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

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.

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。

支气管肺发育不良（BPD）是早产儿常见的严重并发症之一，以肺泡和微血管发育障碍为特征。本项目课题组在前期发现BPD中存在Ⅱ型肺泡上皮细胞AT-II向间质细胞转分化（EMT）这一生物学现象的基础上，通过建立BPD动物模型、原代提取AT-II细胞，及应用IF、Western blot、Real-time PCR等实验技术分别从组织和细胞双水平验证BPD中EMT相关标志蛋白SP-C、α-SMA、E-cad、N-cad的表达变化，进一步证实在BPD中AT-II细胞发生EMT可能是导致肺发育障碍的关键机制。同时发现EMT的重要调控因子Runx3的表达下调，并通过建立Runx3基因沉默和过表达的RLE-6TN细胞模型，测定不同Runx3表达的细胞中EMT相关指标的表达，进一步验证Runx3对ATⅡ细胞发生EMT的调控作用。 随后，本项目课题组进一步证实 BPD中存在DNA甲基化和组蛋白H3K27me3修饰，以及组蛋白甲基转移酶（EZH2）表达改变，并通过染色质免疫共沉淀(CHIP)技术证实Runx3与H3K27me3、EZH2均有结合且呈显著高水平，提示RUNX3蛋白表达下调可能是由DNA甲基化和EZH2介导的H3K27me3的共同作用。并分别使用DNA甲基化抑制剂5-Aza-CdR， H3K27me3抑制剂Jmjd3, EZH2抑制剂DZNep干预AT-II细胞，发现抑制DNA甲基化和组蛋白H3K27me3修饰可以逆转AT-Ⅱ中RUNX3基因的表达下降，进一步证实DNA甲基化和EZH2介导的H3K27me3修饰是Runx3基因的重要调控机制。在此基础上，本课题组检测了EZH2的上游SOX4的表达及其相关性。研究发现，SOX的表达与EZH2水平正相关，但是否存在直接调控作用并不明确。为进一步研究EZH2的调控机制，本课题组通过查阅文献及数据库分析，聚焦长链非编码RNA-H19基因。不仅发现BPD中RNA-H19基因表达与EZH2相关，并通过RNA结合蛋白免疫沉淀技术（RIP）进一步证实H19和EZH2存在直接结合，提示H19可能通过与EZH2的相互结合来调控EZH2的表达，进而介导Runx3调控EMT的发生。 综上所诉，本项目率先提出肺上皮EMT是BPD发生发展的关键环节，从新的角度揭示了BPD的分子调控机制，为临床有关BPD的有效防治途径的重新设计奠定了实验基础。