Spatial transcriptomics reveals gene interactions and signaling pathway dynamics in rat embryos with anorectal malformation.

Anorectal malformation (ARM) is a prevalent early pregnancy digestive tract anomaly. The intricate anatomy of the embryonic cloaca region makes it challenging for traditional high-throughput sequencing methods to capture location-specific information. Spatial transcriptomics was used to sequence libraries of frozen sections from embryonic rats at gestational days (GD) 14 to 16, covering both normal and ARM cases. Bioinformatics analyses and predictions were performed using methods such as WGCNA, GSEA, and PROGENy. Immunofluorescence staining was used to verify gene expression levels. Gene expression data was obtained with anatomical annotations of clusters, focusing on the cloaca region's location-specific traits. WGCNA revealed gene modules linked to normal and ARM cloacal anatomy development, with cooperation between modules on GD14 and GD15. Differential gene expression profiles and functional enrichment were presented. Notably, protein levels of Pcsk9, Hmgb2, and Sod1 were found to be downregulated in the GD15 ARM hindgut. The PROGENy algorithm predicted the activity and interplay of common signaling pathways in embryonic sections, highlighting their synergistic and complementary effects. A competing endogenous RNA (ceRNA) regulatory network was constructed from whole transcriptome data. Spatial transcriptomics provided location-specific cloaca region gene expression. Diverse bioinformatics analyses deepened our understanding of ARM's molecular interactions, guiding future research and providing insights into gene regulation in ARM development.

Rack1-mediated ferroptosis affects hindgut development in rats with anorectal malformations: Spatial transcriptome insights.

Anorectal malformation (ARM), a common congenital anomaly of the digestive tract, is a result of insufficient elongation of the urorectal septum. The cytoplasmic protein Receptor of Activated C-Kinase 1 (Rack1) is involved in embryonic neural development; however, its role in embryonic digestive tract development and ARM formation is unexplored. Our study explored the hindgut development and cell death mechanisms in ARM-affected rats using spatial transcriptome analysis. We induced ARM in rats by administering ethylenethiourea via gavage on gestational day (GD) 10. On GDs 14-16, embryos from both normal and ARM groups underwent spatial transcriptome sequencing, which identified key genes and signalling pathways. Rack1 exhibited significant interactions among differentially expressed genes on GDs 15 and 16. Reduced Rack1 expression in the ARM-affected hindgut, verified by Rack1 silencing in intestinal epithelial cells, led to increased P38 phosphorylation and activation of the MAPK signalling pathway. The suppression of this pathway downregulated Nqo1 and Gpx4 expression, resulting in elevated intracellular levels of ferrous ions, reactive oxygen species (ROS) and lipid peroxides. Downregulation of Gpx4 expression in the ARM hindgut, coupled with Rack1 co-localisation and consistent mitochondrial morphology, indicated ferroptosis. In summary, Rack1, acting as a hub gene, modulates ferrous ions, lipid peroxides, and ROS via the P38-MAPK/Nqo1/Gpx4 axis. This modulation induces ferroptosis in intestinal epithelial cells, potentially influencing hindgut development during ARM onset.

CircJag1 promotes apoptosis of ethylene thiourea-exposed anorectal malformations through sponging miR-137-3p by regulating Sox9 and suppressing Wnt/ß-catenin pathway during the hindgut development of rat embryos.

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 ß-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/ß-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.

miR-141-3p affects ß-catenin signaling and apoptosis by targeting Ubtd2 in rats with anorectal malformations.

Anorectal malformations (ARMs) are the most common gastrointestinal malformations. miR-141-3p was obtained from whole-transcriptome sequencing, and Ub domain-containing protein 2 (Ubtd2) was predicted as the target gene. An ARM rat model was induced using ethylenethiourea. Fluorescence in situ hybridization and immunofluorescence were used to detect the spatiotemporal expression of miR-141-3p and Ubtd2, respectively. A dual-luciferase reporter assay confirmed their targeting relationship, and cell proliferation and apoptosis were investigated after transfection in the intestinal epithelium (IEC-6). Additionally, western blotting and co-immunoprecipitation were used to examine the protein levels and the endogenous binding relationship. miR-141-3p was downregulated in the ARM group, whereas Ubtd2 increased and colocalized with TUNEL-positive cells. After miR-141-3p inhibition, protein expression of USP5 and ß-catenin was affected via Ubtd2, and USP5 could bind to both Ubtd2 and ß-catenin. Flow cytometry analysis and caspase 3/7 staining demonstrated that downregulated miR-141-3p promoted cell apoptosis through Ubtd2. In summary, targeting Ubtd2 decreased in miR-141-3p and promoted apoptosis of intestinal epithelium and regulated ß-catenin expression. This may cause aberrant apoptosis during hindgut development and mediate the imbalance of ß-catenin signaling in the cloaca, further affecting the occurrence of ARMs.