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

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.

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

1、ARM大鼠后肠组织RT-qPCR最佳内参基因研究。选取15个候选参考基因，RT-qPCR检测其mRNA表达。使用geNorm、NormFinder、比较ΔCt和BestKeeper方法评估表达的稳定性和可变性。结果显示，候选基因的RT-qPCR循环阈值（Ct）范围在14.07（Rplp1）和21.89（Sdha）之间，4种方法综合分析后得出 RT-qPCR最稳定的参考基因为Rpl13a、Ywhaz和Rps18。.2、PPPDE1在正常和ARM大鼠后肠发育中的时空表达、增殖和凋亡研究。IHC、RT-qPCR、Western blotting检测PPPDE1、MYC和p53的表达；免疫荧光双染检测PPPDE1与Bax、Cyt-c、TUNEL的共定位；CCK-8、流式凋亡检测细胞功能。结果显示，PPPDE1、P53在ARM组高表达，而MYC表达明显降低。PPPDE1、TUNEL阳性细胞和Bax、 Cyt-c阳性细胞存在共定位。过表达PPPDE1后，细胞增殖减低、凋亡增加。.3、正常和ARM大鼠后肠发育中环状RNA的高通量测序结果分析。构建环状circRNA-miRNA-mRNA调控网络，RT-qPCR进行验证，琼脂糖凝胶电泳、Sanger克隆测序进行环状位点验证。结果显示共鉴定出18295个circRNA，结合miREAP、miRanda和TargetScan预测共有55个circRNA能够与195个miRNA和947个mRNA结合。GO和KEGG分析表明，大量的差异表达circRNA与发育相关。.4、CircJag1/miR-137-3p/Sox9调控Wnt/β-catenin信号通路在大鼠ARM发生中作用机制研究。双荧光素酶实验证明靶向结合位点。使用原位杂交、CCK-8、EdU、流式和TUNEL法检测细胞的表达和功能。结果显示，circJag1在ARM组在后肠组织中表达上调。circJag1/miR-137-3p/Sox9之间存在直接靶向关系。通过一系列功能实验，证明了circJag1作为miR-137-3p的分子海绵，使Sox9表达上调，引起核内β-catenin、cyclin D1和c-myc的表达下调，促进了细胞凋亡、抑制增殖。免疫荧光显示circJag1使核内β-catenin降解，Wnt通路传导受抑制，可能是引起ARM发生的重要原因。