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

Pediatric Research (2025) Cite this article

Abstract

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.

Impact

  • The study identifies circANKRD12 and circTIMMDC1 as pathogenic circRNAs in HSCR, revealing their synergistic effects on regulating ENCC migration.

  • The study reveals that prox1a (human PROX1 homolog) deficiency in zebrafish induces HSCR-like phenotypes.

  • The study elucidates the detailed mechanistic insights into the circANKRD12/circTIMMDC1-miR-181b-5p-PROX1-NOTCH1 axis in ENCC migration.

  • The study contributes to the development of diagnostic tools and therapies targeting the ceRNA network in HSCR, alongside improved prenatal screening methods for early detection.

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Fig. 1: circANKRD12/circTIMMDC1 are downregulated in HSCR and promote human neural cell migration.
The alternative text for this image may have been generated using AI.
Fig. 2: miR-181b-5p is upregulated in HSCR and suppresses human neural cell migration.
The alternative text for this image may have been generated using AI.
Fig. 3: Expression characteristics and function of PROX1/NOTCH1/HES1 pathway in ENS development and HSCR.
The alternative text for this image may have been generated using AI.
Fig. 4: circANKRD12 and circTIMMDC1 function as miR-181b-5p sponges.
The alternative text for this image may have been generated using AI.
Fig. 5: miR-181b-5p suppresses human neural cells migration by targeting PROX1-NOTCH1-HES1 axis.
The alternative text for this image may have been generated using AI.
Fig. 6: circANKRD12/circTIMMDC1 promote neural cell and ENCC migration through miR-181b-5p-PROX1-NOTCH1-HES1 axis.
The alternative text for this image may have been generated using AI.The alternative text for this image may have been generated using AI.

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Data availability

Data supporting the findings of this work are available in the article/Supplementary Materials.

References

  1. Torroglosa, A. et al. Identification of new potential LncRNA biomarkers in Hirschsprung disease. Int. J. Mol. Sci. 21, 5534 (2020).

  2. Hong, M. et al. Hirschsprung’s disease: key microRNAs and target genes. Pediatr. Res. 92, 737–747 (2022).

    Article  CAS  PubMed  Google Scholar 

  3. Tang, W. et al. Exome-wide association study identified new risk loci for Hirschsprung’s disease. Mol. Neurobiol. 54, 1777–1785 (2017).

    Article  CAS  PubMed  Google Scholar 

  4. Amiel, J. et al. Hirschsprung disease, associated syndromes and genetics: a review. J. Med. Genet. 45, 1–14 (2008).

    Article  CAS  PubMed  Google Scholar 

  5. Greenberg, A. et al. Automatic ganglion cell detection for improving the efficiency and accuracy of Hirschsprung disease diagnosis. Sci. Rep. 11, 3306 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Das, K. & Mohanty, S. Hirschsprung disease—current diagnosis and management. Indian J. Pediatr. 84, 618–623 (2017).

    Article  PubMed  Google Scholar 

  7. Shi, Y., Jia, X. & Xu, J. The new function of circRNA: translation. Clin. Transl. Oncol. 22, 2162–2169 (2020).

    Article  CAS  PubMed  Google Scholar 

  8. Zhou, W. Y. et al. Circular RNA: metabolism, functions and interactions with proteins. Mol. Cancer 19, 172 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Caba, L., Florea, L., Gug, C., Dimitriu, D. C. & Gorduza, E. V. Circular RNA-is the circle perfect? Biomolecules 11, 5534 (2021).

  10. Misir, S., Wu, N. & Yang, B. B. Specific expression and functions of circular RNAs. Cell Death Differ. 29, 481–491 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Yang, Q., Li, F., He, A. T. & Yang, B. B. Circular RNAs: expression, localization, and therapeutic potentials. Mol. Ther. 29, 1683–1702 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Kristensen, L. S. et al. The biogenesis, biology and characterization of circular RNAs. Nat. Rev. Genet. 20, 675–691 (2019).

    Article  CAS  PubMed  Google Scholar 

  13. Du, T. & Zamore, P. D. Beginning to understand microRNA function. Cell Res. 17, 661–663 (2007).

    Article  CAS  PubMed  Google Scholar 

  14. Yates, L. A., Norbury, C. J. & Gilbert, R. J. The long and short of microRNA. Cell 153, 516–519 (2013).

    Article  CAS  PubMed  Google Scholar 

  15. Saliminejad, K., Khorram Khorshid, H. R., Soleymani Fard, S. & Ghaffari, S. H. An overview of MicroRNAs: biology, functions, therapeutics, and analysis methods. J. Cell Physiol. 234, 5451–5465 (2019).

