MicroRNA-133a通过caveolin-1调节肺上皮屏障紧密连接开放机制的研究

Our team found that hyperoxia exposure in neonatal rats resulted in downregulation of pulmonary epithelial occludin and ZO-1, tight junction openning and pulmonary edema formation. Caveolin-1 is the major integral membrane protein and is cave-like retracted on cell surface. Studied related to its functional mechanisms in vivo and in vitro found that caveolin-1 regulated the process of occludin and ZO-1 integrated to tight junction (TJ) and adjusted to epithelial permeability. Our previous study found that the expression of caveolin-1 decreased and the expression of its upstream microRNA-133a abnormally increased in bronchopulmonaru dysplasia (BPD) model of neonatal rats induced by hyperoxia. Based on these studies, the aim of this project is to confirm that dynamic change of caveolin-1 expression in hyperoxia-induced pulmonary edema has temporal association with that of the tight junction protein expression, to verify that miR-133a regulates the openning of pulmonary epithelial barrier tight junction via caveolin-1, to clarify that the corrective expression of miR-133a promotes the expression of caveolin-1 and TJ-related proteins and improves pulmonary epithelial barrier function, to explore the molecular mechanisms of miR-133a promoting hyperoxia-induced lung edema, and to provide evidence that miR-133a becomes a new therapeutic targe for early pulmonary edema of BPD.

申请者所在团队发现高氧暴露导致新生鼠肺上皮occludin和ZO-1表达下调，紧密连接开放，诱发肺水肿。Caveolin-1是一种细胞表面穴样内陷的主要膜内在蛋白，对其体内、外功能及相关机制研究发现: caveolin-1调节occludin和ZO-1到紧密连接的整合过程，对上皮通透性有调控作用。课题组前期研究发现高氧致新生鼠BPD模型肺上皮中caveolin-1表达降低，其上游microRNA-133a表达异常增高。本项目拟在上述研究基础上，对高氧致肺水肿过程中caveolin-1与紧密连接相关蛋白表达变化是否有时空关联；miR-133a是否通过caveolin-1调节肺上皮屏障紧密连接开放；纠正miR-133a的表达是否能够改善肺上皮屏障的功能等问题进行系统研究，旨在阐明miR-133a促进高氧致肺水肿形成的分子机制, 为miR-133a成为新生儿BPD早期肺水肿治疗的新靶点奠定基础。

在高氧致BPD肺水肿阶段，伴有肺上皮屏障紧密连接TJ开放及TJ相关蛋白occludin和ZO-1基因和蛋白表达低下。caveolin-1作为一种细胞表面穴样内陷的主要膜内在蛋白，调节occludin和ZO-1到紧密连接的整合过程，对上皮通透性有调控作用。 BPD肺水肿期伴有caveolin-1下调，而其上游microRNA-133a表达异常增高。本课题作为既往工作的深入，以高氧致新生大鼠 BPD 模型和原代培养的新生大鼠肺泡II型上皮细胞为研究对象，通过western blot、realtime PCR、免疫荧光、投射电镜、荧光示踪剂单细胞层流量测定、跨上皮电阻记录模式等检测方法，分别从组织、细胞水平明确高氧对miR-133a及其预测靶基因Cav-1的影响，caveolin-1 的动态变化以及 caveolin-1 与 TJ 相关蛋白表达变化的时空关联；证实 miR-133a 通过 caveolin-1 调节肺上皮屏障 TJ 开放；还证实纠正 miR-133a 的表达能够恢复caveolin-1 和 TJ 相关蛋白的表达，改善肺上皮屏障的功能。本研究明确caveolin-1 及 miR-133a 在高氧致 BPD 早期肺水肿形成中的作用, 并且阐明miR-133a 能够成为临床早产儿 BPD 早期防治的新靶点，为 BPD 早期防治奠定实验基础和提供理论依据。