRNAs in human diseases can be sequestered by anti-miRNA oligonucleotides, miRNA sponges, miRNA masking and small molecule inhibitors [8].

In silico analysis and surveying the literature revealed the deregulation of microRNAs in various pulmonary diseases (Fig. 1). Several lines of evidence suggest a key role for hsa-microRNA-499a (miR-499a) in modulating the immune response, cell proliferation, apoptosis, neuromuscular regulation and neoangiogenesis [11, 12]. Exploring gene targets of miR-499a by computational tools identified inflammatory-related gene targets, including IL-13 and Il-23, which represent important mediators in asthma KEGG pathway [ID hsa05310] (microRNA.​org). A single nucleotide polymorphism (SNP), rs3746444 (A > G), is located in the seed sequence of miR-499a-3p, a region essential for miRNA-mediated silencing mechanism. SNPs within mRNA-binding site of miRNAs may influence mRNA gene set, target binding ability, or pre-miRNA maturation process, which in turn, could alter the susceptibility to develop human diseases. The rs3746444 MIR-499a SNP was found to be associated with higher risk of rheumatoid arthritis [12, 13], coronary artery disease [14], Behcet’s disease [15], and ankylosing Spondylitis [16]. Hence, the current study was conducted to investigate the association between rs3746444 polymorphism and susceptibility to asthma disease in children and adolescents, and to further assess computationally its impact on the clinical outcome and bronchial hyper-responsiveness (BHR) in a group of Egyptian asthmatic children and adolescents.

MicroRNAs are involved in various biological cellular processes. Aberrant transcriptomic signatures of various miRNAs have been detected in the airways and circulation of asthmatic patients [33]. Sequence variations within miRNA genes, especially in the mature miRNA seed region, may have a profound impact on miRNA biogenesis and function [34, 35]. In 2009, an inflammation-related MIR-499A was identified within the intronic sequence of the myosin MYH7B gene [36]. Several lines of evidence suggest that miR-499a play critical roles in orchestrating the immune response in various human disorders [11, 12, 37]. Bioinformatics analysis determined complement base pairing with important cytokines as IL-13 and IL-23, both are recognized effectors in asthma pathway. IL-13 signaling results in mucin hypersecretion, airway remodeling and fibrosis, and bronchial hyper-responsiveness [38]. IL-23 induction regulates allergic asthma through modulation of TH2 cell differentiation and eosinophilic infiltration [39]. Functional enrichment clustering illustrated the involvement of miR-499a-3a and miR-499a-3b in Toll-like receptor (TLC) signaling pathways that are implicated in the initial phase of the host defense against invading pathogens and allergens leading to TH2 activation [40, 41]. In addition, miR-499a and miR-499b are involved in Fc-epsilon signaling pathway; cross-linking of IgE with FcεRI receptors on mast cells, causes the release of leukotrienes and histamines essential for immediate allergic reaction. Other biological processes as gene expression, cellular protein modification process, transcription initiation from RNA polymerase II, and cellular component assembly are significantly mediated by miR-499 gene family.

Using various computational tools, miR-499a and miR499b were predicted to target key molecules in asthma-related KEGG pathways. They could modulate 18 targets in mucin type O-biosynthesis pathway related to polypeptide N-Acetylgalactosaminyltransferase (GALNT) genes. Hyperglycosylated mucins are commonly found in chronic inflammatory airway diseases such as asthma, for which inhibition of mucin and their glycosylation could contribute in controlling the disease [42]. The next most significant pathway was fatty acid biosynthesis with 5 target genes. Recent studies demonstrated the vital role of fatty acids in the formation of inflammatory mediators relevant to the pathophysiology of asthma [43]. Another identified pathway that is highly impacted by miR-499 gene family is glycosphingolipid (GSL) biosynthesis. β-Glycosphingolipids have emerged as a family of potential ligands for natural killer (NK) cells. Extensive infiltration of NK cells in the airway bronchial mucosa is considered one of the prominent driver in asthma development via inducing the secretion of TH2 cytokines that results in airway hyper-responsiveness and inflammation [44]. Lowering GSL levels in mast cells in an animal model of asthma was found to ameliorate disease manifestations [45]. Both mature forms of miR-499a and miR-499b were predicted to target about 40 and 60 genes in TGF-beta and TNF signaling pathways, important mediators contributing in lung tissue fibrosis and proliferation of smooth muscle cells with subsequent airway remodeling [46]. We also identified several targets for miR-499 gene family in adherence junction and focal adhesion pathways. Tight junctions not only seal the pulmonary epithelium and maintain the structural integrity of the airway walls, but also act as key regulators for epithelial cell proliferation and differentiation. Dysfunction of this homeostasis could participate in airway wall remodeling in asthma disease [47].

