Runx3基因DNA甲基化介导BPD肺上皮细胞转分化的作用及机制研究

早产儿支气管肺发育不良（BPD）肺泡化障碍的病理机制，现认为与氧化应激反应、细胞因子等介导的Ⅱ型肺上皮细胞（ATⅡ）直接损伤密切相关。本项目在前期工作发现并证实存在ATⅡ向间质细胞转分化（EMT）生物学现象的基础上，提出ATⅡ损伤并非影响BPD肺泡化的唯一机制，EMT可能是又一关键环节，并以肺发育相关基因Runx3为切入点：构建Runx3缺失和过表达细胞模型，阐明Runx3是调节EMT的重要基因；应用亚硫酸氢盐测序PCR等技术，证实启动子区CpG岛DNA甲基化是导致Runx3表达沉默的关键靶点，并在整体和细胞水平，通过阻断甲基化，揭示Runx3介导EMT干扰BPD肺泡发育的分子机制；采用芯片杂交等技术，筛选靶向调节的微小RNA（miRNA），探索并验证miRNA调控DNA 甲基化、上调Runx3表达机制，最终阐明miRNA/DNA甲基化/Runx3/EMT 是改善BPD肺泡发育又一新途径。

The pathological mechanism of alveolar disorder is now considered closely correlated with diretc injury of Type Ⅱ alveolar epithelial cells (ATⅡ) mediated by oxidative stress, cytokines and other factors in preterms with bronchopulmonary dysplasia. On the basis of the biological phenomena which ATⅡtransdifferentiate to mesenchymal cells (EMT) proved in our previous researches, it is proposed that the injury of ATⅡ is not the only way affecting alveolarization while EMT may be another key aspect. Runx3, which participates in lung development and being taken as the access point, is elucidated as the key regulator of EMT by preparing cell models of Runx3 gene deletion and overexpression. The result of bisulfite sequencing PCR shows hypermethylation of DNA located in the CpG islands is the critical target leading to Runx3 silencing. Meanwhile by blocking DNA methylation in both invo and vitro, we reveal the molecular mechanisms of EMT mediated by Runx3 in BPD alveolar disorder. We screen certain target regulating small RNA (miRNA) By utilization of chip hybridization，search and verify the mechanism of DNA methylation and upregulation of Runx3 expression regulated by miRNA. Finally we conclude that miRNA / DNA methylation / Runx3/EMT is a new way to improve alveolar development.