基于单细胞核测序的共表达网络分析人静脉畸形发生与侵袭相关的内皮细胞关键基因  

Identification of endothelial cell key genes associated with pathogenesis and invasion of human venous malformations using single-nucleus RNA sequencing-based co-expression network analysis

作　　者：刘文博[1] 林俊杰 张媚娟 苑春杰 冯小娟 焦文婷 乔军波 王雯秋 方斌 陈长宽 Liu Wenbo;Lin Junjie;Zhang Meijuan;Yuan Chunjie;Feng Xiaojuan;Jiao Wenting;Qiao Junbo;Wang Wenqiu;Fang Bin;Chen Changkuan(Department of Hemangioma Surgery,The Third Affiliated Hospital of Zhengzhou University,Henan Provincial Key Department of Hemangioma and Vascular Malformation Medicine,Henan Provincial Key Laboratory of Hemangioma and Vascular Malformation,Zhengzhou 450052,China)

机构地区：[1]郑州大学第三附属医院血管瘤外科,河南省血管瘤与血管畸形医学重点学科,河南省血管瘤与血管畸形医学重点实验室,郑州450052

出　　处：《中华预防医学杂志》2025年第4期458-467,共10页Chinese Journal of Preventive Medicine

基　　金：河南省重点研发专项(231111311400)。

摘　　要：目的利用生物信息学分析筛选内皮细胞(endothelial cell,EC)中与人静脉畸形(venous malformations,VMs)发生、侵袭相关的关键基因,为VMs早期筛查和靶向干预提供依据。方法采用病例对照研究方法,收集2021年9月至2023年9月郑州大学第三附属医院VMs患者术中切除的组织标本,其中畸形静脉组织为实验组,切缘远端正常的静脉组织为对照组。纳入4例VMs患者(包含每例的实验组与对照组)进行单细胞核测序(single-nucleus RNA sequencing,snRNA-seq),通过高维加权基因共表达网络分析(high-dimensional weighted gene correlation network analysis,hdWGCNA),结合基因本体论(gene ontology,GO)和京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)及蛋白质-蛋白质互作网络(protein-protein interaction,PPI)筛选关键基因。另纳入15例VMs患者(包含每例的实验组与对照组),通过实时荧光定量聚合酶链反应(reverse transcription quantitative polymerase chain reaction,RT-qPCR)、免疫组织化学、蛋白质印迹法(Western blot)对关键基因进行验证。结果snRNA-seq共捕获55430个细胞核,其中实验组30391个,对照组25039个,聚类分析得到22个细胞群,注释为8种细胞类型。hdWGCNA筛选出4个侵袭相关模块,GO分析显示富集于血管生成、整合素信号和细胞黏附;KEGG提示PI3K-AKT与局部黏附为核心通路。PPI网络联合cytoscape分析鉴定出EGFL7、TEK、FLT1为关键基因。据RT-qPCR结果,实验组中3个关键基因的mRNA相对表达量(6.66±2.31、1.86±0.62、3.49±0.58)明显高于对照组(1.05±0.14、1.00±0.14、1.06±0.25),差异均有统计学意义(t=9.37、4.27、11.20,P<0.05);据免疫组织化学结果,实验组胞质中3个关键基因的蛋白相对表达量(0.84±0.15、0.68±0.14、0.85±0.12)明显高于对照组(0.19±0.05、0.23±0.06、0.30±0.05),差异均有统计学意义(t=16.62、5.93、11.68,P<0.05);据Western blot结果,实验组3个关键基因蛋白相对表达量(ObjectiveThis study aimed to identify key genes in endothelial cell(EC)associated with the pathogenesis and progression of human venous malformations(VMs)through bioinformatics analysis,providing potential biomarkers for early screening and targeted therapy of VMs.MethodsA case-control study was conducted using surgically resected tissue specimens from VMs patients at the Third Affiliated Hospital of Zhengzhou University(from September 2021 to September 2023),with malformed venous tissues as the experimental group and distal normal venous tissues as controls.Single-nucleus RNA sequencing(snRNA-seq)was performed on paired experimental and control samples from four VM patients.High-dimensional weighted gene co-expression network analysis(hdWGCNA),combined with gene ontology(GO),Kyoto encyclopedia of genes and genomes(KEGG),and protein-protein interaction(PPI)network analysis,identified critical genes.Validation experiments included 15 additional VM cases and controls using reverse transcription quantitative polymerase chain reaction(RT-qPCR),immunohistochemistry(IHC),and Western blot.ResultsA total of 55430 nuclei were captured using snRNA-seq,with 30391 nuclei from the experimental group and 25039 nuclei from the control group.Cluster analysis identified 22 distinct cell populations,which were annotated into 8 cell types.hdWGCNA revealed four modules associated with invasion,which were enriched in angiogenesis,integrin signaling,and cell adhesion according to GO analysis.KEGG pathway analysis indicated that the PI3K-AKT signaling pathway and focal adhesion are key regulatory mechanisms.PPI network analysis combined with cytoscape identified EGFL7,TEK,and FLT1 as key genes.RT-qPCR results demonstrated that the relative mRNA expression levels of these three genes in the experimental group(6.66±2.31,1.86±0.62,3.49±0.58)were significantly higher than those in the control group(1.05±0.14,1.00±0.14,1.06±0.25),with statistically significant differences(t=9.37,4.27,11.20,P<0.05).Immunohistochemical analysis showed t

关 键 词：静脉畸形 单细胞核转录组测序 高维加权基因共表达网络分析 关键基因 

分 类 号：R654.3[医药卫生—外科学]