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.
Subject(s)
Arthritis, Rheumatoid , Exosomes , MicroRNAs , Synoviocytes , Humans , Arthritis, Rheumatoid/metabolism , Cell Proliferation , Exosomes/genetics , Exosomes/metabolism , Exosomes/pathology , Fibroblasts/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/pathology , Kruppel-Like Factor 6/metabolism , Kruppel-Like Factor 6/pharmacology , MicroRNAs/genetics , MicroRNAs/metabolism , Synoviocytes/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Tumor Necrosis Factor-alpha/metabolism , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor A/pharmacologyABSTRACT
Transcription factors (TFs) act as powerful levers to regulate neural physiology and can be targeted to improve cellular responses to injury or disease. Because TFs often depend on cooperative activity, a major challenge is to identify and deploy optimal sets. Here we developed a bioinformatics pipeline, centered on TF co-occupancy of regulatory DNA, and used it to predict factors that potentiate the effects of pro-regenerative Klf6 in vitro. High content screens of neurite outgrowth identified cooperative activity by 12 candidates, and systematic testing in a mouse model of corticospinal tract (CST) damage substantiated three novel instances of pairwise cooperation. Combined Klf6 and Nr5a2 drove the strongest growth, and transcriptional profiling of CST neurons identified Klf6/Nr5a2-responsive gene networks involved in macromolecule biosynthesis and DNA repair. These data identify TF combinations that promote enhanced CST growth, clarify the transcriptional correlates, and provide a bioinformatics approach to detect TF cooperation.
Subject(s)
Axons/metabolism , Spinal Cord Injuries/genetics , Spinal Cord Injuries/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Animals , Computational Biology , DNA , DNA Repair , Female , Gene Expression Regulation , Gene Regulatory Networks , Kruppel-Like Factor 6/genetics , Kruppel-Like Factor 6/pharmacology , Male , Mice , Mice, Inbred C57BL , Neurons/metabolism , Pyramidal Tracts/metabolism , Rats, Sprague-Dawley , Receptors, Cytoplasmic and Nuclear/metabolism , TranscriptomeABSTRACT
Krüppel-like factors (KLFs) are a highly conserved family of transcription factors. We analysed expression profile data of KLFs and identified KLF6 as a new potential regulator of erythropoiesis. Knocking down the expression of KLF6 significantly raised γ-globin mRNA and protein levels in the erythroid cell line HUDEP-2 and haematopoietic progenitor (CD34+ ) cells. We found that overexpression of microRNA (miR)-2355-5p in HUDEP-2 and CD34+ cells correlated with increased γ-globin synthesis by suppressing expression of KLF6. Our discovery that the interaction between miR-2355-5p and KLF6 affects the expression of γ-globin may provide more information for the clinical management of ß-thalassaemia patients.