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
10.3760/cma.j.cn112150-20241230-01059
- VernacularTitle: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
- Author:
Wenbo LIU
1
;
Junjie LIN
1
;
Meijuan ZHANG
1
;
Chunjie YUAN
1
;
Xiaojuan FENG
1
;
Wenting JIAO
1
;
Junbo QIAO
1
;
Wenqiu WANG
1
;
Bin FANG
1
;
Changkuan CHEN
1
Author Information
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
- Publication Type:Journal Article
- Keywords:
Venous malformations;
Single-nucleus RNA sequencing;
High-dimensional weighted correlation network analysis;
Key gene
- From:
Chinese Journal of Preventive Medicine
2025;59(4):458-467
- CountryChina
- Language:Chinese
-
Abstract:
Objective:This 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.Methods:A 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.Results:A total of 55 430 nuclei were captured using snRNA-seq, with 30 391 nuclei from the experimental group and 25 039 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 that the relative protein expression levels of these three genes in the cytoplasm of the experimental group (0.84±0.15, 0.68±0.14, 0.85±0.12) were also significantly higher than those in the control group (0.19±0.05, 0.23±0.06, 0.30±0.05), with statistically significant differences ( t=16.62, 5.93, 11.68, P<0.05). Western blot analysis confirmed that the relative protein expression levels of these three genes in the experimental group (0.35±0.04, 0.36±0.09, 0.31±0.04) were significantly higher than those in the control group (0.19±0.01, 0.13±0.02, 0.14±0.04), with statistically significant differences ( t=7.05, 4.61, 5.93, P<0.05). Conclusion:EGFL7, FLT1, and TEK in EC may play crucial roles in the occurrence and invasion of VMs.
