Xue Yuzhou, Hu Yu, Yu Shikai, Zhu Wenyan, Liu Lin, Luo Minghao, Luo Suxin, Shen Jian, Huang Longxiang, Liu Jie, Lv Dingyi, Zhang Wenming, Wang Jingyu, Li Xiang
Department of Cardiology and Institute of Vascular Medicine, NHC Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides, State Key Laboratory of Vascular Homeostasis and Remodeling, Peking University Beijing Key Laboratory of Cardiovascular Receptors Research, Peking University Third Hospital, Beijing, China.
Department of Cardiology, the First Affiliated Hospital of Chongqing Medical University, Chongqing, China.
Heliyon. 2024 Jan 4;10(2):e24103. doi: 10.1016/j.heliyon.2024.e24103. eCollection 2024 Jan 30.
Inflammatory macrophages play a crucial role in atherosclerosis development. The long non-coding RNA growth arrest-specific 5 (GAS5) regulates THP-1 macrophage inflammation by sponging microRNAs. The purpose of this study was to investigate the regulatory mechanism of GAS5 in atherosclerosis development. GSE40231, GSE21545, and GSE28829 datasets from the Gene Expression Omnibus database were integrated after adjusting for batch effect. Differential analysis was performed on the integrated dataset and validated using the Genotype-Tissue Expression and GSE57691 datasets. Potential biological functions of GAS5 and annexin A2 (ANXA2) were identified using gene set enrichment analysis (GSEA). ssGSEA, CIBERSORTx, and ImmuCellAI algorithms were used to identify immune infiltration in plaque samples. GAS5 and ANXA2 expression levels in RAW264.7 cells treated with oxidized low-density lipoprotein (ox-LDL) were measured by qRT-PCR and Western blot. Small interfering and short hairpin RNA were used to silence GAS5 expression. Plasmids of ANXA2 were used to establish ANXA2 overexpression. Apoptosis and inflammatory markers in macrophages were detected by Western blot. Aortic samples from APOE mice were collected to validate the expression of GAS5 and ANXA2. GAS5 expression was significantly increased during atherosclerosis. GAS5 expression was positively correlated with macrophage activation and ANXA2 expression in plaques. Furthermore, ANXA2 upregulation was also related to the activation of macrophage. GSEA indicated similar biological functions for GAS5 and ANXA2 in plaques. Moreover, experiments showed that both GAS5 and ANXA2 contributed to macrophage apoptosis and inflammation. Rescue assays revealed that the inflammatory effects of GAS5 on macrophages were ANXA2-dependent. In vivo experiments confirmed the highly expression of Gas5 and Anxa2 in the plaque group. We identified the atherogenic roles of GAS5 and ANXA2 in the inflammatory response of macrophages. The inflammatory response in ox-LDL-treated macrophages was found to be mediated by GAS5-ANXA2 regulation, opening new avenues for atherosclerosis therapy.
炎症性巨噬细胞在动脉粥样硬化发展中起关键作用。长链非编码RNA生长停滞特异性5（GAS5）通过海绵化微小RNA来调节THP-1巨噬细胞炎症。本研究旨在探讨GAS5在动脉粥样硬化发展中的调控机制。对来自基因表达综合数据库的GSE40231、GSE21545和GSE28829数据集进行批次效应校正后进行整合。对整合后的数据集进行差异分析，并使用基因型-组织表达和GSE57691数据集进行验证。使用基因集富集分析（GSEA）确定GAS5和膜联蛋白A2（ANXA2）的潜在生物学功能。使用单样本基因集富集分析（ssGSEA）、CIBERSORTx和免疫细胞AI算法来识别斑块样本中的免疫浸润。通过qRT-PCR和蛋白质免疫印迹法测量氧化低密度脂蛋白（ox-LDL）处理的RAW264.7细胞中GAS5和ANXA2的表达水平。使用小干扰RNA和短发夹RNA沉默GAS5表达。使用ANXA2质粒建立ANXA2过表达。通过蛋白质免疫印迹法检测巨噬细胞中的凋亡和炎症标志物。收集载脂蛋白E（APOE）小鼠的主动脉样本以验证GAS5和ANXA2的表达。在动脉粥样硬化过程中，GAS5表达显著增加。GAS5表达与斑块中的巨噬细胞活化和ANXA2表达呈正相关。此外，ANXA2上调也与巨噬细胞活化有关。GSEA表明斑块中GAS5和ANXA2具有相似的生物学功能。此外，实验表明GAS5和ANXA2均促成巨噬细胞凋亡和炎症。拯救实验表明GAS5对巨噬细胞的炎症作用依赖于ANXA2。体内实验证实了斑块组中Gas5和Anxa2的高表达。我们确定了GAS5和ANXA2在巨噬细胞炎症反应中的致动脉粥样硬化作用。发现ox-LDL处理的巨噬细胞中的炎症反应由GAS5-ANXA2调节介导，为动脉粥样硬化治疗开辟了新途径。
