新疆医科大学学报

目的 通过缺血性脑卒中患者外周血单个核细胞(Peripheral blood mononuclear cell, PBMC)的RNA-seq测序结果对可变剪接事件进行分析，探究缺血性脑卒中关键核心基因与可变剪接事件的关系。方法 选取2024年3-11月就诊于新疆医科大学第一附属医院神经内科的10例缺血性脑卒中患者为病例组，纳入10例正常体检者为对照组，采集两组受试者的PBMC样本，利用转录组测序技术对受试者样本剪接位点进行分析，采用rMATS软件获取可变剪接调控基因(Regulated alternative splicing genes, RASG)。通过韦恩图获取RASG和差异表达基因(Differentially expressed gene, DEG)的共表达基因。对RASG和共表达基因进行基因本体论(Gene ontology, GO)功能分析与京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes, KEGG)通路富集分析，对共表达基因进行蛋白互作分析获取关键核心基因。利用AutoDock软件对关键核心基因进行分子对接，探究其对接活性。定量反转录聚合酶链式反应(Quantitative reverse transcription polymerase chain reaction, qRT-PCR)验证关键核心基因在缺血性脑卒中患者PBMC mRNA的表达水平。结果 病例组及对照组在基线资料及临床检测指标等方面的差异无统计学意义，具有可比性。对病例组和对照组的转录组测序数据进行分析，共检测出10种不同类型的可变剪接事件，其中内含子保留(Intron retention, IntronR)可变剪接事件发生数目最高。RASG有808个基因，DEG有118个基因，共筛选出52个共表达基因，GO功能分析结果显示，发现共表达基因主要存在于细胞质，分子功能主要为蛋白结合，KEGG通路分析显示共表达基因主要富集在烟酸和烟酰胺代谢。共表达基因蛋白互作分析获取3个关键核心基因，分别为TMEM255B、SEPTIN5及SEPTIN14。通过AutoDock分子对接发现SEPTIN5与SEPTIN14的对接评分为-92.5 kcal/mol,有良好的亲和作用。qRT-PCR验证结果显示，与对照组相比，病例组PBMC的SEPTIN5、SEPTIN14 mRNA表达水平升高(P<0.01)。结论 SEPTIN5与SEPTIN14为缺血性脑卒中关键核心基因且存在相互作用，可能经烟酸和烟酰胺代谢通路调控可变剪接，诱发缺血性脑卒中。

Objective The alternative splicing(AS) events were analyzed through the RNA-seq sequencing results of peripheral blood mononuclear cells(PBMC) in patients with ischemic stroke to explore the relationship between the key core genes of ischemic stroke and the AS event. Methods 10 patients with ischemic stroke from the hospital, between March 2024 and November 2024, were selected as the case group, and 10 healthy individuals undergoing routine physical examinations were enrolled as the control group. PBMC samples were collected from all participants. Transcriptome sequencing was performed to analyze splicing sites, and the rMATS software was used to identify regulated alternative splicing genes(RASG). Venn diagrams were used to identify co-expressed genes between RASGs and differentially expressed genes(DEG). Gene ontology(GO) and kyoto encyclopedia of genes and genomes(KEGG) pathway enrichment analyses were conducted on both RASGs and the co-expressed genes. A protein-protein interaction(PPI) network was constructed from the co-expressed genes to identify hub genes. Molecular docking of the key hub genes was performed using AutoDock software to explore their binding affinity. The mRNA expression levels of the key hub genes in PBMCs from ischemic stroke patients were validated using quantitative reverse transcription polymerase chain reaction(qRT-PCR). Results No statistically significant differences were observed in the baseline characteristics and clinical indicators between the case and control groups, indicating that the groups were comparable. Analysis of the transcriptomic sequencing data from the case group and control group detected a total of 10 different types of AS events. Among these, intron retention(IntronR) was the most abundant. There search identified 808 RASGs and 118 DEGs, from which 52 co-expressed genes were screened. GO analysis revealed that the co-expressed genes were primarily localized in the cytoplasm and were mainly involved in protein binding. KEGG pathway analysis indicated significant enrichment of these genes in the nicotinate and nicotinamide metabolism pathway. PPI network analysis of the co-expressed genes identified 3 key hub genes: TMEM255B, SEPTIN5 and SEPTIN14. Molecular docking using AutoDock demonstrated a strong binding affinity between SEPTIN5 and SEPTIN14, with a docking score of-92.5 kcal/mol. qRT-PCR validation confirmed that the mRNA expression levels of SEPTIN5 and SEPTIN14 in patient PBMCs were significantly higher than those in the control group(P<0.01). Conclusion SEPTIN5 and SEPTIN14 are key genes in ischemic stroke that interact with each other. They may contribute to the pathogenesis of ischemic stroke by regulating AS, potentially through the nicotinate and nicotinamide metabolism pathway.