Microrna And Pre-microRNA Related Genetic Variants Contribute To The Pathogenesis Of Allergic Rhinitis

Background : AR is one of the most common chronic allergic inflammatory disorders,induced by an Ig E-mediated reaction,following exposure to an allergen.It is a global health problem,with a high prevalence especially in industrialized countries,and has exhibited a fairly rapid upward trend,and which is a multifactorial disorder produced by the interaction of genes and the environment.AR is also known to be a risk factor for the development of asthma.The prevalence of AR and asthma are markedly increasing,and it is known that a very high comorbid rate of these two diseases.AR and asthma has been reported different combinations of genes or SNPs increase the risk of phenotypic expression,resulting in allergic inflammation.These reports suggest that genetic factors may play a role in this disease.To date,despite significant findings regarding susceptibility regions and genes,these studies barely detect the link between exposure to allergen and genetic regulation,and have only provided us with a very limited understanding of allergic diseases.MiRNAs are single-stranded RNA molecules of 19-25 nucleotides in length that have been recently recognized as an important regulator in immune homeostasis and are highly conserved throughout evolution.Recent studies have demonstrated that the functions of miRNAs to the pathomechanism of chronic inflammatory conditions,allergic disease,such as asthma,atopic dermatitis,and allergic rhinitis have already been emphasized.Mi RNAs can regulate gene expression by translational inhibition and destabilization of m RNAs,and have been implicated in the regulation of maturation,proliferation,differentiation and activation of immune cells.Previous studies have shown miRNA-associated SNPs would likely improve our understanding of miRNA biogenesis and the potential contribution of these SNPs to normal human variation and disease pathogenesis.Therefore,SNPs located in the pre-miRNA regions in mature miRNA processing may contribute to autoimmune or autoinflammatory diseases.Among them,mi R-21,mi R-126 and mi R-155 were demonstrated to play a regulatory role in balancing the Thl/Th2 immune response.These SNPs located in the pre-miRNA regions of mi R-146 a,mi R-196a2,mi R-499,mi R-149 and mi R-585 have been shown to play a critical role in the pathogenesis of various human diseases including various inflammation related diseases.But these studies barely detected the interaction between the miRNAs single polymorphisms and the environment.Therefore,in consideration of the important role of seven SNPs of miRNAs in the development of inflammation-related and autoimmune diseases,we aimed to determine whether the polymorphisms of seven miRNA genes were associated with AR and AR-A,if so,whether the variant affected gene expression and relevant cytokine production.Then,we also investigated function of miRNAs polymorphisms from different genotypes in response to HDM allergen,in order to study the role of allergen in genetic regulation.More importantly,the underlying mechanisms of miRNAs in the regulation of immune responses in AR against varied pathogens or stimuli remain poorly understood,which await further investigation.Part Ⅰ Gene variant of miRNA-149 confers risk for allergic rhinitis and comorbid asthma in Chinese childrenPurpose: The prevalence of allergic rhinitis（AR）and asthma are markedly increasing,and it is known that a very high comorbid rate of these two diseases.Our intention was to assess the association of mi R-146 a,mi R196a2,mi R-499,mi R-149 and mi R-585 single-nucleotide polymorphisms（SNPs）with AR comorbid with asthma.Methods: We included 124 AR patients,176 AR with asthma（AR-A）patients and 206 control Chinese children subjects in a case-control comparison.7 SNPs,mi R-146a（rs2910164,rs57095329 and rs6864584）,mi R-196a2（rs11614913）,mi R-499（rs3746444）,mi R-149（rs2292832）and mi R-585（rs62376935）were genotyped by polymerase chain reactionrestriction fragment length polymorphism（PCR-RFLP）method.Results: A substantially growing prevalence of the homozygous mi R-149（rs2292832）CC genotype and C allele appeared in AR and AR-A patients,compared with controls.AR-A groups showed significantly increased frequencies of the CC genotype and C allele of mi R-149（rs2292832）than AR group.No significant difference concerning the genotypic and allelic frequencies of mi R-146a（rs2910164,rs57095329 and rs6864584）,mi R-196a2（rs11614913）and mi R-499（rs3746444）polymorphisms could be found aomng patients with AR,AR-A and the normal controls.Conclusions: To the best of our knowledge,this is the first study to show a vital association between single polymorphisms in miRNAs and AR,AR-A in the Chinese population.Our data indicate a gene variant of mi R-149 rs2292832 confers risk for comorbid of AR and asthma.Part Ⅱ Association between miRNA gene polymorphisms and allergic rhinitis in ChinesePurpose: The current study aimed to explore whether singe nucleotide polymorphisms（SNPs）of seven genes mi R-146a（rs2910164,rs57095329 and rs6864584）,mi R-196a2（rs11614913）,mi R-499（rs3746444）,mi R-149（rs2292832）and mi R-585（rs62376935）were associated with allergic rhinitis（AR）in Chinese.Methods: We included 610 AR patients and 720 control Chinese subjects in a case-control comparison.The study involved determination of 7 selected SNPs in miRNAs by performing PCR restriction fragment length polymorphism analysis（PCR-RFLP）.In order to validate the method used in this study,Invitrogen Biotechnology Company was assigned to execute direct sequencing by using randomly selected subjects（20% of all the blood samples）.Results: A substantially growing prevalence of the homozygous mi R-149 rs2292832 CC genotype and C allele appeared in the AR patients unlike that observed in the control individuals(P= 2.60×10-9,OR 2.66;P= 4.11×10^-11,OR 1.70,respectively).Conclusions: This study shows that a gene variant of mi R-149 confers risk for AR.Part Ⅲ A functional variant of miRNA-149 with and without allergen exposure in allergic rhinitisPurpose: Since we found that polymorphisms of mi R-149 showed an association with susceptibility to AR,we investigated whether the variant affected gene expression and relevant cytokine production.Then,we also investigated function of miRNAs polymorphisms from different genotypes in response to HDM allergen,in order to study the role of allergen in genetic regulation.Methods: Peripheral blood mononuclear cells（PBMCs）were prepared from health people.For antigen stimulation,PBMCs were incubated with house dust mite extracts（HDM）for 48 h.Real-time PCR was used to detect m RNA expression from PBMCs with or without stimulation by house dust mite extracts（HDM）,and Levels of interleukin（IL）-4,IL-5,IL-6,IL-17,IL-10,and INF-γ in culture supernatants were measured by ELISA.Results: Functional experiments showed a decreased mi R-149 expression,an increased production of IL-4 and a decreased production of INF-γ in CC cases compared to TT cases in response to HDM allergen（P<0.05,respectively）.Conclusions: This study shows that a functional variant of mi R-149 confers risk for AR by modulating the mi R-149 gene expression and regulating Th1/Th2 inflammatory IL-4 and INF-γ production following exposure to allergen.