novel_circ_008138/rno-miR-374-3p/SFRP4调控Wnt信号通路参与先天性肛门直肠畸形发生的分子机制研究

先天性肛门直肠畸形(ARM)是小儿外科最常见的消化道畸形，具体发病机制不清。本项目拟采用ARM大鼠胎鼠动物模型，检测novel_circ_008138、rno-miR-374-3p和SFRP4基因在ARM胎鼠后肠的时空表达；应用双荧光素酶报告实验，验证novel_circ_008138/rno-miR-374-3p/SFRP4之间的靶向关系；下调大鼠肠上皮细胞中novel_circ_008138的表达，检测rno-miR-374-3p和SFRP4表达以及细胞增殖、凋亡和EMT；利用CRISPR/Cas9技术，获得novel_circ_008138基因敲除胎鼠，检测胎鼠ARM类型和畸形率、后肠神经和肌肉发育情况。目的在于明确novel_circ_008138/rno-miR-374-3p/SFRP4调控Wnt信号通路影响肛门直肠发育的作用机制，为ARM的产前诊断、遗传干预和畸形防治奠定基础。

Anorectal malformations (ARMs) are among the most common congenital anomalies and adversely influence patient quality of life. As ARM is a multigenerational complex disease, its etiology, embryology, and pathogenesis are poorly understood and controversial. Our preliminary studies have suggested that the Wnt pathway was closely related to the occurrence of ARM. The expression of novel_circ_008138 was down-regulated in the hindgut of ARM fetal rats, whereas rno-mir-374-3p was up-regulated in the hindgut of ARM fetal rats, and the expression levels of them were negatively correlated. Software predicted rno-mir-374-3p was the target miRNA of novel_circ_008138, and one of the target mRNAs of rno-mir-374-3p was SFRP4. In this study, we will analyze the spatiotemporal distribution of novel_circ_008138, rno-miR-374-3p and SFRP4 genes in normal and ethylenethiourea (ETU)-induced ARM rat embryos by Fluorescence in situ hybridization, Immunohistochemistry, Real-time PCR and Western blot methods with emphasis on embryonic stages GD13 to GD16, which are the critical time-points in anorectal development. Dual-luciferase reporter assay system was used to verify targeting relationship between novel_circ_008138 and rno-miR-374-3p, rno-miR-374-3p and SFRP4. The expression of novel_circ_008138 was down regulated in rat intestinal epithelial cells and the expression of rno-miR-374-3p, SFRP4, and target genes in Wnt pathway was analyzed, as well as cell proliferation, apoptosis and epithelial-mesenchymal transition. By using CRISPR/Cas9 technology, novel_circ_008138 gene was knocked out in the intestinal tract at the critical time-points in anorectal development. After obtaining novel_circ_008138 gene conditional knockout fetal rats, ARM type and deformity rate, anorectal nerve and muscle development were detected in the fetal rats. The aim of this study is to clear the influence of novel_circ_008138/rno-miR-374-3p/SFRP4’s regulation of wnt signal pathway on anorectal development and establish a foundation for prenatal diagnosis, genetic intervention, prevention and treatment of ARM.