DNA氧化—甲基化耦联调控哮喘气道上皮整合素β4低表达机制研究

前期研究揭示气道上皮整合素β4低表达与哮喘气道上皮脱落、局部免疫和炎症紊乱有关。明确哮喘气道上皮整合素β4低表达的机制具有理论和临床意义。本项目提出DNA氧化-甲基化耦联调控哮喘气道上皮整合素β4低表达假说：环境或变应原激发气道上皮氧化应激，通过氧化损伤碱基8-oxoG—OGG1途径选择性调节气道上皮整合素β4基因转录调控区胞嘧啶甲基化，阻碍应激条件下转录因子的作用，降低整合素β4表达。拟研究：①临床哮喘标本及哮喘动物模型气道上皮整合素β4基因甲基化检测、甲基化序列的定位及分布；②炎症、免疫刺激下整合素β4基因的8-oxoG位点及OGG1结合活性；③8-oxoG—OGG1途径对整合素β4基因的特异性序列识别及其对整合素β4基因转录调控机制分析；④炎症、免疫刺激下整合素β4基因转录因子谱鉴定，分析转录因子结合位点及活性与甲基化序列及8-oxoG—OGG1作用的耦联关系。

In our previous study we found that decreased expression of integrin β4 in brochial epithelial cells (BECs) led to shedding of BECs and disturbed local immune responses in asthmatic airways. A better understanding of molecular mechanisms underlying the decreased expression of integrin β4 will provide potential noval therapeutic targets for asthma. We hypothesized that the expression of integrin β4 is under the control of coupled DNA oxidation and methylation in asthmatic BECs. Environmental stimli or allergens may trigger the oxidative responses in BECs and then lead to the methylation of cytosines within the promoter region of ITGB4 gene which encodes integrin β4 in BECs via an 8-oxoG-OGG1 pathway. The epigenetic changes within the promoter region of ITGB4 gene consequently blocks the transcription sites and ultimately prevent the stress induced transcriptional activation of ITGB4. To test our hypothesis, we will conduct the following experiments: ① to examine the pattern of methylation of ITGB4 in BECs from both asthmatic patients and asthmatic animal models; ② to examine the 8-oxoG containing sites within the promoter region of ITGB4 and determine the binding affinity of those sites to OGG1 in response to various immune stimuli; ③ to explore the mechanisms underlying the recognition of specific sequences of ITGB4 by 8-oxoG-OGG1 pathway and analyze the regulatory role of DNA-RNA hybrid in transcriptional activation of ITGB4; ④ to characterize the transcription factors in control of ITGB4 expression and determine the corresponding relationships between methylated sequences and oxidized sequences recognized by 8-oxoG-OGG1 pathway, and to provide evidence to the regulation of ITGB4 expression by coupling of oxidation-methylation pathways within its promoter region.