CircHIPK3 regulates cementoblast differentiation via the miR-10b-5p/DOHH/NF-κB axis

Background

Intact cementum is vital for tooth stability and health. Cementoblasts, which line the root surface, are responsible for cementum formation. Recent evidence suggests that circular RNAs (circRNAs) are involved in various cellular functions and may have clinical applications. Although circHIPK3 has been shown to participate in osteogenesis, its role in cementoblast differentiation and mineralization is not well understood.

Methods

The ring structure of circHIPK3 was confirmed using Sanger sequencing and an actinomycin D assay. Subcellular localization of circHIPK3 was assessed using a nucleus-cytoplasm separation assay. RT-qPCR was employed to analyze circHIPK3 expression during cementoblast differentiation and following TNF-α treatment. In vivo, periapical lesions were induced in mouse mandibular first molars to mimic an inflammatory environment, and circHIPK3 expression was evaluated. The interaction of the circHIPK3/miR-10b-5p/DOHH axis was explored through RNA pull-down assays, bioinformatics analysis, and dual-luciferase reporter assays. The effects on cementoblast differentiation and mineralization were assessed by measuring osteogenic markers, alkaline phosphatase (ALP) activity, ALP staining, and alizarin red S staining.

Results

CircHIPK3 was predominantly located in the cytoplasm of cementoblasts, and its expression was significantly upregulated during cementoblast differentiation. Knockdown of circHIPK3 inhibited cementoblast differentiation and mineralization, whereas its overexpression promoted these processes. Mechanistically, circHIPK3 upregulated DOHH expression by sponging miR-10b-5p, thereby enhancing cementoblast differentiation and mineralization. The NF-κB pathway was found to act downstream of the circHIPK3/miR-10b-5p/DOHH axis in these processes. Additionally, circHIPK3 expression was significantly downregulated in inflammatory environments both in vitro and in vivo. Forced overexpression of circHIPK3 mitigated the inhibitory effects of TNF-α on cementoblast differentiation and mineralization.

Conclusion

CircHIPK3 acts as a positive regulator of cementoblast differentiation and mineralization through the miR-10b-5p/DOHH/NF-κB axis, playing a crucial role in both normal and pathological cementogenesis.