Wang Song, Zhou Zifu, Li Jing, Wang Yu, Li Hongwen, Lv Renrong, Xu Guangqi, Zhang Jian, Bi Jianhai, Huo Ran
Department of Burn and Plastic Surgery, Shandong Provincial Hospital, Cheeloo College of Medicine, Shandong University, Jinan, China.
Department of Burn and Plastic Surgery, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China.
Front Cell Dev Biol. 2021 Nov 8;9:755409. doi: 10.3389/fcell.2021.755409. eCollection 2021.
Proteomics and high connotation functional gene screening (HCS) were used to screen key functional genes that play important roles in the pathogenesis of venous malformation. Furthermore, this study was conducted to analyze and explore their possible functions, establish a gene mutation zebrafish model, and perform a preliminary study to explore their possible pathogenic mechanisms in venous malformation. Pathological and normal tissues from patients with disseminated venous malformation were selected for Tandem Mass Tag (TMT) proteomics analysis to identify proteins that were differentially expressed. Based on bioinformatics analysis, 20 proteins with significant differential expression were selected for HCS to find key driver genes and characterize the expression of these genes in patients with venous malformations. experiments were then performed using human microvascular endothelial cells (HMEC-1). A gene mutant zebrafish model was also constructed for experiments to explore gene functions and pathogenic mechanisms. The TMT results showed a total of 71 proteins that were differentially expressed as required, with five of them upregulated and 66 downregulated. Based on bioinformatics and proteomics results, five highly expressed genes and 15 poorly expressed genes were selected for functional screening by RNAi technology. HCS screening identified ACTA2 as the driver gene. Quantitative polymerase chain reaction (qPCR) and western blot were used to detect the expression of ACTA2 in the pathological tissues of patients with venous malformations and in control tissues, and the experimental results showed a significantly lower expression of ACTA2 in venous malformation tissues ( < 0.05). Cell assays on the human microvascular endothelial cells (HMEC-1) model showed that cell proliferation, migration, invasion, and angiogenic ability were all significantly increased in the ACTA2 over-expression group ( < 0.05), and that overexpression of ACTA2 could improve the inhibitory effect on vascular endothelial cell proliferation. We constructed an ACTA2-knockdown zebrafish model and found that the knockdown of ACTA2 resulted in defective vascular development, disruption of vascular integrity, and malformation of micro vein development in zebrafish. Further qPCR assays revealed that the knockdown of ACTA2 inhibited the Dll4/notch1 signaling pathway, Ephrin-B2 signaling pathway, and vascular integrity-related molecules and activated the Hedgehog signaling pathway. This study revealed that ACTA2 deficiency is an important factor in the pathogenesis of venous malformation, resulting in the disruption of vascular integrity and malformed vascular development. ACTA2 can be used as a potential biomarker for the treatment and prognosis of venous malformations.
蛋白质组学和高内涵功能基因筛选（HCS）被用于筛选在静脉畸形发病机制中起重要作用的关键功能基因。此外，本研究旨在分析和探索它们可能的功能，建立基因突变斑马鱼模型，并进行初步研究以探索它们在静脉畸形中可能的致病机制。选择散发型静脉畸形患者的病理组织和正常组织进行串联质谱标签（TMT）蛋白质组学分析，以鉴定差异表达的蛋白质。基于生物信息学分析，选择20种差异表达显著的蛋白质进行HCS，以寻找关键驱动基因并表征这些基因在静脉畸形患者中的表达。然后使用人微血管内皮细胞（HMEC-1）进行实验。还构建了基因敲除斑马鱼模型用于实验，以探索基因功能和致病机制。TMT结果显示共有71种蛋白质按要求差异表达，其中5种上调，66种下调。基于生物信息学和蛋白质组学结果，选择5个高表达基因和15个低表达基因通过RNAi技术进行功能筛选。HCS筛选确定ACTA2为驱动基因。采用定量聚合酶链反应（qPCR）和蛋白质免疫印迹法检测ACTA2在静脉畸形患者病理组织和对照组织中的表达，实验结果显示ACTA2在静脉畸形组织中的表达明显降低（<0.05）。在人微血管内皮细胞（HMEC-1）模型上进行的细胞实验表明，ACTA2过表达组的细胞增殖、迁移、侵袭和血管生成能力均显著增加（<0.05），并且ACTA2的过表达可以增强对血管内皮细胞增殖的抑制作用。我们构建了ACTA2敲低斑马鱼模型，发现敲低ACTA2导致斑马鱼血管发育缺陷、血管完整性破坏和微静脉发育畸形。进一步的qPCR分析显示，敲低ACTA2会抑制Dll4/Notch1信号通路、Ephrin-B2信号通路以及与血管完整性相关的分子，并激活Hedgehog信号通路。本研究表明，ACTA2缺陷是静脉畸形发病机制中的一个重要因素，导致血管完整性破坏和血管发育畸形。ACTA2可作为静脉畸形治疗和预后的潜在生物标志物。
