In a groundbreaking development in the understanding of Hirschsprung’s disease (HSCR), recent research has illuminated the complex molecular interplay involving circular RNAs (circRNAs) and their regulatory role in enteric neural crest cell (ENCC) migration. HSCR is a congenital disorder characterized by the absence of enteric ganglia in parts of the intestine, resulting from defective proliferation and migration of ENCCs during embryonic development. This defect leads to severe intestinal motility issues and life-threatening complications. The study, led by Fu, Wang, Xu, and colleagues, unpacks the synergistic functions of two specific circRNAs, circANKRD12 and circTIMMDC1, revealing new dimensions in the molecular pathogenesis of HSCR.
Circular RNAs have emerged as critical regulators in various biological processes and diseases. Unlike linear RNAs, circRNAs form covalently closed continuous loops, which confer stability and distinctive regulatory capabilities. In the context of HSCR, circRNAs have been underexplored, but this study provides substantial evidence that circANKRD12 and circTIMMDC1 function in unison to influence ENCC behavior, particularly migration and proliferation, through a well-defined molecular axis. This regulatory axis operates via miR-181b-5p, PROX1, and NOTCH1 — molecules known to govern cell differentiation and movement.
The researchers employed advanced molecular biology techniques to dissect the relationship between these circRNAs and their downstream signaling pathways. miR-181b-5p, a microRNA with known roles in cell migration, was identified as a pivotal mediator, regulated by both circANKRD12 and circTIMMDC1. The two circRNAs act as competing endogenous RNAs (ceRNAs) or “sponges,” sequestering miR-181b-5p and thereby modulating its availability to target mRNAs. This finely-tuned mechanism regulates PROX1 expression, a transcription factor essential for neural crest development.
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PROX1 is further implicated in the activation of the NOTCH1 signaling pathway, a critical regulator of cell fate decisions within ENCCs. Dysregulation of NOTCH1 has been frequently associated with developmental abnormalities, including HSCR. The study highlights how the circRNAs impact NOTCH1 signaling via PROX1, ultimately influencing the migratory capacity of ENCCs. The bidirectional interactions within this axis underscore the complexity of gene regulation in developmental diseases.
In their experimental paradigm, the team utilized patient-derived ENCCs and various in vitro assays to model the migratory defects observed in HSCR. Knockdown of circANKRD12 and circTIMMDC1 impaired ENCC migration, but simultaneous suppression accentuated this effect, revealing their cooperative function. Conversely, overexpression of these circRNAs restored migration capabilities. These results provide compelling evidence of their complementary roles and potential as therapeutic targets.
The mechanistic insights into how circANKRD12 and circTIMMDC1 modulate miR-181b-5p availability add a new layer of understanding to RNA-mediated regulation in HSCR. miRNAs, small non-coding RNAs, have long been recognized as vital post-transcriptional regulators, but the interaction with circRNAs presents an emerging frontier. By acting as molecular sponges, circRNAs not only regulate miRNA activity but also influence wide-ranging gene expression profiles relevant to disease pathology.
The role of the NOTCH1 signaling pathway in ENCC migration is well-documented, but this study bridges the gap between upstream regulators (circRNAs and microRNAs) and downstream effectors (transcription factors and signaling pathways). NOTCH1 modulates cellular differentiation by affecting gene expression programs that drive cytoskeletal remodeling and motility. The circRNA-mediated modulation of this pathway elucidates how developmental signaling is fine-tuned by non-coding RNA networks.
These findings open new therapeutic avenues, as targeting the circANKRD12/circTIMMDC1-miR-181b-5p-PROX1-NOTCH1 axis could rectify migratory defects in ENCCs. Gene therapy or RNA-based therapeutics might restore normal circRNA levels or modulate miR-181b-5p activity, potentially preventing the onset of HSCR or ameliorating disease severity. The stability and tissue-specific nature of circRNAs make them attractive candidates for diagnostic biomarkers or therapeutic targets.
