circANKRD12/circTIMMDC1 synergistically regulates enteric neural crest cell migration via miR-181b-5p-PROX1-NOTCH1 axis in Hirschsprung's disease

Background: Circular RNAs (circRNAs) are implicated in Hirschsprung's disease (HSCR), a genetic disorder caused by defective migration and proliferation of enteric neural crest cells (ENCCs). Methods: Expression patterns of circANKRD12 and circTIMMDC1, and related molecules in the miR-181b-5p-PROX1-NOTCH1 axis were analyzed in human and mouse fetal intestines and HSCR patient tissues. Functional assays, including in vitro neural cell experiments, ex vivo ENCC explant, and in vivo zebrafish models, were conducted to assess the effects on neural cell behavior. Results: circANKRD12 and circTIMMDC1 were significantly downregulated in HSCR patient tissues. Single-cell analysis confirmed PROX1, NOTCH1, and HES1 expression in ENCCs from human and mouse fetal intestines. Both circRNAs synergistically regulated PROX1 by sponging miR-181b-5p, activating the NOTCH1-HES1 signaling pathway, and enhancing neural cell migration. Knockdown of these circRNAs impaired ENCC proliferation and migration. Zebrafish lacking prox1a showed reduced enteric neurons. Conclusions: circANKRD12 and circTIMMDC1 synergistically modulate ENCC function via the miR-181b-5p-PROX1-NOTCH1 axis, providing insights into HSCR pathogenesis and potential therapies.