Within MIR499A and MIR499B genes, bioinformatics analysis revealed the presence of a common polymorphism at the position 20:34,990,448 within the seed region of mature miR-499a-3p (rs3746444; AC[A/G]UCAC). In the current study, we genotyped rs3746444 polymorphism in asthmatic children and adolescents compared to controls. Genotype frequencies did not deviate from the Hardy-Weinberg equilibrium in the study groups. G variant was associated with increased susceptibility to develop bronchial asthma under all genetic association models. Despite the MAF (G) represented 0.26 in the control group, it accounted for 0.59 among patients. The same allele was found to be associated with poor pre-bronchodilator FEV₁ in asthmatic patients. In addition, homozygotes for G had the worst bronchodilator response after Salbutamol inhalation. That was consistent with prior studies; G allele was also associated with increased risk of developing of several autoimmune and inflammatory diseases. miR-499a*G carriers showed higher susceptibility to RA in Egyptians [12] and Iranian people [48], Behcet’s disease in Turks [15], ulcerative colitis in Japanese [49], ankylosing spondylitis [16], idiopathic recurrent spontaneous abortion in Koreans [50], and coronary artery disease [51, 52]. In addition, individuals with rs3746444*G allele showed a more active phenotype of RA in Egyptian female patients [53] and a severe course of multiple sclerosis [54].

Though our in silico data analysis revealed that the substitution of A > G has no dramatic effect on the folding pattern of the hairpin loop structure, each allele was predicted to have different set of gene targets. A total of 1890 genes is influenced by mature miR-499a with rs3746444*A variant. In contrast, rs3746444*G allele could delete 41.8% of these genes and create new 763 targets. By conducting functional annotation clustering and enrichment analysis, our results illustrated that gene sets for each allele only shared the same 37.8%, 40.1%, and 52.8% for biological process, molecular function, and cellular components, respectively. In particular, G allele targets were significantly involved in two new KEGG pathways (other glycan degradation and glycolysis/gluconeogenesis). In the former pathway, miR-499 can target both aspartylglucosaminidase (AGA) gene involved in the catabolism of N-linked oligosaccharides of glycoproteins in the lysosomes, and galactosidase beta 1 (GLB1) gene, which plays functional roles in the formation of extracellular elastic fibers and in the development of connective tissue. In the other pathway for glycolysis/gluconeogenesis, miR-499 mainly target aldehyde dehydrogenase 1 family member A3 (ALDH1A3) and alcohol dehydrogenase 1 beta polypeptide (ADH1B) genes, members of the enzyme family which metabolize a wide variety of substrates as multiple inositol-polyphosphate phosphatase 1, an important second messenger in eukaryotic cells. ALDH1A3 gene was found to be over-expressed more than two times in asthmatic patients and 3.5 times during exacerbations [55]. Further, in vivo functional analysis is warranted to explore the mechanistic regulation of these targets in the etiopathogenesis of asthma.

In some previous studies, computational methods were devised to study the effects of air-borne dust through high fidelity computational simulation of different particle sizes over several breathing cycles [56], modeling lung pressures [57], and developing 3D Euler Lagrangian models to obtain the regional deposition of the poly-dispersed drug Budesonide [58]. It would be a beneficial next step to assess the pharmacogenomics effect of variants with the regional deposition of dust particles and drug delivery to the respiratory tract during respiration.

To the best of our knowledge, this is the first study highlighting the role of miR-499 rs3746444 in bronchial asthma disease. However, some limitations need to be addressed. First, the small relative sample size in the current study might underestimate the synergistic effect of miR-499 SNP with environmental exposure. Second, the impact of the SNP was not correlated with the miRNA expression profile. Third, functional assessment of targets involved in bronchodilator response and methacholine challenge test is mandatory to develop more efficient therapeutic strategies.

In conclusion, our study suggested that the rs3746444 (A > G) polymorphism in miR-499 gene family might contribute to the susceptibility of asthma in children and adolescents with bronchial asthma. Additional studies, including larger cohorts with diverse ethnic background, and functional tests are warranted to explore the immunomodulatory mechanism of miR-499 genes in bronchial asthma for developing more specific theranostic agents.