TNF-α stimulated exosome derived from fibroblast-like synoviocytes isolated from rheumatoid arthritis patients promotes HUVEC migration, invasion and angiogenesis by targeting the miR-200a-3p/KLF6/VEGFA axis

Abstract

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 via the miR-200a-3p/KLF6/VEGFA axis.

Keywords:

• TNF-α
• exosomes
• angiogenesis
• miR-200a-3p
• KLF6

Authors’ contributions

Bin Zhang, Mingfeng Yang: Conceptualisation, Methodology, Software, Validation, Formal analysis, Investigation, Data Curation, Writing - Original Draft, Funding acquisition. Juanfang Gu: Methodology, Software, Validation, Formal analysis, Investigation, Visualisation, Funding acquisition. Linfeng Guo: Methodology, Software, Validation, Investigation, Data Curation. Jiangzhen Xie: Software, Investigation, Resources, Funding acquisition.

Ethics approval and consent to participate

The studies involving human participants were reviewed and approved by the Institutional Research Ethics Committee of the Ethics Committee of The First Hospital of Jiaxing (Approve number: LS2020-073). The study was conducted in accordance with the Helsinki declaration of the world medical association.

Disclosure statement

The authors declare no competing interests.

Additional information

Funding

This work was supported by Natural Science Foundation of Zhejiang [grant numbers LQ19H010001], Science and Technology Foundation of Jiaxing [grant numbers 2021AD10022], Jiaxing Key Discipiline of Medcine, Rheumatology and Autoimmunology (Supporting Subject2023-ZC-016).