miR-21 may Act as a Potential Mediator Between Inflammation and Abnormal Bone Formation in Ankylosing Spondylitis Based on TNF-α Concentration-Dependent Manner Through the JAK2/STAT3 Pathway

Abstract

Objective: To explore the role of microRNA (miR-21) in new bone formation in ankylosing spondylitis (AS) as mediated by different concentration of tumor necrosis factor-α (TNF-α). Methods: Fibroblasts isolated from the hips of patients with AS were induced to osteogenesis. These cells were then stimulated with varying concentrations of TNF-α. MicroRNA-21 expressions were evaluated using reverse transcription–polymerase chain reaction (RT-PCR) and osteogenesis was detected via Alizarin Red S (ARS) staining and measurement of alkaline phosphatase (ALP) activity. Relative expressions of p-STAT3, Nuclear STAT3, cytoplasm STAT3, Runx2, BMP2, osteopontin, osteocalcin, and LC3B in AS fibroblasts were measured after exposure to different concentrations of TNF-α. The STAT3-inhibiting small interfering RNA allowed further exploration on its impact on miR-21 and primary miR-21 expressions. A proteoglycan-induced arthritis (PGIA) Balb/c mouse model was established in order to monitor sacroiliac joint (SIJ) inflammation and subsequent damage through magnetic resonance image. Serum miR-21 and TNF-α expressions were evaluated using RT-PCR and enzyme-linked immunosorbent assay. At week 16, mice models were transfected intravenously with miR-21 overexpressing agomir and miR-21 inhibiting antagomir for 7 successive days. The rate of abnormal bone formation at SIJ was evaluated using microcomputed tomography and hematoxylin and eosin staining at week 24. Western blot analysis enabled quantification of STAT-3, JAK-2, and interleukin (IL)-17A expressions present in the SIJ. Results: The in vitro miR-21 expression and osteogenesis activity were noted to be augmented in the setting of low TNF-α concentrations (0.01-0.1 ng/mL) while they were depressed in settings with higher TNF-α concentrations (1-10 ng/mL). Samples with the most distinct ARS manifestation and ALP activity as well as the highest miR-21 expressions were those who received 0.1 ng/mL of TNF-α. Primary miR-21 was found to be notable raised by Si-STAT3, while the converse effect was seen in mature miR-21 expressions. Intravenous injection of exogenous miR-21 contributed to new bone formation and significantly elevated expressions of STAT3, JAK2, and IL-17 in PGIA mice. Conclusions: The results revealed that miR-21 may act as a potential mediator between new bone formation and inflammation in AS.

Long non‐coding RNA NEAT1 and its targets (microRNA‐21 and microRNA‐125a) in rheumatoid arthritis: Altered expression and potential to monitor disease activity and treatment outcome

“…The possible reasons might be that: (a) elevated lnc-NEAT1 and decreased miR-21 and miR-125a could promote inflammatory cytokines and immune response, which consequently resulted in elevated inflammation in RA patients 7,19,20,23 ; and (b) increased lnc-NEAT1 and declined miR-21 and miR-125a might inhibit new bone formation and accelerate bone degradation through regulating several signaling pathways (such as Janus kinase 2/signal transducer, activator of transcription-3, and E26 transformation-specific-1 pathways), which could lead to high disease activity in RA patients. [24][25][26] Therefore, elevated lnc-NEAT1 but declined miR-21 and miR-125a were correlated with inflammation and disease activity in RA.…”

Exploring the Extracellular Vesicle MicroRNA Expression Repertoire in Patients with Rheumatoid Arthritis and Ankylosing Spondylitis Treated with TNF Inhibitors

“…Balzano et al hypothesized that low expression of miR-21-5p was a result of corticosteroids that inhibit NF- κ B [ 94 ]. Further analyses revealed that miR-21 may be implicated in several signalling pathways such as the IL-34/STAT3/miR-21 pathway, essential for synovial fibroblast survival in RA [ 95 ], as well as a mediator between inflammation and bone formation through the JAK2/STAT3 pathway in AS [ 96 ]. Additionally, miR-21 plays a role in mediation of RANKL-induced osteoclastogenesis and downregulation of programmed cell death 4 (PDCD4) protein levels [ 97 ].…”

Difference in serum miRNA expression between immunoglobulin‑sensitive and ‑insensitive incomplete Kawasaki disease patients

“…RevertAid™ H Minus First Strand cDNA Synthesis Kit (Fermentas) was used to prepare the mRNA reverse transcription system to synthesize cDNA by reverse transcription. The primer sequences of miRNA-21, miRNA-145, miRNA-155, and miRNA-199b-5p are as previously documented ( 17-20 ).…”