Circ_0000396 suppresses the proliferation and inflammation of rheumatoid arthritis synovial fibroblasts by targeting miR-574-5p/RSPO1 axis

• Published in: Journal of orthopaedic surgery and research 2023-09, Vol.18 (1), p.718-11, Article 718
• Main Authors: Yu, Hongchao, Yang, Jin, Chen, Kun, Kang, Wulin, Zhu, Fengfeng
• Format: Article
• Language: English
• Subjects: 3' Untranslated Regions; Analysis; Antigens; Apoptosis; Arthritis; Arthritis, Rheumatoid - genetics; Autoimmune diseases; BAX protein; Bcl-2 protein; Cardiovascular disease; Cell growth; Cell proliferation; Cell Proliferation - genetics; Circ_0000396; Circular RNA; Cyclin D1; Enzyme-linked immunosorbent assay; Enzymes; Fibroblasts; Flow cytometry; Genetic transcription; Humans; IL-1β; Inflammation; Inflammation - genetics; Interleukins; Lymphocytes B; MicroRNAs; MicroRNAs - genetics; miR-574-5p; miRNA; Orthopedics; Pathology; Patients; Proliferating Cell Nuclear Antigen; Proteins; Reagents; Reverse transcription; Rheumatoid arthritis; Rheumatoid factor; RNA; RNA, Circular - genetics; RSPO1; Synovial fibroblasts; Thrombospondins; Tumor Necrosis Factor-alpha; Tumor necrosis factor-TNF; Tumor necrosis factor-α; Variance analysis
• ISSN: 1749-799X; 1749-799X
• DOI: 10.1186/s13018-023-04117-5

Circular RNAs (circRNAs) are important regulators on the onset and progression of rheumatoid arthritis (RA). Our purpose is to explore the role and underpin mechanism of circ_0000396 in RA progression. RA patients (n = 39) and healthy volunteers (n = 33) were recruited from the Affiliated Hospital of Shaanxi University of Chinese Medicine for the present work. Circ_0000396, microRNA-574-5p (miR-574-5p) and R-spondin 1 (RSPO1) RNA levels were analyzed by reverse transcription-quantitative polymerase chain reaction. Cell proliferation was analyzed by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony formation assay, and 5-ethynyl-2'-deoxyuridine (EDU) assay. Cell apoptosis was assessed by flow cytometry. Protein expression levels of proliferating cell nuclear antigen (PCNA), Cyclin D1, Cyclin E1, BCL2-associated × protein (Bax), B-cell lymphoma-2 (Bcl2), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and RSPO1 were detected by western blot assay. Enzyme-linked immunosorbent assay (ELISA) was conducted to analyze the secretion of pro-inflammatory cytokines including IL-1β and TNF-α. The interaction between miR-574-5p and circ_0000396 or RSPO1 was confirmed by dual-luciferase reporter assay and RNA-pull down assay. Circ_0000396 expression was notably down-regulated in RA patients compared with healthy controls. Circ_0000396 overexpression suppressed the proliferation and inflammatory response and triggered the apoptosis of RA synovial fibroblasts (RASFs), accompanied by decreases in PCNA, Cyclin D1, Cyclin E1, Bcl2, IL-1β and TNF-α protein expression and an increase in Bax protein expression. Circ_0000396 acted as a molecular sponge for miR-574-5p, and circ_0000396 overexpression-mediated protective effects on RASFs dysfunction were largely reversed by the introduction of miR-574-5p mimics. miR-574-5p interacted with the 3' untranslated region (3'UTR) of RSPO1, and miR-574-5p negatively regulated RSPO1 expression in RASFs. Circ_0000396 could up-regulate the expression of RSPO1 by sponging miR-574-5p in RASFs. RSPO1 interference largely overturned circ_0000396 overexpression-mediated effects in RASFs. Circ_0000396 restrained the proliferation and inflammation and induced the apoptosis of RASFs by mediating miR-574-5p/RSPO1 axis, which provided novel potential targets for RA treatment.