Lin Jinquan, Wei Qiongying, Fang Zhipeng
Department of Trauma Center and Emergency Surgery, National Regional Medical Center, Binhai Campus of the First Affiliated Hospital, Fujian Medical University, Fuzhou City, Fujian Province, PR China; Department of Trauma Center and Emergency Surgery, The First Affiliated Hospital, Fujian Medical University, Fuzhou City, Fujian Province, PR China.
Department of Pulmonary and Critical Care Medicine, Fujian Medical University Union Hospital, Fuzhou City, Fujian Province, PR China; Department of Geriatrics, Fujian Medical University Union Hospital, Fuzhou City, Fujian Province, PR China.
Clinics (Sao Paulo). 2024 Dec 11;80:100550. doi: 10.1016/j.clinsp.2024.100550. eCollection 2025.
The study was to investigate circRBM33 in septic acute lung injury (ALI).
Treatment of Murine Lung Epithelial-12 cells (MLE-12) cells was performed using 10 ng/mL Lipopolysaccharide (LPS). circRBM33, miR-15a-5p, and Enhancer of zeste homolog 1 (EZH1) were ascertained through RT-qPCR or Western blot analysis. The viability of MLE-12 cells was measured using the MTT assay, and their rate of apoptosis was ascertained through flow cytometry. B-cell lymphoma-2 (Bcl-2), and Bcl-2-associated X (Bax) were determined using Western blot analysis. Oxidative stress levels were assessed with ELISA kits, and levels of malondialdehyde(MDA) content, Superoxide Dismutase (SOD) activity, and glutathione (GSH) were detected. Dual luciferase reporter gene and RIP assays verified the targeting link between miR-15a-5p and circRBM33 or EZH1. The role of circRBM33 in ALI in vivo was determined by performing cecum ligation-perforation (CLP) surgery. HE staining, W/D pulmonary edema, and histological damage scores were taken to assess the extent of lung tissue damage. ELISA was performed to determine proinflammatory factors in lung tissue and cells.
CircRBM33 downregulation ameliorated ALI-induced edema, apoptotic, and inflammatory reactions in mouse lung tissues. In addition, apoptosis and inflammation mediated by LPS in MLE-12 cells were ameliorated by circRBM33 downregulation, whereas miR-15a-5p knockdown or EZH1 elevation eliminated the action of silencing circRBM33. circRBM33 mediated EZH1 expression by competitive adsorption of miR-15a-5p.
CircRBM33 improves ALI in septic mice by targeting the miR-15a-5p/EZH1 axis.
本研究旨在探究环状RNA结合蛋白33（circRBM33）在脓毒症急性肺损伤（ALI）中的作用。
采用10 ng/mL脂多糖（LPS）处理小鼠肺上皮细胞-12（MLE-12）。通过逆转录定量聚合酶链反应（RT-qPCR）或蛋白质免疫印迹法检测circRBM33、微小RNA-15a-5p（miR-15a-5p）和zeste同源物增强子1（EZH1）的表达。采用MTT法检测MLE-12细胞的活力，通过流式细胞术确定细胞凋亡率。采用蛋白质免疫印迹法检测B细胞淋巴瘤-2（Bcl-2）和Bcl-2相关X蛋白（Bax）的表达。使用酶联免疫吸附测定（ELISA）试剂盒评估氧化应激水平，检测丙二醛（MDA）含量、超氧化物歧化酶（SOD）活性和谷胱甘肽（GSH）水平。双荧光素酶报告基因和RNA免疫沉淀（RIP）实验验证miR-15a-5p与circRBM33或EZH1之间的靶向关系。通过盲肠结扎穿孔（CLP）手术确定circRBM33在体内ALI中的作用。采用苏木精-伊红（HE）染色、肺组织湿干重比（W/D）及组织学损伤评分评估肺组织损伤程度。通过ELISA检测肺组织和细胞中的促炎因子。
circRBM33表达下调可改善ALI诱导的小鼠肺组织水肿、凋亡和炎症反应。此外，circRBM33表达下调可改善LPS介导的MLE-12细胞凋亡和炎症，而敲低miR-15a-5p或上调EZH1可消除circRBM33沉默的作用。circRBM33通过竞争性吸附miR-15a-5p介导EZH1表达。
circRBM33通过靶向miR-15a-5p/EZH1轴改善脓毒症小鼠的ALI。
