Jin Tingyu, Gao Han, Meng Danyang, Luo Man, Hu Jin
Department of Neurology, Affiliated Hospital of Jiaxing University, The First Hospital of Jiaxing, Jiaxing, Zhejiang, China.
Department of Neurology, Affiliated Hospital of Jiaxing University, The First Hospital of Jiaxing, 1882 Zhonghuan South Road, Chengnan Street, Jiaxing, Zhejiang, China.
Open Med (Wars). 2024 Oct 24;19(1):20241072. doi: 10.1515/med-2024-1072. eCollection 2024.
Ischemic stroke associated with atherosclerosis is globally named atherothrombotic stroke. Presently, the underlying pathogenic genes promoting carotid atherosclerotic plaques transfer from a stable to unstable state remains elusive. This study aims to find the hub genes disturbing the stability of plaques and explore the primary cells affected by these hub genes.
The optimal hub genes from five datasets for unstable plaques were identified by overlapping genes derived from Boruta and LASSO algorithms. The hub genes' expression levels in stroke patients were confirmed through RT-qPCR. Visualization of the hub genes' expression across various cell clusters was achieved with the aid of the Seurat R package. Then, hub genes were overexpressed or knock-down by lentivirus and siRNA, respectively. The inflammatory factors in the culture medium were detected using an ELISA assay.
Eight genes (APOD, ASXL1, COL4A5, HTR7, INF2, NSUN6, PDSS2, and RBBP7) were identified and confirmed by RT-qPCR. The prognostic model was built upon this eight-gene composite foundation, and the area under the curve was 0.98. Based on CIBERSORT findings, unstable plaques displayed a higher macrophage proportion compared to stable ones ( < 0.05). These eight genes also correlated with infiltrated immune cells, especially macrophages. Then, according to single-cell RNA-seq analysis, we found that the eight hub genes mainly expressed in macrophages. The cellular localization of two hub genes (NSUN6 and HTR7) with high distinguishability was confirmed, and gene set enrichment analysis also clarified the possible biological pathways regulated by them. The findings from the investigation revealed that TNF-α and IL-6 were reduced in macrophages with NSUN6 overexpression or HTR7 knockdown.
Eight hub genes, especially NSUN6 and HTR7, were found to promote the progression of plaques by regulating the immune responses of macrophages.
与动脉粥样硬化相关的缺血性中风在全球范围内被称为动脉粥样硬化血栓形成性中风。目前，促进颈动脉粥样硬化斑块从稳定状态转变为不稳定状态的潜在致病基因仍不清楚。本研究旨在寻找干扰斑块稳定性的关键基因，并探索受这些关键基因影响的主要细胞。
通过重叠来自Boruta和LASSO算法的基因，从五个不稳定斑块数据集识别出最佳关键基因。通过RT-qPCR确认关键基因在中风患者中的表达水平。借助Seurat R包实现关键基因在各种细胞簇中的表达可视化。然后，分别通过慢病毒和siRNA对关键基因进行过表达或敲低。使用ELISA测定法检测培养基中的炎症因子。
通过RT-qPCR鉴定并确认了八个基因（载脂蛋白D、ASXL1、IV型胶原α5链、5-羟色胺受体7、肌动蛋白解聚因子INF2、NOP2/Sun RNA甲基转移酶6、泛醌生物合成蛋白2和视网膜母细胞瘤结合蛋白7）。基于这八个基因的组合建立了预后模型，曲线下面积为0.98。根据CIBERSORT结果，与稳定斑块相比，不稳定斑块的巨噬细胞比例更高（<0.05）。这八个基因也与浸润的免疫细胞相关，尤其是巨噬细胞。然后，根据单细胞RNA测序分析，我们发现这八个关键基因主要在巨噬细胞中表达。确认了两个具有高区分度的关键基因（NOP2/Sun RNA甲基转移酶6和5-羟色胺受体7）的细胞定位，基因集富集分析也阐明了它们可能调控的生物学途径。研究结果表明，过表达NOP2/Sun RNA甲基转移酶6或敲低5-羟色胺受体7可使巨噬细胞中的肿瘤坏死因子-α和白细胞介素-6减少。
发现八个关键基因尤其是NOP2/Sun RNA甲基转移酶6和5-羟色胺受体7通过调节巨噬细胞的免疫反应促进斑块进展。
