Overexpression of Circ_0004496 is Associated with Pathological Bone Formation in Ankylosing Spondylitis via the miR-145/ACTG1 Axis.

BACKGROUND

The functional role of circular ribonucleic acids in ankylosing spondylitis (AS) remains poorly understood. In our previous study, circ_0004496 and actin gamma 1 (ACTG1) were found to be upregulated in ossified tissues from patients with AS.

AIMS: To investiagate the regulatory role of circ_0004496 in pathological bone formation through the miR-145/ACTG1 axis.

STUDY

DESIGN

Combined in vitro and in vivo experimental study.

METHODS

Tissue samples were obtained from 15 patients with AS and hip ankylosis and 15 control patients with femoral neck fractures. Quantitative real-time polymerase chain reaction was performed to measure circ_0004496 and miR-145 expression. Western blot analysis was used to determine the protein levels of alkaline phosphatase (ALP), osteocalcin (OCN), runt-related transcription factor 2 (Runx2), and ACTG1. Cell proliferation was evaluated using Cell Counting Kit-8 and 5-ethynyl-2'-deoxyuridine assays. Osteogenic differentiation was assessed by measuring ALP activity and performing Alizarin Red S staining. Interactions among circ_0004496, miR-145, and ACTG1 were examined using RNA immunoprecipitation (RIP), RNA pull-down, dual-luciferase reporter, and fluorescence in situ hybridization (FISH) assays.

RESULTS

Circ_0004496 and ACTG1 expression levels were significantly elevated in AS hip capsule tissues, whereas miR-145 expression was reduced. Overexpression of circ_0004496 in AS-derived fibroblasts enhanced cell proliferation, accelerated cell cycle progression, increased calcium deposition, and upregulated ALP, OCN, and Runx2 protein levels. Mechanistically, circ_0004496 functioned as a molecular sponge for miR-145, which directly targets ACTG1. These regulatory interactions were confirmed by dual-luciferase reporter, RIP, RNA pulldown, and FISH assays. In vivo, circ_0004496 overexpression was associated with sacroiliac joint fusion in proteoglycan-induced arthritis mice.

CONCLUSION

Circ_0004496 overexpression promotes pathological bone formation in AS by regulating the miR-145/ACTG1 axis both in vitro and in vivo.