Abstract
Anorectal malformations (ARMs) are common birth defects involving congenital structural anomalies of the gastrointestinal tract. As an important component of non-coding RNAs, circular RNAs (circRNAs) widely participate in the digestive system development; however, the specific molecular mechanism of their involvement in ARM occurrence remains obscure. Herein, we generated rat models of ARMs induced by ethylene thiourea. A novel circRNA (circJag1) was screened and identified by RNA-Seq, which is remarkably upregulated in hindgut tissues of ARM rat embryos. In vivo experiments, colocation analysis via fluorescence in situ hybridization, and immunofluorescence further demonstrated that the disordered circJag1/miR-137-3p/Sox9 expression caused a spatiotemporal imbalance in the urorectal septum (URS) of ARMs. In vitro, functional assays confirmed that circJag1 upregulation resulted in the degradation of nuclear β-catenin, C-myc, and Cyclin D1 in rat intestinal epithelial cells, as well as the promotion of apoptosis and suppression of cell proliferation. Mechanistically, dual-luciferase reporter assay and RNA immunoprecipitation assay indicated that circJag1 acted as a miR-137-3p sponge, thereby inhibiting its repressive effect on its target Sox9. Further experiments showed that a loss of Sox9 abolished the circJag1-mediated increase in apoptosis. In conclusion, aberrantly high circJag1 expression promotes epithelial apoptosis by suppressing the canonical Wnt/β-catenin pathway via the miR-137-3p/Sox9 axis, which leads to fusion failure of the URS and cloacal membrane, and eventually contributed to ARMs. Our achievements might boost the comprehension of ARM pathogenesis and could provide a novel candidate target for the development of therapies for ARMs to complement surgical treatment.
Graphical abstract
Highlights
• CircJag1 was remarkably upregulated in ARM hindgut tissues during rat embryo development.
• CircJag1 acts as a competing endogenous RNA to sequester miR-137-3p, resulting in activation of miR-137-3p-target Sox9, and then promote degradation of β-catenin and turn off Wnt pathway.
• Abnormal expression of circjag1 may result in fusion failure of URS and CM by reducing epithelial proliferation and promoting apoptosis.
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Data availability
Data available on request from the authors. The original data of RNA-Seq are available in the Gene Expression Omnibus database (GSE159306).
Code availability
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Funding
The present work was supported by the National Natural Science Foundation of China (grant number 81770511, 82070530 and 82170530), the Liaoning Revitalization Talents Program (grant number XLYC1908008), and the Outstanding Scientific Fund of Shengjing Hospital (grant number ME56).
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S. Y. L. and Y. Z. B. designed the experiments. S. Y. L. performed the experiments and wrote the manuscript. C. Y. W. and X. G. W. analyzed the data. X. B. T and Y. Z. B. performed development of review and revision of the paper. Z. W. Y. provided technical and material support.
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Supplementary Table 1
The sequences of the primers pairs. (DOCX 16 kb)
Supplementary Fig. 1
A The knockdown efficiency of three circJag1 siRNAs. B The knockdown efficiency of three Sox9 siRNAs. C A cytofluorogram (a 2D histogram of the two channels) of Fig. 3C. D Dual luciferase reporter experiment of circJag1 and miR-873-5p. **P < 0.01. (PNG 351 kb)
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Li, S.Y., Wang, C.Y., Wei, X.G. et al. CircJag1 promotes apoptosis of ethylene thiourea–exposed anorectal malformations through sponging miR-137-3p by regulating Sox9 and suppressing Wnt/β-catenin pathway during the hindgut development of rat embryos. Cell Biol Toxicol 39, 1593–1610 (2023). https://doi.org/10.1007/s10565-022-09750-0
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DOI: https://doi.org/10.1007/s10565-022-09750-0