后肠
细胞凋亡
流式细胞术
连环素
生物
污渍
细胞生物学
转染
分子生物学
标记法
报告基因
信号转导
癌症研究
Wnt信号通路
细胞培养
基因
基因表达
遗传学
植物
幼虫
中肠
作者
Chen Yi Wang,Si Ying Li,Yun Xiao,Zhen Lin,Xiao Gao Wei,Xiao Tang,Zheng Wei Yuan,Yu Bai
摘要
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.
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