基于阿勒泰羊饥饿模型的circRNAs响应尾脂沉积与动员的分子机制及其circRNA-miRNA-mRNA网络调控环路研究

Fat-rumped trait is one of stress tolerance traits which ensure that Altay survive in adverse situations. However, the molecular mechanism of rapid deposition and mobilization of tail-fat remains unclear. Differentially expressed non-coding RNAs were screened by whole-exome sequencing in our previous studies, of them, some circRNAs were significantly correlated with the expression of miRNAs and mRNAs. According to above, we speculated that the regulatory pathways of circRNA-miRNA-mRNA probably responded to rapid fat deposition and mobilization. This project intends to set a starvation model which imitates rump-fat deposition and mobilizes in two extreme states, and select circRNAs, miRNAs and mRNAs which were differentially expressed in two extreme states, and then predict their target relationship and the regulatory networks by using bioinformatics methods. q-PCR, Northern blot and other methods were employed to detect the correlation of the expression of circRNA, miRNA and mRNA, and the reporter gene technology was used to verify the targeted among them accurately, and finally ove-rexpression and RNAi methods were applied to identify the biological function of the predicted circRNA-miRNA-mRNA in fat metabolism at the cellular level. The implementation of this project will help to elucidate the molecular mechanism of rapid fat deposition and mobilization of rump-fat in Altay sheep, and to lay a theoretical foundation for breeding low-fat Altay sheep in future.

臀脂性状是阿勒泰羊逆境生存的耐逆性状，其臀脂快速沉积与动员的分子机制仍未解析。课题组前期通过组学测序筛选出差异表达的非编码RNA,其中部分circRNA与miRNA及其靶标mRNA的表达存在显著相关性，由此我们推测circRNA-miRNA-mRNA的调控通路在响应阿勒泰羊尾脂快速沉积与动员中可能扮演着重要角色。基于此，本项目拟构建阿勒泰羊饥饿模型，并在此基础上筛选出两种极端状态下差异表达的circRNAs、miRNAs和mRNAs；预测三者之间的靶标关系以及相互之间调控网络；通过q-PCR、Northern Blot等方法检测三者之间的表达相关性，利用报告基因技术进行靶标调控的初步验证，最后应用过表达和干扰手段在细胞水平上验证所预测的circRNA-miRNA-mRNA在脂质代谢中的生物学功能。本项目的实施将有助于揭示阿勒泰羊尾脂快速沉积与动员的分子机制，为培育低绵羊新品种奠定理论基础。

绵羊脂尾（臀）性状是一种逆境生存所必需的生物性状。研究表明脂肪发育受众多功能基因、信号通路以及表观修饰组调控。本研究利用全转录组测序技术深度挖掘绵羊尾脂不同发育节点中基因与非编码RNA的表达特征，筛选出各节点尾脂中差异表达的mRNA、miRNA、circRNA、lncRNA并绘制出其相互间的互作网路图，从时间、空间上系统阐明以上差异表达的基因与非编码RNA调控脂肪发育的机制。高通量测序在阿勒泰羊尾脂组织发生中监测到29,300个mRNAs、1,167个miRNAs、14,309个circRNAs 和44,167个lncRNAs，其中新发现基因4,862个；miRNA152个。 差异表达的mRNA、miRNA、circRNA、lncRNA分别为3,297、255、190和1,369个。我们筛选到了ANXA2、PSAP、oar-miR-23a、oar-miR-191circZEB1-1、circFER、MSTRG.29942.1、MSTRG.30892.1等在 4 个节点差异表达且表达量排名前 30 的基因和表观调控子；差异mRNA的WGCNA分析显示，两时期中MEblack 模块的差异最显著（R2= 0.92， P = 2e-04），该模块包含了OLFML3、NCALD、CACNA1A等差异表达基因。GO和KEGG分析结果显示差异表达基因和表观调控子靶基因在诸多与脂肪沉积密切相关的GO词条中显著富集，涉及到lipopolysaccharide receptor activity、regulation of glucose transport、lipid droplet等途径。此外，互作网络图显示诸多mRNA与ncRNA参与脂肪沉积密切相关的PI3K-AKT signaling pathway、 MAPK signalingpathway和Ras signaling pathway等信号通路。最终锁定NC_019468.2:28918137 |29112121/NC_019477.2:27499541|27665107/NC_019478.2:48778852|48909994-oar-miR-432-TTN ceRNA等调控网络进行后续研究。以上研究工作为进一步阐明阿勒泰羊尾脂沉积的分子调控机制奠定基础。