miR-130a靶向NRBF2调控细胞自噬在纳米氧化锌诱发中性粒细胞炎症中的作用

Zinc oxide nanoparticles (ZnO-NPs) are one of the most abundantly produced metal oxide nanoparticles and have been widely used in industries. Exposure to engineered ZnO-NPs induced airway inflammation, represented as increased neutrophil and pulmonary inflammatory mediators. miRNA played a pivotal role in inflammation and autophagy. We have recently screened aberrantly expressed miRNA in exosomes from rats serum with ZnO-NPs -induced pulmonary inflammation based on miRNA microarray. miR-130a was dramatically down-regulated and NRBF2 was predicted as a target gene which was the key autophagic regulators and the protein expression was negatively related to miR-130a level. However, effect of miR-130a and NRBF2 on the airway inflammation induced by ZnO-NPs has been unclear. This project is proposed to explore the mechanism whether miR-130a mediates cell autophagy via inhibiting NRBF2 and leads to promote inflammation induced by ZnO-NPs. Up or down regulation of miR-130a expression by transfection with miR-130a mimics or inhibitor is applied to observe the effect on NRBF2, cell autophagy and inflammatory reaction. The regulatory effect of autophagy on inflammatory response is detected after treated with autophagy promoter and inhibitor. Secondly, wild type (WT) mice, and GFP-LC3, miR-130a-/-，and NRBF2-/- mice with acute respiratory inflammation induced by ZnO-NPs will be utilized to investigate and compare the difference in neutrophil differentiation, autophagy and inflammation response. In general the effect of miR-130a mediated autophagy via targeting NRBF2 on respiratory inflammation induced by ZnO-NPs will be explored combining with in vitro and in vivo experiments. This study is expected to provide new ideas and target on effective prevention and treatment of ZnO-NP-induced health hazards.

吸入纳米氧化锌（ZnO-NPs）可诱发以中性粒细胞浸润为特征的呼吸道炎症。miRNA对炎症反应和自噬起着重要的调控作用，课题组前期发现miR-130a是与ZnO-NPs诱发呼吸道炎症相关的miRNA，其靶基因NRBF2是调控自噬的重要基因，二者表达呈负相关，但在ZnO-NPs致呼吸道炎症中的作用尚不清楚。本研究拟用ZnO-NPs刺激中性粒细胞，上调/下调miR-130a表达，检测其对NRBF2、细胞分化成熟、自噬与炎性反应的调控，利用自噬促进剂和抑制剂检测细胞自噬改变对炎性反应的影响；利用自噬报告小鼠、miR-130a敲除和NRBF2敲除小鼠，检测miR-130a及NRBF2表达变化对中性粒细胞分化、细胞自噬和呼吸道炎症的影响。从不同层次探讨miR-130a靶向NRBF2调控细胞自噬在ZnO-NPs致呼吸道炎症中的作用，为ZnO-NPs毒性研究和预防措施提供新思路和靶标。

吸入纳米氧化锌（ZnO-NPs）可诱发以中性粒细胞浸润为特征的呼吸道炎症，课题组前期发现miR-130a是与ZnO-NPs诱发呼吸道炎症相关的miRNA，其靶基因NRBF2是调控自噬的重要基因，但miR-130a和NRBF2在ZnO-NPs致呼吸道炎症中的作用尚不清楚。本研究采用ZnO-NPs刺激中性粒细胞，可抑制miR-130a表达，促进NRBF2表达，同时出现细胞自噬及炎性反应。通过上调/下调miR-130a表达，观察其对靶基因NRBF2、中性粒细胞分化成熟、细胞自噬与炎性反应的调控，发现上调miR-130a表达，可以显著降低NRBF2的表达，降低ZnO-NPs诱发的ROS、自噬及炎症因子的高表达。利用细胞自噬促进剂和抑制剂检测细胞自噬改变对炎性反应的影响，发现自噬激动剂RAPA预处理可使纳米氧化锌诱发的ROS、LDH、IL-1β、IL-6、TNF-α升高，同时可影响中性粒细胞的分化成熟从而影响细胞功能。利用miR-130a敲除和NRBF2敲除小鼠，检测miR-130a及NRBF2表达变化对中性粒细胞分化、细胞自噬和呼吸道炎症的影响，发现自噬增强可加重ZnO-NPs诱发的肺部炎症，敲除miR-130a可显著增强炎症，敲除NRBF2可抑制炎症。敲除miR-130a可增加CEBP α、γ、εmRNA高表达，增加MPO和MMP-9表达，从而影响中性粒细胞的分化成熟。从体内细胞实验和体外动物实验不同层次阐明miR-130a通过抑制NRBF2的表达，影响其调控的细胞自噬，从而促进炎性反应调控，并在此过程中影响中性粒细胞分化成熟。miR-130a的表达水平可预测机体接触ZnO-NPs诱发呼吸道炎症的水平并发挥重要的调控作用，可作为ZnO-NPs毒性作用的标志和靶点，为预防和控制ZnO-NPs毒性损伤提供重要依据。