microRNA and Pre-microRNA Related Genetic Variants Contribute to the Pathogenesis of Behcet' Diseases

Background：Uveitis is a general term for uveal tract inflammationdue to any cause and usually affecting not only the uvea but also the retina,vitreous and retinal vessel. Majority of uveitis have occurred in young adultpopulation. It is one of the main global causes of blindness. Behcet’sdisease （BD） is one of the most prevalent uveitis entities seen in China. Ithas long been understood that certain infectious events and/orenvironmental factors may trigger BD in individuals with particular geneticvariants. Although it was initially thought that Th1cells were the main cellsmediating BD, recent studies have focused on the critical role for Th17cells. The pathways involved in the triggering of Th17cells are not fullyunderstood. Previous studies suggested that the Notch pathway played acritical role in Th1and/or Th2-mediated immune responses, but lately acritical role for Notch pathway in the differentiation of Th17cells hasemerged. As a novel gene regulator, microRNA （miRNA） has beeninvolved in the regulation of Notch pathway. Furthermore, miRNA has alsobeen shown to play a critical role in immune regulation and the pathogenesis of autoimmune diseases.Considering the background mentioned above, the present study wasdesigned to investigate the questions as follows:1.Whether Notch pathwayand its related miRNAs are involved in the pathogenesis of Behcet’sdisease.2. Whether common pre-miRNA Single nucleotide polymorphismswere associated with Behcet’s disease （BD）, Vogt-Koyanagi-Harada （VKH）syndrome and acute anterior uveitis （AAU） associated with Ankylosingspondylitis （AS）.Our study may not only provide the novel insights into thepathogenesis of BD but also offer a novel therapeutic approach for Behcet’sdisease.Part Ⅰ Increased Notch pathway activation due to down-regulatedmiR-23b is involved in the pathogenesis of Behcet’s diseasePurpose: Behcet’s disease （BD） is a refractory inflammatory disorderwith unknown causes. Since the Notch pathway is critically involved in theimmune response, the present study was undertaken to investigate the roleof this pathway in BD.Methods: Hes-1and Notch1-4expression, frequency of IFN-γ andIL-17expressing T helper cells, Notch intracellular domain （NICD）, phosphorylation of STAT3and the production of IFN-γ and IL-17wereexamined by real-time PCR, flow cytometry and ELISA. Notch blockadewas performed using the γ-secretase inhibitor DAPT. Transfection withmiR-23b mimics and inhibitor was used to examine the effect of miR-23bon Notch pathway activation.Results: Active BD patients showed an increased activation of theNotch pathway in association with a higher Th17and Th1response. Notchblockade significantly inhibited both Th17and Th1responses. The effect ofNotch blockade on the Th17response was associated with a lower level ofSTAT3phosphorylation. miR-23b was significantly decreased in CD4+Tcells from active BD patients. CD4+T cells transfected with miR-23bshowed a reduced expression of NICD and a reduced frequency of IL-17and IFN-γ expressing T cells.Conclusions: The present study suggests that an increased activationof the Notch pathway may contribute to the pathogenesis of Behcet’sdisease. Decreased expression of miR-23b may be involved in theactivation of the Notch pathway in BD. Manipulation of the Notch pathwayor miR-23b may offer a novel therapeutic approach for Behcet’s disease. Part Ⅱ A functional variant of pre-miRNA-196a2confers risk forBehcet’s disease but not for Vogt-Koyanagi-Harada syndrome or Acuteanterior uveitis （AAU） associated with Ankylosing spondylitisPurpose: This study aimed to investigate the predisposition ofcommon pre-miRNA Single nucleotide polymorphisms （SNPs） withBehcet’s disease （BD）, Vogt-Koyanagi-Harada （VKH） syndrome and acuteanterior uveitis （AAU） associated with Ankylosing spondylitis （AS）.Methods: A two-stage association study was conducted in859BD,400VKH syndrome,209AAU+AS+patients and1685controls among theChinese Han population. Genotyping, the expression of miR-196a andBach1（the target gene of miR-196a）, cell proliferation, cytokineproduction were examined by PCR-RFLP, Real-time PCR, CCK8andELISA.Results: In first stage study, the results showed a significantlyincreased frequencies of the miR-196a2/rs11614913TT genotype and Tallele but a decreased frequency of the CC genotype in BD patients（PBonferroni correction（Pc）=0.027, OR=1.58; P-4c=6×10, OR=1.45; Pc=0.018,OR=0.56, respectively）. However, No significant association of the testedSNPs with VKH and AAU+AS+patients was observed. The second stage and combined studies confirmed the association of rs11614913with BD(TT genotype: Pc=9.0×10^-4, OR=1.41; T allele: Pc=7.5×10^-5, OR=1.29; CCgenotype: Pc=0.023, OR=0.71). A stratified analysis showed the associationof rs11614913TT genotype and T allele with arthritis subgroup of BD(Pc=5.3×10^-3, OR=1.89; Pc=0.015, OR=1.56, respectively). Functionalexperiments showed a decreased miR-196a expression, increased Bach1expression and increased production of IL-1β and MCP-1in TT casescompared to CC cases（P=0.023; P=0.0073; P=0.012; P=0.002，respectively）.Conclusions: This study shows that a functional variant of miR-196a2confers risk for BD but not VKH syndrome or AAU+AS+in a ChineseHan population through modulating the expression of miR-196a andregulating cytokine production such as IL-1β and MCP-1.