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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

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

摘要

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。

关键词： 慢性阻塞性肺疾病; miR-155; PIK3R1; 气道平滑肌细胞; 增殖; 迁移

Effect and mechanism of microRNA-155 in chronic obstructive pulmonary disease by targeting PIK3R1 

CHEN Ye^1,*, 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

Abstract

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. 

Key words: chronic obstructive pulmonary disease; miR-155; PIK3R1; airway smooth muscle cells; proliferation; migration

收稿日期：　　录用日期：

通讯作者：陈晔　　E-mail: 20094012@zcmu.edu.cn

DOI: 10.13294/j.aps.2022.0072

引用本文：

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).