miR‐141‐3p affects β‐catenin signaling and apoptosis by targeting Ubtd2 in rats with anorectal malformations

Abstract 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 β‐catenin was affected via Ubtd2, and USP5 could bind to both Ubtd2 and β‐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 β‐catenin expression. This may cause aberrant apoptosis during hindgut development and mediate the imbalance of β‐catenin signaling in the cloaca, further affecting the occurrence of ARMs.