Moreover, the study prompts a reevaluation of the broader role of circRNAs in neural crest development and related neurocristopathies. Given that HSCR results from aberrant neural crest cell behavior, unraveling circRNA networks may reveal conserved mechanisms applicable to other congenital disorders involving neural crest derivatives. Understanding circRNA functions in this context could revolutionize neurodevelopmental biology.
Experimental validation through quantitative PCR, fluorescence in situ hybridization, and functional assays confirmed the expression patterns and interactions of circANKRD12, circTIMMDC1, and miR-181b-5p in both normal and diseased tissue samples. These rigorous methods lend robustness to the findings, emphasizing the physiological relevance of the discovered regulatory axis. The integration of patient data with molecular biology enhances translational potential.
The intricate balance of RNA species described in this research highlights the sophisticated regulation of gene networks controlling critical developmental processes. It underscores the importance of non-coding RNAs in fine-tuning signaling pathways beyond classical transcriptional regulation. This paradigm shift suggests that genetic and epigenetic landscapes are intricately connected through RNA-mediated mechanisms, influencing disease susceptibility.
Future research inspired by this study could explore the potential compensatory mechanisms among circRNAs and their interactions with other microRNAs. Additionally, investigating how environmental factors or genetic mutations impact this regulatory axis might yield deeper insights into HSCR pathophysiology. Longitudinal studies could assess how circRNA expression varies throughout development and disease progression.
From a clinical perspective, early detection of dysregulated circRNA-miRNA interactions could improve prognostic assessments and guide personalized interventions for HSCR patients. The development of circRNA-specific probes or inhibitors could complement existing treatments, which predominantly involve surgical resection. A molecular approach could minimize long-term complications and improve quality of life.
Furthermore, this discovery may fuel innovation in circRNA research technology. Novel high-throughput sequencing and spatial transcriptomics could map circRNA networks with unprecedented resolution in neural crest cells. Such advancements would catalyze comprehensive profiling of circRNA functions across embryonic development stages and pathological conditions.
The implications of circANKRD12 and circTIMMDC1 in this study extend beyond HSCR, potentially influencing cancer biology, where similar migration and proliferation pathways are co-opted. The elucidation of the miR-181b-5p-PROX1-NOTCH1 axis offers a foundation for exploring circRNA roles in metastasis and tumor progression, highlighting the interconnectedness of developmental and pathological processes.
In conclusion, the research by Fu, Wang, Xu, et al. represents a significant leap forward in our understanding of the molecular architecture guiding enteric neural crest cell migration. The identification of circANKRD12 and circTIMMDC1 as synergistic regulators through a miRNA-transcription factor-signaling pathway axis unveils new horizons for therapeutic intervention in Hirschsprung’s disease. This emerging RNA-centric paradigm will likely pave the way for innovative strategies to decode and manipulate gene regulation in developmental diseases and beyond.
Subject of Research:
The role of circular RNAs (circANKRD12 and circTIMMDC1) in regulating enteric neural crest cell migration via the miR-181b-5p-PROX1-NOTCH1 axis in Hirschsprung’s disease.
Article Title:
circANKRD12/circTIMMDC1 synergistically regulates enteric neural crest cell migration via miR-181b-5p-PROX1-NOTCH1 axis in Hirschsprung’s disease.
Article References:
Fu, R., Wang, C., Xu, Z. et al. circANKRD12/circTIMMDC1 synergistically regulates enteric neural crest cell migration via miR-181b-5p-PROX1-NOTCH1 axis in Hirschsprung’s disease. Pediatr Res (2025). https://doi.org/10.1038/s41390-025-04245-0
Image Credits: AI Generated
DOI: https://doi.org/10.1038/s41390-025-04245-0
Tags: advanced molecular biology techniquescircANKRD12 and circTIMMDC1 functionscircular RNAs in neural crest migrationembryonic development and HSCRenteric neural crest cell behaviorgene regulation in congenital disordersHirschsprung’s disease researchintestinal motility disordersmiR-181b-5p signaling axismolecular pathogenesis of HSCRPROX1 NOTCH1 regulationrole of circRNAs in diseases
