Circular ZBTB44-encoded ZBTB44-342aa alleviates osteogenic differentiation of human aortic valve interstitial cells by inhibiting the cGAS-STING pathway

Background Circular RNAs (circRNAs) are implicated in various physiopathologic activities and play a crucial role in calcific aortic valve disease (CAVD) progression. However, the role of coding circRNAs in CAVD remains unclear. In this study, we aimed to characterize coding circRNAs and explore their functions in CAVD. Methods We identified circular Zinc finger and BTB domain-containing protein 44 (CircZBTB44) through transcriptome sequencing and characterized its coding capacity through a series of experiments. We investigated the biological role of circZBTB44 and its encoded peptide, ZBTB44-342aa, in CAVD, as well as its mechanism of action through in vivo and in vitro experiments. Results We found that circZBTB44 promotes the translation of ZBTB44-342aa through N6-methyladenosine modifications. Functionally, ZBTB44-342aa alleviates mitochondrial DNA (mtDNA) release into the cytoplasm, suppresses the activation of the Cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway, and alleviates the osteogenic differentiation of human aortic valve interstitial cells (HAVICs). In vivo, circZBTB44 adeno-associated virus and STING deprivation alleviated the aortic valve lesions induced in an aortic valve injury (AVI) model. In vitro, the overexpression of circZBTB44 or the exogenous addition of the ZBTB44-342aa recombinant protein inhibited the osteogenic response of HAVICs. Conversely, the siRNA-mediated knockdown of circZBTB44 enhanced this response. Furthermore, H-151-induced inhibition of STING alleviated the osteogenic response of HAVICs, whereas STING activation by dimeric amidobenzimidazole exacerbated this response. ZBTB44-342aa inhibits mitochondrial fission by binding to Insulin-like growth factor 2 mRNA-binding protein 3, which reduces the leakage of mtDNA into the cytoplasm and promotes the stability of calcium/calmodulin-dependent protein kinase 1. This reduction inhibited STING activation and suppressed downstream osteogenic responses. Conclusion This study demonstrates that circZBTB44 encodes a novel peptide, ZBTB44-342, which alleviates CAVD progression by inhibiting the activation of the cGAS-STING signaling pathway. Therefore, circZBTB44 and STING may be potential targets for future therapeutic interventions in CAVD.