    Article  CAS  PubMed  Google Scholar 

  16. Cao, Y. Z., Sun, J. Y., Chen, Y. X., Wen, C. C. & Wei, L. The roles of CircRNAs in cancers: perspectives from molecular functions. Gene 767, 145182 (2021).

    Article  CAS  PubMed  Google Scholar 

  17. Jarlstad Olesen, M. T. & L, S. K. Circular RNAs as microRNA sponges: evidence and controversies. Essays Biochem. 65, 685–696 (2021).

    Article  PubMed  Google Scholar 

  18. Panda, A. C. Circular RNAs act as miRNA sponges. Adv. Exp. Med. Biol. 1087, 67–79 (2018).

    Article  CAS  PubMed  Google Scholar 

  19. Xia, R. P. et al. Circ-itch overexpression promoted cell proliferation and migration in Hirschsprung disease through mir-146b-5p/Ret axis. Pediatr. Res. 92, 1008–1016 (2022).

    Article  CAS  PubMed  Google Scholar 

  20. Wen, Z. et al. Circular RNA Ccdc66 targets Dcx to regulate cell proliferation and migration by sponging mir-488-3p in Hirschsprung’s disease. J. Cell Physiol. 234, 10576–10587 (2019).

    Article  CAS  PubMed  Google Scholar 

  21. Kaltezioti, V. et al. Prox1 regulates the notch1-mediated inhibition of neurogenesis. PLoS Biol. 8, e1000565 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Miyoshi, G. et al. Prox1 regulates the subtype-specific development of caudal ganglionic eminence-derived GABAergic cortical interneurons. J. Neurosci. 35, 12869–12889 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Sugiyama, T., Osumi, N. & Katsuyama, Y. A novel cell migratory zone in the developing hippocampal formation. J. Comp. Neurol. 522, 3520–3538 (2014).

    Article  CAS  PubMed  Google Scholar 

  24. Margarido, A. S. et al. Prox1 is a specific and dynamic marker of sacral neural crest cells in the chicken intestine. J. Comp. Neurol. 528, 879–889 (2020).

    Article  CAS  PubMed  Google Scholar 

  25. Mead, T. J. & Yutzey, K. E. Notch pathway regulation of neural crest cell development in vivo. Dev. Dyn. 241, 376–389 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Cao, J. et al. A human cell atlas of fetal gene expression. Science 370, eaba7721 (2020).

  27. Morarach, K. et al. Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing. Nat. Neurosci. 24, 34–46 (2021).

    Article  CAS  PubMed  Google Scholar 

  28. Li, J. H., Liu, S., Zhou, H., Qu, L. H. & Yang, J. H. Starbase V2.0: decoding miRNA-ceRNA, miRNA-ncRNA and protein-RNA interaction networks from large-scale clip-seq data. Nucleic Acids Res. 42, D92–D97 (2014).

    Article  CAS  PubMed  Google Scholar 

  29. Kameda, Y. et al. Hes1 is required for the development of pharyngeal organs and survival of neural crest-derived mesenchymal cells in pharyngeal arches. Cell Tissue Res. 353, 9–25 (2013).

    Article  CAS  PubMed  Google Scholar 

  30. Wang, Z., Lei, X. & Wu, F. X. Identifying cancer-specific circRNA-Rbp binding sites based on deep learning. Molecules 24, 4035 (2019).

  31. Liu, C. X. & Chen, L. L. Circular RNAs: characterization, cellular roles, and applications. Cell 185, 2016–2034 (2022).

    Article  CAS  PubMed  Google Scholar 

  32. D’Anca, M., Buccellato, F. R., Fenoglio, C. & Galimberti, D. Circular RNAs: emblematic players of neurogenesis and neurodegeneration. Int. J. Mol. Sci. 23, 4134 (2022).

  33. Lee, E. C. S. et al. The roles of circular RNAs in human development and diseases. Biomed. Pharmacother. 111, 198–208 (2019).

    Article  CAS  PubMed  Google Scholar 

  34. Li, J., Sun, D., Pu, W., Wang, J. & Peng, Y. Circular RNAs in cancer: biogenesis, function, and clinical significance. Trends Cancer 6, 319–336 (2020).

    Article  CAS  PubMed  Google Scholar 

  35. Chen, W. et al. Circular RNA mtcl1 targets Smad3 by sponging Mir-145-5p for regulation of cell proliferation and migration in Hirschsprung’s disease. Pediatr. Surg. Int. 40, 25 (2023).

    Article  CAS  PubMed  Google Scholar 

  36. Zhou, L. et al. Down-regulation of circ-Prkci inhibits cell migration and proliferation in Hirschsprung disease by suppressing the expression of Mir-1324 target Plcb1. Cell Cycle 17, 1092–1101 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Lake, J. I. & Heuckeroth, R. O. Enteric nervous system development: migration, differentiation, and disease. Am. J. Physiol. Gastrointest. Liver Physiol. 305, G1–G24 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Jevans, B. et al. Human enteric nervous system progenitor transplantation improves functional responses in Hirschsprung disease patient-derived tissue. Gut 73, 1441–1453 (2024).

