Methylation Array And Gene Expression Profiles For Screening Of Differential Genes Associated With Allergic Rhinitis And Partial Validation

BackgroundAllergic Rhinitis(AR)is one of the most common airway inflammatory diseases,and the incidence of AR is increasing annually.The pathogenesis of AR has not yet been fully understood,making the effective and accurate treatments challenging to be developed.Studies have shown that DNA methylation changes and abnormal gene expression levels of RNA may have implicated in the pathogenesis of AR.By using high-throughput microarray,some potentially pathogenic genes and methylation sites in patients with AR can be identified.The combination with bioinformatic analysis and biological assays for verification may provide novel ideas and theoretical basis for the pathogenesis of AR.MethodsWe sampled the inferior turbinate mucosae from patients with AR(n = 19)and without AR(n = 11).Through whole-genome methylation chip and genome-wide expression microarray assay,we screened the abnormal methylation sites and differentially expressed genes in patients with AR.The genes were then analyzed with Gene Ontology enrichment,KEGG pathway enrichment database,combinatorial chips(methylation and expression profiling)and literature search.Finally,we selected samples from patients with AR(n = 55)and without AR(n = 40),and validated for partially differential genes which were screened by performing genome-wide expression microarray assays at both mRNA and protein levels.ResultsThere were 94 aberrant methylation sites in patients with AR.No statistically significant methylation genes were identified with Gene Ontology enrichment,KEGG pathway enrichment,and both.Through literature search,TLR6 and IL-4R gene abnormal methylation sites might be closely related to the pathogenesis of AR.Gene expression profiling revealed a total of 160 differentially expressed genes in patients with AR;Gene Ontology analysis demonstrated 32 significant functional enrichment groups,of which 20 were directly related to ciliary structure or function;KEGG analysis identified 8 significant pathways,of which one was directly related to the ciliary structure and mostly enriched.Based on the literature search and the characteristics of ciliary structure and function,we selected the following differentially expressed genes related to the ciliary function(FOXJ1)and structure(DNAI1,DNALI1 and DNAH9),respectively.The expression of FOXJI1 DNAI1,DNALI1 and DNAH9 in the inferior turbinate mucosa from patients with AR was also confirmed by using real-time PCR.The protein expression levels of FOXJ1 and DNAI1 were detected with immunofluorescence staining.We have identified four localization patterns of FOXJ1 protein.Furthermore,we have established a scoring system,and in combination with cell experiments,to evaluate the localization characteristics of FOXJ1 and DNAI1 protein.We found significantly increased abnormal localization patterns of FOXJ1 and DNAI1 protein in inferior turbinate mucosa from patients with AR.ConclusionIn our study,we have screened the abnormal methylation sites and differentially expressed genes in the inferior turbinate mucosa from patients with AR,which provided important clues of the pathogenesis in AR.The down-regulation of ciliary structure-and function-related genes and abnormal localization of protein markers are important features in the inferior turbinate mucosa sampled from patients with AR,and may be closely related to the occurrence and development of AR.Further studies on this mechanism are warranted for further confirmation.Finally,we have established a localization scoring system for ciliary marker proteins,which may provide an important reference for future studies.