Chen Ye, Li Min-Jing, Wang Wei-Si, Ye Bai-Chun, Qiu Chen-Hui
The Second Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou 310015, China.
The Second Clinical Medical School, Zhejiang Chinese Medical University, Hangzhou 310053, China.
Sheng Li Xue Bao. 2022 Oct 25;74(5):737-750.
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.
本研究旨在通过靶向磷酸肌醇-3-激酶调节亚基1（PIK3R1）来调节PTEN/PI3K/Akt信号通路，探讨微小RNA-155（miR-155）在慢性阻塞性肺疾病（COPD）及香烟烟雾提取物（CSE）处理的气道平滑肌细胞（ASMCs）中的作用。采用脂多糖（LPS）联合被动吸烟诱导建立COPD小鼠模型。建模后，使用miR-155模拟物和miR-155抑制剂进行干预治疗。用EMKA检测仪检测各组的肺功能。采用苏木精-伊红（HE）染色观察肺组织的病理变化及评分。用酶联免疫吸附测定（ELISA）法检测支气管肺泡灌洗液（BALF）中肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）和白细胞介素-1β（IL-1β）的表达。分离原代ASMCs并在贴壁组织培养中进行培养。用CCK-8和EdU检测法检测增殖活性。用Transwell和伤口愈合检测法检测ASMCs的迁移。通过双荧光素酶报告基因检测法验证miR-155与PIK3R1之间的靶向关系。用定量逆转录-聚合酶链反应（qRT-PCR）检测各组小鼠肺组织中miR-155和PIK3R1 mRNA的表达水平。用蛋白质免疫印迹法检测肺组织和ASMCs中Ki67、增殖细胞核抗原（PNCA）、PTEN、磷酸化PI3K（p-PI3K）、PI3K、p85α、磷酸化Akt（p-Akt）和Akt的表达水平。结果显示，miR-155模拟物组肺功能显著降低，PIK3R1水平显著升高；而miR-155抑制剂组肺功能显著改善。HE染色结果显示，与模型组相比，miR-155模拟物组有明显的炎性细胞浸润。miR-155抑制剂组肺组织病理损伤明显减轻，同时伴有Ki67、PNCA、PI3K、p-Akt表达降低，PTEN和p85α蛋白水平升高，BALF中TNF-α、IL-6和IL-1β水平降低。双荧光素酶报告基因检测结果显示，miRNA-155可靶向结合PIK3R1。与CSE+miR-155阴性对照（NC）组相比，CSE+miR-155组ASMCs的增殖和迁移明显增加。与CSE+miR-155组相比，CSE+miR-155+pcDNA PIK3R1组ASMCs的增殖和迁移明显减弱，同时伴有Ki67、PNCA、p-Akt表达降低，PTEN和p85α蛋白水平升高。这些结果表明，miR-155通过靶向PIK3R1激活PTEN/PI3K/Akt信号通路，促进COPD的发生发展，为miR-155用于COPD治疗提供了新的证据。
