陈晔1,*, 李敏静1, 王维斯1, 叶柏春1, 裘晨晖2
1浙江中医药大学附属第二医院，杭州 310015；2浙江中医药大学第二临床医学院，杭州 310053
本研究旨在探讨microRNA-155 (miR-155)靶向磷脂酰肌醇-3激酶调节亚基1 (phosphoinositide-3-kinase regulatory subunit 1, PIK3R1)基因调控PTEN/PI3K/Akt信号通路对慢性阻塞性肺疾病(chronic obstructive pulmonary disease, COPD)小鼠及香烟提取物(cigarette smoke extract, CSE)刺激的气道平滑肌细胞(airway smooth muscle cells, ASMCs)的影响。采用脂多糖(lipopolysaccharide, LPS)联合被动吸烟构建小鼠COPD模型，建模后采用miR-155 mimics和miR-155 inhibitor进行干预治疗。运用EMKA检测仪检测小鼠肺功能；苏木精-伊红(HE)染色观察肺组织病理变化；酶联免疫吸附法检测支气管肺泡灌洗液(bronchial alveolar lavage fluid, BALF)中TNF-α、IL-6、IL-1β 水平。组织贴壁法分离和培养原代ASMCs，CSE刺激后，转染miR-155 mimics或pcDNA PIK3R1，CCK-8及EdU染色法检测ASMCs增殖活性；Transwell及细胞划痕实验检测ASMCs迁移水平；双荧光素酶报告基因实验验证miR-155与PIK3R1的靶向关系；实时荧光定量PCR (qRT-PCR)检测各组小鼠肺组织miRNA-155、PIK3R1 mRNA表达水平；免疫印迹法检测肺组织及ASMCs中Ki67、PNCA、PTEN、p-PI3K、PI3K、p85α、p-Akt、Akt蛋白表达水平。结果显示，miR-155 mimics组小鼠肺功能显著降低，且肺组织PIK3R1表达水平显著增加，而miR-155 inhibitor组上述指标则显著改善；HE染色结果显示miR-155 mimics组较模型组炎性细胞浸润更为明显；miR-155 inhibitor组肺组织病理损伤显著降低，Ki67、PNCA、PI3K及p-Akt蛋白表达显著降低，而PTEN及p85α的蛋白表达显著升高；BALF中TNF-α、IL-6、IL-1β含量显著降低。双荧光素酶报告基因实验分析结果显示，miR-155能够靶向结合PIK3R1；与CSE+miR-155 NC组相比，CSE+miR-155组CSE介导的ASMCs增殖及迁移显著增加，而CSE+miR-155+pcDNA PIK3R1组较miR-155组ASMCs增殖及迁移能力显著减弱，Ki67、PNCA及p-Akt蛋白表达显著降低，而PTEN及p85α的蛋白表达显著升高。以上结果提示，miR-155通过靶向PIK3R1激活PTEN/PI3K/Akt信号通路促进COPD发生和发展，这些发现为miR-155用于治疗COPD提供了新的科学依据和方向。
Effect and mechanism of microRNA-155 in chronic obstructive pulmonary disease by targeting PIK3R1
CHEN Ye1,*, LI Min-Jing1, WANG Wei-Si1, YE Bai-Chun1, QIU Chen-Hui2
1he Second Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou 310015, China；2The Second Clinical Medical School, Zhejiang Chinese Medical University, Hangzhou 310053, China
This study aimed to investigate the effect of microRNA-155 (miR-155) in chronic obstructive pulmonary disease (COPD) and cigarette smoke extract (CSE)-treated airway smooth muscle cells (ASMCs) by targeting phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) to regulate the PTEN/PI3K/Akt signaling pathway. The COPD mouse model was induced by lipopolysaccharide (LPS) combined with passive smoking. After modeling, miR-155 mimics and miR-155 inhibitor were used for intervention treatment. The pulmonary function of each group was detected by an EMKA detector. Hematoxylin-eosin (HE) staining was used to observe the pathological changes and scores of lung tissues. The expression of TNF-α, IL-6, and IL-1β in bronchial alveolar lavage fluid (BALF) was detected by ELISA. Primary ASMCs were isolated and cultured in adherent tissue culture. The proliferation activity was detected by CCK-8 and EdU assays. Transwell and wound healing assays were used to measure the migration of ASMCs. The targeting relationship between miR-155 and PIK3R1 was validated by a double luciferase reporter gene assay. The expression levels of miR-155 and PIK3R1 mRNA in lung tissues of mice in each group were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Western blot was used to detect the expression levels of Ki67, PNCA, PTEN, p-PI3K, PI3K, p85α, p-Akt, and Akt in lung tissues and ASMCs. The results showed that lung function was significantly reduced in the miR-155 mimic group, and the levels of PIK3R1 were significantly increased; while lung function in the miR-155 inhibitor group was significantly improved. The results of HE staining showed that there was obvious inflammatory cell infiltration in the miR-155 mimics group compared to that of the model group. Lung histopathological injury was significantly reduced in the miR-155 inhibitor group, accompanied by decreased expression of Ki67, PNCA, PI3K, p-Akt, increased PTEN and p85α protein levels, and reduced levels of TNF-α, IL-6, and IL-1β in BALF. The results of the double luciferase reporter gene assay showed that miRNA-155 could target bind to PIK3R1. Compared with those in the CSE+miR-155 NC group, the proliferation and migration of ASMCs were significantly increased in the CSE+miR-155 group. The proliferation and migration of ASMCs were significantly attenuated in the CSE+miR-155+pcDNA PIK3R1 group compared with those in the CSE+miR-155 group, accompanied by decreased expression of Ki67, PNCA, p-Akt and increased PTEN and p85α protein levels. These results suggest that miR-155 activates the PTEN/PI3K/Akt signaling pathway by targeting PIK3R1 to promote the occurrence and development of COPD, which provides new evidence for the use of miR-155 in the treatment of COPD.
通讯作者：陈晔 E-mail: email@example.com
陈晔, 李敏静, 王维斯, 叶柏春, 裘晨晖. MicroRNA-155通过靶向调控PIK3R1在慢性阻塞性肺疾病中的作用及机制[J]. 生理学报 2022; 74 (5): 737-750.
CHEN Ye, LI Min-Jing, WANG Wei-Si, YE Bai-Chun, QIU Chen-Hui. Effect and mechanism of microRNA-155 in chronic obstructive pulmonary disease by targeting PIK3R1 . Acta Physiol Sin 2022; 74 (5): 737-750 (in Chinese with English abstract).