Department of Neurosurgery, Second Affiliated Hospital of Zhejiang University School of Medicine, Zhejiang University, 88 Jiefang Road, Hangzhou, 310009 Zhejiang, China.

Department of Orthopedic Surgery, Second Affiliated Hospital of Zhejiang University School of Medicine, Zhejiang University, 88 Jiefang Road, Hangzhou, 310009 Zhejiang, China.

Department of Sports Medicine, Huashan Hospital, Fudan University, Shanghai, China.

背景 。本研究旨在利用多重加权基因共表达网络分析（WGCNA）和单细胞分析，在单细胞水平上研究缺血性卒中（IS）后相关通路的变化和相关基因表达的差异表达。 方法 . IS样本的转录组表达数据集和脑血管组织的单细胞RNA测序（scRNA-seq）谱通过搜索基因表达综合（GEO）数据库获得。首先，通过基因集变异分析（GSVA）计算基因通路评分，并将其导入多个WGCNA以获得关键通路和通路相关的枢纽基因。此外，SCENIC 还用于使用 scRNA-seq 数据识别调节这些核心基因的转录因子 (TF)。最后，使用伪时间轨迹分析来分析这些 TF 在缺氧和常氧条件下对各种细胞类型的作用。 结果 . 使用 40 个样本的微阵列表达谱通过 GSVA 获得 186 条 KEGG 通路的分数。KEGG通路的WGCNA揭示了以下两条通路：钙信号通路和神经活性配体-受体相互作用通路。随后，样本基因表达矩阵的WGCNA揭示了钙信号通路相关基因（ AC079305.10 、 BCL10 、 BCL2A1 、 BRE-AS1 、 DYNLL2 、 EREG 和 PTGS2 ），通过相关分析确定为核心基因。此外，SCENIC 和伪时间分析显示 JUN 、 IRF9 、 ETV5 和 PPRA 评分基因相关 TF。 发现Jun 与内皮细胞中的缺氧有关，而 Irf9 和 Etv5 被鉴定为与小鼠大脑皮层中氧浓度相关的星形胶质细胞特异性 TF。 结论 。钙信号通路相关基因（ AC079305 .10、 BCL10 、 BCL2A1 、 BRE-AS1 、 DYNLL2 、 EREG 和 PTGS2 ）和 TF（ JUN 、 IRF9 、 ETV5 和 PPARA ） ) 被确定在 IS 中发挥关键作用。该研究为研究IS的发病机制和开发新的治疗方法提供了新的视角和基础。 Background . This study is aimed at investigating the changes in relevant pathways and the differential expression of related gene expression after ischemic stroke (IS) at the single-cell level using multiple weighted gene coexpression network analysis (WGCNA) and single-cell analysis. Methods . The transcriptome expression datasets of IS samples and single-cell RNA sequencing (scRNA-seq) profiles of cerebrovascular tissues were obtained by searching the Gene Expression Omnibus (GEO) database. First, gene pathway scoring was calculated via gene set variation analysis (GSVA) and was imported into multiple WGCNA to acquire key pathways and pathway-related hub genes. Furthermore, SCENIC was used to identify transcription factors (TFs) regulating these core genes using scRNA-seq data. Finally, the pseudotemporal trajectory analysis was used to analyse the role of these TFs on various cell types under hypoxic and normoxic conditions. Results . The scores of 186 KEGG pathways were obtained via GSVA using microarray expression profiles of 40 specimens. WGCNA of the KEGG pathways revealed the two following pathways: calcium signaling pathway and neuroactive ligand-receptor interaction pathways. Subsequently, WGCNA of the gene expression matrix of the samples revealed the calcium signaling pathway-related genes ( AC079305.10 , BCL10 , BCL2A1 , BRE-AS1 , DYNLL2 , EREG , and PTGS2 ) that were identified as core genes via correlation analysis. Furthermore, SCENIC and pseudotemporal analysis revealed JUN , IRF9 , ETV5 , and PPARA score gene-related TFs. Jun was found to be associated with hypoxia in endothelial cells, whereas Irf9 and Etv5 were identified as astrocyte-specific TFs associated with oxygen concentration in the mouse cerebral cortex. Conclusions . Calcium signaling pathway-related genes ( AC079305 .10, BCL10 , BCL2A1 , BRE-AS1 , DYNLL2 , EREG , and PTGS2 ) and TFs ( JUN , IRF9 , ETV5 , and PPARA ) were identified to play a key role in IS. This study provides a new perspective and basis for investigating the pathogenesis of IS and developing new therapeutic approaches.