    Article  CAS  PubMed  Google Scholar 

  39. Russo, I. et al. The culture of gut explants: a model to study the mucosal response. J. Immunol. Methods 438, 1–10 (2016).

    Article  CAS  PubMed  Google Scholar 

  40. Marchetti, M., Zhang, C. & Edgar, B. A. An improved organ explant culture method reveals stem cell lineage dynamics in the adult Drosophila intestine. Elife 11, e76010 (2022).

  41. Zhao, X., Zhong, Y., Wang, X., Shen, J. & An, W. Advances in circular RNA and its applications. Int J. Med. Sci. 19, 975–985 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Ma, Z., Kuang, Z. & Deng, L. Ngcicm: a novel deep learning-based method for predicting circRNA-miRNA interactions. IEEE/ACM Trans. Comput. Biol. Bioinform. 20, 3080–3092 (2023).

    Article  CAS  PubMed  Google Scholar 

  43. Kulcheski, F. R., Christoff, A. P. & Margis, R. Circular RNAs are miRNA sponges and can be used as a new class of biomarker. J. Biotechnol. 238, 42–51 (2016).

    Article  CAS  PubMed  Google Scholar 

  44. Zhong, S. et al. Decoding the development of the human hippocampus. Nature 577, 531–536 (2020).

    Article  CAS  PubMed  Google Scholar 

  45. Hidalgo, A. & Griffiths, R. Coupling glial numbers and axonal patterns. Cell Cycle 3, 1118–1120 (2004).

    Article  CAS  PubMed  Google Scholar 

  46. Yu, D. X. et al. Modeling hippocampal neurogenesis using human pluripotent stem cells. Stem Cell Rep. 2, 295–310 (2014).

    Article  CAS  Google Scholar 

  47. Okamura, Y. & Saga, Y. Notch signaling is required for the maintenance of enteric neural crest progenitors. Development 135, 3555–3565 (2008).

    Article  CAS  PubMed  Google Scholar 

  48. Pawolski, V. & Schmidt, M. H. H. Neuron-glia interaction in the developing and adult enteric nervous system. Cells 10, 47 (2020).

  49. Lo, L. & Anderson, D. J. Postmigratory neural crest cells expressing C-ret display restricted developmental and proliferative capacities. Neuron 15, 527–539 (1995).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank all the members for their contributions to the current study.

Funding

This work was supported by the Clinical Research Plan of SHDC (SHDC2020CR2010A), the Natural Science Foundation of Shanghai (22ZR1451500), and Shanghai Key Laboratory of Pediatric Gastroenterology and Nutrition (17DZ2272000). No funder has any roles in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author information

Authors and Affiliations

  1. Department of Pediatric Surgery, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Runxi Fu, Ze Xu, Yuling Zhao, Xiaowei Liu, Ying Zhou, Weihua Pan, Yang Wang & Wei Cai

  2. Shanghai Institute for Pediatric Research, Shanghai, China

    Runxi Fu, Ze Xu, Yuling Zhao, Xiaowei Liu, Peilin Gu, Yang Wang & Wei Cai

  3. Shanghai Key Laboratory of Pediatric Gastroenterology and Nutrition, Shanghai, China

    Runxi Fu, Ze Xu, Yuling Zhao, Peilin Gu, Yang Wang & Wei Cai

  4. Department of Pediatric Neurosurgery, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Chen Wang

  5. Department of Gastroenterology, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Jinyang Zhai

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  1. Runxi Fu
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  2. Chen Wang
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  3. Ze Xu
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  11. Wei Cai
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Contributions

Conceptualization: R.F., Y.W., and W.C.; Funding acquisition: Y.W. and W.C.; Methodology: R.F., C.W., Z.X,. and J.Z.; Formal analysis: R.F., C.W., Z.X., J.Z., Y.Z,. and X.L.; Investigation: R.F. and C.W.; Data curation: R.F., Y.W., and W.C.; Resources: Y.Z., P.G., and W.P.; Writing—original draft: R.F. and Y.W.; Supervision: Y.W. and W.C.; Project administration: W.C. All authors have read and agreed to the published version of the manuscript.

Corresponding authors

Correspondence to Yang Wang or Wei Cai.

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Competing interests

The authors declare no competing interests.

Ethics

The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of Xinhua Hospital of Shanghai Jiao Tong University. The animal study protocol was approved by the Animal Care and Use Committees of Xinhua Hospital of Shanghai Jiao Tong University School of Medicine.

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Written informed consents were obtained from all patients.

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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

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