Department of Rheumatology and Immunology, The Affiliated Hospital of Jiaxing University (The First Hospital of Jiaxing), Jiaxing, Zhejiang, China.
Jiaxing Key Laboratory of Osteoporosis and Bone Metabolism, The Affiliated Hospital of Jiaxing University (The First Hospital of Jiaxing), Jiaxing, Zhejiang, China.
Autoimmunity. 2023 Dec;56(1):2282939. doi: 10.1080/08916934.2023.2282939. Epub 2023 Nov 17.
The pathogenesis of rheumatoid arthritis (RA) is heavily impacted by the inflammation and activation of fibroblast-like synoviocytes (FLS). The objective of this investigation is to clarify the involvement of exosomes derived from FLS stimulated by tumour necrosis factor α (TNF-α) in angiogenesis and the underlying mechanisms. FLS cells were obtained from synovial fluid of RA patients and exosomes were obtained from FLS cell supernatant with TNF-α stimulation by ultracentrifugation. Exosomes were subsequently analysed using transmission electron microscopy, nanoparticle tracking analysis, and western blotting. The functional effects of exosomes with TNF-α stimulation on human umbilical vein endothelial cells (HUVEC) migration, invasion, and angiogenesis was evaluated using wound scratch healing test, transwell invasion assay, and tube formation assay. DNA nanoball-seq (DNBSEQ) sequencing platform was utilised to analysis different expression miRNA from exosomes, miRNA and mRNA from HUVEC. The expression level of miR-200a-3p was determined through quantitative real-time polymerase chain reaction (qRT-PCR). The quantification of KLF6 and VEGFA expression levels were performed by qRT-PCR and western blot analysis. The validation of the association between miR-200a-3p and KLF6 was established through a fluorescence enzyme reporting assay. In comparison to exosome induced by PBS, exosome induced by TNF-α exhibited a substantial exacerbation of invasion, migration, and angiogenesis in HUVEC. 4 miRNAs in exosomes and HUVEC cells, namely miR-1246, miR-200a-3p, miR-30a-3p, and miR-99b-3p was obtained. MiR-200a-3p maintained high consistency with the sequencing results. We obtained 5 gene symbols, and KLF6 was chose for further investigation. The expression of miR-200a-3p in exosomes induced by TNF-α and in HUVEC treated with these exosomes demonstrated a significantly increase. Additionally, HUVEC cells displayed a notable decrease in KLF6 expression and a significant elevation in VEGFA expression. This was further confirmed by the fluorescence enzyme report assay, which provided evidence of the direct targeting of KLF6 by miR-200a-3p. Exosomes induced by TNF-α have the ability to enhance the migration, invasion, and angiogenesis of HUVEC cells the miR-200a-3p/KLF6/VEGFA axis.
类风湿关节炎（RA）的发病机制受到成纤维样滑膜细胞（FLS）炎症和激活的严重影响。本研究的目的是阐明肿瘤坏死因子α（TNF-α）刺激的 FLS 衍生的外泌体在血管生成中的作用及其潜在机制。从 RA 患者的滑膜液中获得 FLS 细胞，并通过超速离心从 FLS 细胞上清液中获得 TNF-α刺激的外泌体。然后使用透射电子显微镜、纳米颗粒跟踪分析和 Western blot 分析外泌体。使用划痕愈合试验、Transwell 侵袭试验和管形成试验评估 TNF-α刺激的外泌体对人脐静脉内皮细胞（HUVEC）迁移、侵袭和血管生成的功能影响。利用 DNA 纳米球测序（DNBSEQ）测序平台分析外泌体和 HUVEC 中的不同表达 miRNA，以及 HUVEC 中的 miRNA 和 mRNA。通过定量实时聚合酶链反应（qRT-PCR）测定 miR-200a-3p 的表达水平。通过 qRT-PCR 和 Western blot 分析测定 KLF6 和 VEGFA 表达水平的定量。通过荧光酶报告测定验证 miR-200a-3p 与 KLF6 之间的关联。与 PBS 诱导的外泌体相比，TNF-α 诱导的外泌体在 HUVEC 中显著加剧了侵袭、迁移和血管生成。在 exosomes 和 HUVEC 细胞中获得了 4 个 miRNA，即 miR-1246、miR-200a-3p、miR-30a-3p 和 miR-99b-3p。miR-200a-3p 与测序结果具有高度一致性。我们获得了 5 个基因符号，选择 KLF6 进行进一步研究。TNF-α 诱导的外泌体和用这些外泌体处理的 HUVEC 中的 miR-200a-3p 表达显著增加。此外，HUVEC 细胞中 KLF6 的表达明显降低，VEGFA 的表达显著升高。荧光酶报告测定进一步证实了 miR-200a-3p 对 KLF6 的直接靶向作用。TNF-α 诱导的外泌体能够增强 HUVEC 细胞的迁移、侵袭和血管生成，该过程涉及 miR-200a-3p/KLF6/VEGFA 轴。
