环状RNA及Wnt3a在肛门直肠畸形发生发展中的作用及机制的研究

Anorectal malformations(ARMs) are one of the most common congenital anomalies in the newborn. The gene regulation mechanism associated with ARMs is complicated. On the basis of our previous research, the Wnt is one of the main pathways in ARMs .Wnt3a is an important signal gene in classical pathway and the more detailed regulatory mechanisms of Wnt3a in ARMs are still not clear. We have confirmed much circRNAs and mir-128-1/mir-216a are closely related with Wnt3a by forecast using Targetscan and Starbasev2.0 software.. The design of our study is to use ethylenethiourea (ETU)-exposed rat embryos with ARMs，enteric neural stem cell and the virus vectors of Wnt3a’s 3’UTR with immune luciferase. The effect and regulation mechanism of circRNAs will be confirmed by using real-time PCR (RT-PCR), western blot and trans- planceta interference of siRNAs to test the relationship between mir-128-1/mir-216a、circRNAs and ARMs and their regulation mechanism. . This study will give a new point of view to learn the mechanism of circRNAs and Wnt3a in ARMs and more comprehensive understanding the pathogenesis of circRNAs and Wnt3a in the ARMs.This will concentrate to guide the ARMs’ clinical treatments.

先天性肛门直肠畸形(anorectal malformation ARMs)的基因表达调控机制复杂，涉及多种信号通路。课题组前期工作已证实Wnt通路为调控ARMs发生的主要途径之一 ，同时证实Wnt3a与ARMs的发生及进一步的病理改变密切相关，经生物学预测分析在其表达过程中涉及到mir-128-1/mir-216a及多种环状RNA（circRNAs），但其具体的调控机制仍然未知。本课题拟利用乙烯硫脲致畸的ARMs大鼠模型、肠神经干细胞系，Wnt3a的3’UTR荧光素酶表达载体，利用RT-PCR、Western-blot、激光捕获以及跨胎盘siRNAs干扰等技术，验证mir-128-1/mir-216a及多种circRNAs与Wnt3a之间的相互调控作用及对ARMs发生的调控机制，为ARMs的发病调控机制绘制出更全面、详细的网络框架及开拓疾病新的治疗思路。

先天性肛门直肠畸形(anorectal malformation ARMs)是小儿外科最常见的消化道畸形，由于胚胎发育期后肠发育障碍所致，其病理改变复杂，是一种复杂的多基因疾病，关于其遗传机制的研究众多，但其深入的调控机制仍未明确，Wnt通路作为调控ARMs发生的主要途径之一，Wnt通路的上游信号调控机制对ARMs发病对其进行早期诊断及干预具有重大的社会意义。本课题主要通过乙烯硫脲致畸的ARMs大鼠模型于畸形的发生/发育过程（E14-E21）、肠神经干细胞，肠上皮IECs细胞系，利用qRT-PCR、Western-blot、双荧光素酶实验、质粒转染、siRNAs干扰、细胞增殖及凋亡等实验证实经全基因组测序结果筛选出差异表达的circRNAs：rno_circ_0006880，rno_circ_0011386表达上调, rno_circ_0000436, rno_circ_0004002, rno_circ_0005139, rno_circ_0009285, rno_circ_0014367表达下调，miR-128-3p/ miR-324-3p/ miR-342-5p在ARMs E17-E21表达上调及Wnt信号通路中Wnt3a/ Wnt5a/ Wnt2b/ Wnt10b在ARMs发生发展过程中（E14-E21）表达量为下调，通过双荧光素酶实验证实rno_circ_0004002-miR-342-5p-Wnt3a之间以及rno_circ_0005139- miR-324-3p-Wnt5a存在结合位点以及 rno_circ_0004002、rno_circ_0005139、miR-324-3p、miR-342-5p、Wnt3a及Wnt5a均参与ARMs中增殖及凋亡功能的调控，并通过细胞系转染rno_circ_0004002/rno_circ_0005139的细胞质粒及干扰siRNA后发现miR-342-5p、miR-324-3p、Wnt3a及Wnt5a表达量改变，并且增殖及凋亡功能发生改变。与此同时根据lncRNA测序结果，我们发现与 Wnt5a相关的lncRNA AARB07048878.1在ARMs中表达下调，并对其干扰及过表达后Wnt5a表达及细胞增殖、凋亡功能均受到影响。为ARMs的发病调控机制绘制出更全面、详细的网络框架及开拓疾病新的治疗