polyA+ RNA sequencing on FACS sorted alveolar macrophages (CD45+SiglecF+CD11c+) from air and cigarette-smoke exposed wild type and miR-155 KO mice

Chronic obstructive pulmonary disease (COPD) is a highly prevalent respiratory disease characterized by airflow limitation and chronic inflammation. MiR-155 is described as an ancient regulator of the immune system. Our objective was to establish a role for miR-155 in cigarette smoke (CS)-induced inflammation and COPD. We demonstrate increased miR-155 expression by RT-qPCR in lung tissue of smokers without airflow limitation and patients with COPD compared to never smokers and in lung tissue and alveolar macrophages of CS-exposed mice compared to air-exposed mice. In addition, we exposed wild type and miR-155 deficient mice to CS and show an attenuated inflammatory profile in the latter. Alveolar macrophages were sorted by FACS from the different experimental groups and their gene expression profile was analyzed by RNA sequencing. This analysis revealed increased expression of miR-155 targets (including the NF-?B inhibitor rassf6) and an attenuation of the CS-induced increase in inflammation-related genes in miR-155 deficient mice. Finally, intranasal instillation of a specific miR-155 inhibitor significantly attenuated the CS-induced pulmonary inflammation in mice. In conclusion, we highlight a role for miR-155 in CS-induced inflammation and the pathogenesis of COPD, implicating miR-155 as a new therapeutic target in COPD. Overall design: poly-A+ RNA sequencing was performed on 5 mice per group (4 groups: air_wild-type, air_miR-155-KO, CS_wild-type, CS_miR-155-KO)

慢性阻塞性肺疾病（Chronic obstructive pulmonary disease, COPD）是一种高患病率的呼吸系统疾病，以气流受限与慢性炎症为核心特征。miR-155被认为是免疫系统的古老调控因子。本研究旨在明确miR-155在香烟烟雾（cigarette smoke, CS）诱导的炎症及慢性阻塞性肺疾病发生发展中的作用。
我们通过逆转录定量PCR（RT-qPCR）检测发现，与从未吸烟者相比，无气流受限的吸烟者与慢性阻塞性肺疾病患者的肺组织中miR-155表达水平显著升高；与空气暴露小鼠相比，香烟烟雾暴露小鼠的肺组织与肺泡巨噬细胞中miR-155表达水平同样升高。
此外，我们将野生型小鼠与miR-155基因敲除小鼠暴露于香烟烟雾中，观察到后者的炎症反应谱显著减弱。我们通过荧光激活细胞分选术（Fluorescence-Activated Cell Sorting, FACS）从各实验组中分选肺泡巨噬细胞，并通过RNA测序（RNA-seq）分析其基因表达谱。分析结果显示，miR-155的靶基因（包括核因子κB抑制剂rassf6）表达上调，且miR-155基因敲除小鼠中香烟烟雾诱导的炎症相关基因表达升高现象得到显著缓解。
最后，经鼻滴注特异性miR-155抑制剂可显著减轻小鼠体内香烟烟雾诱导的肺部炎症。综上，本研究证实了miR-155在香烟烟雾诱导的炎症及慢性阻塞性肺疾病发病机制中的关键作用，提示miR-155可作为慢性阻塞性肺疾病的新型治疗靶点。
总体实验设计：对每组5只小鼠（共4组：空气暴露野生型组、空气暴露miR-155敲除组、香烟烟雾暴露野生型组、香烟烟雾暴露miR-155敲除组）进行poly-A+ RNA测序。