Table_2_Identification and exploration of novel M2 macrophage-related biomarkers in the development of acute myocardial infarction.DOCX

BackgroundAcute myocardial infarction (AMI), one of the most severe and fatal cardiovascular diseases, is a major cause of morbidity and mortality worldwide. Macrophages play a critical role in ventricular remodeling after AMI. The regulatory mechanisms of the AMI progression remain unclear. This study aimed to identify hub regulators of macrophage-related modules and provide translational experiments with potential therapeutic targets.
Materials and methodsThe GSE59867 dataset was downloaded from the Gene Expression Omnibus (GEO) database for bioinformatics analysis. The expression patterns of 22 types of immune cells were determined using CIBERSORT. GEO2R was used to identify differentially expressed genes (DEGs) through the limma package. Then, DEGs were clustered into different modules, and relationships between modules and macrophage types were analyzed using weighted gene correlation network analysis (WGCNA). Further functional enrichment analysis was performed using significantly associated modules. The module most significantly associated with M2 macrophages (Mϕ2) was chosen for subsequent analysis. Co-expressed DEGs of AMI were identified in the GSE123342 and GSE97320 datasets and module candidate hub genes. Additionally, hub gene identification was performed in GSE62646 dataset and clinical samples.
ResultsA total of 8,760 DEGs were identified and clustered into ten modules using WGCNA analysis. The blue and turquoise modules were significantly related to Mϕ2, and 482 hub genes were discerned from two hub modules that conformed to module membership values > 0.8 and gene significance values > 0.25. Subsequent analysis using a Venn diagram assessed 631 DEGs in GSE123342, 1457 DEGs in GSE97320, and module candidate hub genes for their relationship with Mϕ2 in the progression of AMI. Finally, four hub genes (CSF2RB, colony stimulating factor 2 receptor subunit beta; SIGLEC9, sialic acid-binding immunoglobulin-like lectin 9; LRRC25, leucine-rich repeat containing 25; and CSF3R, colony-stimulating factor-3 receptor) were validated to be differentially expressed and to have high diagnostic value in both GSE62646 and clinical samples.
ConclusionUsing comprehensive bioinformatics analysis, we identified four novel genes that may play crucial roles in the pathophysiological mechanism of AMI. This study provides novel insights into the impact of macrophages on the progression of AMI and directions for Mϕ2-targeted molecular therapies for AMI.

背景
急性心肌梗死（Acute Myocardial Infarction, AMI）是全球范围内发病率与死亡率最高的严重致死性心血管疾病之一。巨噬细胞在急性心肌梗死后的心室重构过程中发挥关键作用，而AMI进展的调控机制目前仍未明确。本研究旨在筛选巨噬细胞相关模块的核心调控因子，并为潜在治疗靶点的转化实验提供依据。

材料与方法
本研究从基因表达综合数据库（Gene Expression Omnibus, GEO）下载GSE59867数据集用于生物信息学分析。采用CIBERSORT算法分析22种免疫细胞的浸润表达模式；通过limma数据包结合GEO2R工具筛选差异表达基因（Differentially Expressed Genes, DEGs）。随后，将筛选得到的DEGs进行共表达模块聚类，并采用加权基因共表达网络分析（Weighted Gene Correlation Network Analysis, WGCNA）分析模块与巨噬细胞亚型之间的关联关系。对显著关联的共表达模块进行进一步的功能富集分析，选取与M2型巨噬细胞（M2 macrophages, Mϕ2）关联最为显著的模块开展后续研究。在GSE123342与GSE97320数据集中筛选AMI的共表达DEGs，并结合模块候选核心基因进行分析；此外，在GSE62646数据集及临床样本中开展核心基因的验证分析。

结果
经WGCNA分析，本研究共筛选得到8760个DEGs，并将其聚类为10个共表达模块。其中蓝色模块与绿松石模块与Mϕ2显著相关，从符合模块成员值>0.8且基因显著性值>0.25的两个核心模块中，共筛选得到482个核心基因。后续通过韦恩图分析，对GSE123342中的631个DEGs、GSE97320中的1457个DEGs以及模块候选核心基因与AMI进展中Mϕ2的关联关系进行评估。最终，在GSE62646数据集及临床样本中验证得到4个核心基因：集落刺激因子2受体亚基β（Colony Stimulating Factor 2 Receptor Subunit Beta, CSF2RB）、唾液酸结合免疫球蛋白样凝集素9（Sialic Acid-Binding Immunoglobulin-Like Lectin 9, SIGLEC9）、富含亮氨酸重复序列蛋白25（Leucine-Rich Repeat Containing 25, LRRC25）以及集落刺激因子3受体（Colony-Stimulating Factor-3 Receptor, CSF3R），上述基因均呈差异表达且具备较高的诊断价值。

结论
本研究通过综合生物信息学分析，筛选得到4个可能在AMI病理生理机制中发挥关键作用的全新基因。本研究为阐明巨噬细胞在AMI进展中的调控作用提供了新的视角，并为AMI的Mϕ2靶向分子治疗提供了潜在方向。