miR-27b调控Nrf2/NOX4信号通路在肠缺血再灌注急性肺损伤病理机理中作用

Oxidative stress induced injury and apoptosis of pulmonary vascular endothelial cells(PVECs) plays a central role in intestine ischemia reperfusion(I/R) induced acute lung injury(ALI). MicroRNA-27b plays a protective role against oxidative insults. As a family member of Cap'n'collar/basic region leucine zipper (CNC-bZIP) transcription factors that is activated by diverse oxidants, pro-oxidants, antioxidants and chemo preventive agents,Nrf2 regulates the expression of NOX4 and downstream factors in PVECs and defects oxidative stress induced injury. However，the particular mechanism of miR-27b in regulating Nrf2/NOX4 pathway is still unclear. Our preliminary work found that the level of miR-27b increased,while the expression of Nrf2/NOX4 decreased, which correlated negatively, according to miRNA chip and mRNA expression profiling screen in mice suffering from intestine ischemia reperfusion induced acute lung injury; MicroRNA target gene prediction softwares, such as miRanda,TargetScan and picTar, prognose that Nrf2 mRNA is the target gene of miR-27b. Furthermore, luciferase gene assay validates that there is binding sites in 3'UTR of Nrf2 gene. Therefore, we hypothesize that miR-27b influences the development of ALI by regulating Nrf2/NOX4 pathway. The project intends to use Nrf2 gene knockout mice, kindly approved by Professor Masayuki Yamamoto from the University of Tsukuba and transferred by RIKEN BioResource Center,to make intestine ischemia reperfusion induced ALI model and oxygen-glucose deprivation model of PVEC,and explore the role of miR-27b in the development of ALI by molecular, tissue and animal level. The research will look for molecular diagnostic markers and provide therapeutic targets for intestine I/R induced ALI.

氧化应激引起肺血管内皮细胞(PVEC)损伤和凋亡是肠I/R后ALI中心环节。研究显示miR-27b与氧化应激密切相关，Nrf2调控NOX4及下游因子在PVEC中表达防御氧化应激损伤。目前miR-27b调控Nrf2/NOX4分子机制不清。我们前期miRNA芯片和mRNA表达谱筛查发现肠I/R后ALI中miR-27b高表达，Nrf2/NOX4低表达，两者负相关；软件预测Nrf2是miR-27b靶基因，荧光素酶报告基因检测验证miR-27b在Nrf2基因3'UTR有结合位点。据此假设miR-27b调控Nrf2/NOX4通路是影响ALI中心环节和关键因素。本项目拟用RIKEN BRC转让Nrf2基因敲除小鼠制备肠I/R后ALI模型及PVEC氧糖剥夺/复氧模型，从分子-组织-动物整体水平研究miR-27b在调控Nrf2及ALI发病机制中作用，为寻找肠I/R后ALI分子诊断标记和治疗靶标提供科学依据。

核因（Nrf2）是调控缺血血管成形中主要的氧化应激调控因子。我们发现沉默信息调节因子2同源物 1(SIRT1 )通过调控Nrf2在缺血再灌注急性肺损伤（IIR）模型小鼠中调节血管重塑。在Nrf2基因敲除小鼠中，IIR模型肺组织中白细胞浸润现象显著增加，内皮细胞生长因子CD31表达水平显著下降；过表达SIRT1 激活Nrf2并使其向细胞核转录，抑制肺组织病理学改变，促进内皮细胞标记物CD31的表达，且进一步增加其下游NADPH氧化酶4（NOX4）、低氧诱导因子1α（HIF-1α）及血管内皮生长因子（VEGF）的表达。人肺毛细血管内皮细胞氧糖剥夺复氧模型中，下调Nrf2可抑制细胞多样性、成管及细胞迁移。下调NOX4可减少血管成形及VEGF与HIF1alpha的水平。SIRT1-Nrf2在通过NOX4介导的信号通路维持成熟的人毛细血管内皮细胞形成血管潜能中发挥重要作用。