miR-190调控wnt信号通路在先天性肛门直肠畸形发生中作用机制的研究

先天性肛门直肠畸形(ARM)是小儿外科最常见的消化道畸形，严重影响儿童的生活质量。ARM是复杂的多基因疾病，发病机制不清。前期研究发现，wnt通路与ARM的发生密切相关，miR-190在ARM胎鼠后肠表达上调，软件预测wnt通路中Ppp3r1、Plcb1、Rock2基因是miR-190的作用靶点。本研究将进一步研究miR-190调控wnt信号通路在ARM发生中的作用。检测Ppp3r1、Plcb1、Rock2基因在ARM胎鼠后肠的时空表达；验证miR-190与Ppp3r1、Plcb1、Rock2的3’-UTR是否存在直接结合；双向调控细胞中miR-190的表达，检测Ppp3r1、Plcb1、Rock2基因表达；在后肠发育关键时期上调miR-190表达，检测胎鼠肛门直肠发育、ARM类型及靶基因表达，明确miR-190调控wnt信号通路在ARM发生中作用机制，为ARM的产前诊断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 up-regulation of miR-190 in ARM rat embryo. Software predicted Ppp3r1、Plcb1、Rock2 genes of wnt signaling pathway are target genes of miR-190. In this study we will further study the influence of miR-190’s regulation of wnt signal pathway on anorectal development. In this study, we will analyze the spatiotemporal distribution of Ppp3r1, Plcb1 and Rock2 genes in normal and ethylenethiourea (ETU)-induced ARM rat embryos by 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. By using luciferase reporter gene, demonstrate the direct bonding of miR-190 and 3’-UTR of Ppp3r1, Plcb1 and Rock2 genes. Up regulate and down regulate the expression of miR-190 in cells and analyze the expression of Ppp3r1、Plcb1、Rock2 genes. By using virus infection technology, inject virus plasmid via uterus of pregnant Wistar rats at the critical time-points in anorectal development to up-regulate the expression of miR-190 in offspring fetal rats. Anorectal development, ARM type and deformity rate, Ppp3r1, Plcb1 and Rock2 genes expression were detected in offspring fetal rats. The aim of this study is to clear the influence of miR-190’s regulation of wnt signal pathway on anorectal development and ARM and establish a foundation for genetic intervention, prevention and treatment of ARM.