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

中文翻译：

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CircJag1在大鼠胚胎后肠发育过程中通过调节Sox9和抑制Wnt/β-catenin通路，通过海绵miR-137-3p促进亚乙基硫脲暴露的肛门直肠畸形细胞凋亡

肛门直肠畸形（ARM）是常见的出生缺陷，涉及胃肠道先天性结构异常。环状RNA（circRNA）作为非编码RNA的重要组成部分，广泛参与消化系统的发育；然而，它们参与ARM发生的具体分子机制仍不清楚。在此，我们建立了由亚乙基硫脲诱导的 ARM 大鼠模型。通过RNA-Seq筛选并鉴定出一种新的circRNA（circJag1），它在ARM大鼠胚胎的后肠组织中显着上调。体内实验中，通过荧光原位杂交和免疫荧光进行的共定位分析进一步证明，circJag1/miR-137-3p/Sox9表达紊乱导致ARM的尿直肠隔膜（URS）时空失衡。体外功能测定证实，circJag1上调导致大鼠肠上皮细胞核β-catenin、C-myc和Cyclin D1降解，并促进细胞凋亡和抑制细胞增殖。从机制上讲，双荧光素酶报告基因测定和RNA免疫沉淀测定表明，circJag1充当miR-137-3p海绵，从而抑制其对其靶标Sox9的抑制作用。进一步的实验表明，Sox9 的缺失消除了 circJag1 介导的细胞凋亡增加。总之，异常高表达的 circJag1 通过抑制 miR-137-3p/Sox9 轴的经典 Wnt/β-catenin 通路来促进上皮细胞凋亡，从而导致 URS 和泄殖腔膜的融合失败，并最终导致 ARM。我们的成就可能会增进对 ARM 发病机制的理解，并可能为开发 ARM 疗法以补充手术治疗提供新的候选靶点。

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