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. 2022 Aug 5. doi: 10.1007/s10565-022-09750-0. Online ahead of print.

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.

Keywords: Anorectal malformations; Apoptosis; CircJag1; Circular RNA; Wnt/β-catenin pathway.