中国美利奴（军垦型）细毛羊毛囊发育关键circRNA的筛选及功能初探

Hair follicle in sheep is the most complex and unique micro-organ that is characterized by a constant growth in the whole lifecycle. It is reported that the development of hair follicle is regulated by multiple functional genes, signaling pathways, as well as non-coding RNAs. The latest study shows that circRNAs play important roles in regulating the development of multiple tissues, however, the roles of circRNAs regulating the hair follicle development in sheep is not reported. Accordingly, this project intends to study the function of circRNAs in hair follicle tissue development by using deep high-throughput sequencing, using target skin samples from Chinese Merino sheep (Junken type) in three key development stages (preparation period, primary follicles stage and secondary follicle stage); use the bioinformatics techniques, q-PCR assay and northern blot to detect circRNAs expressions in hair follicle tissue, and identify specific or highly expressed circRNAs; integrate dataset of transcriptome and small RNAs sequencing to make the network-based pathway analysis, and select these circRNAs with potential regulatory roles to verify their biological functions at cellular level. The research is to identify these circRNAs that have regulatory effects on hair follicle development, and provide scientific data for revealing the molecular mechanism of hair follicle development.

毛囊是绵羊最为复杂独特、终生周期性生长的微型器官。研究表明毛囊发育受众多功能基因、信号通路以及非编码RNA调控。最新研究表明，circRNAs在众多组织的发育中均扮演着重要调控角色，但迄今为止，未见相关调控绵羊毛囊发育的circRNA文献报道。基于此，本项目以中国美利奴羊毛囊发育的准备期、初级毛囊盛期、次级毛囊盛期三个关键发育时期的胎儿皮肤组织为样本，采用circRNA高通量测序的方法对以上三个关键时期皮肤组织进行深度测序；采用生物信息、q-PCR、Northernblot的方法鉴定出毛囊组织中表达的circRNA，并鉴定出毛囊组织中特异或高表达的circRNA；结合毛囊组织转录组和小RNA测序数据进行网络化调控通路分析，挑选若干潜在调控毛囊发育的circRNA在细胞水平进行功能验证。本研究将鉴定出对毛囊发育具有调控作用的circRNAs，将为揭秘毛囊发育的分子机制提供科学数据。

羊毛是绵羊毛囊细胞增殖和分化的产物，毛囊是一种呈周期性生长的微小器官。研究表明毛囊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毛囊不同发育节点中基因与非编码RNA的表达特征，筛选出各节点毛囊中差异表达的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调控毛囊发育的机制。毛皮组织的切片及染色结果显示胚胎期90天及120天是中国美利奴细毛羊初级毛囊和次级毛囊发育的关键时间节点，初级毛囊和次级毛囊分别在这两个时期数量激增。高通量测序在毛囊发生中监测到28,514个mRNAs、1,579个miRNAs、18,281个circRNAs 和17,547个lncRNAs，其中新发现基因8,355个；miRNA146个。 差异表达的mRNA、miRNA、circRNA、lncRNA分别为8,263、365、975和957个。差异基因及非编码RNA呈现明显的时间节点差异性，在E50和成年皮肤组织中差异表达的mRNA、miRNA和lncRNA数目最多，分别为6116个，327个和548个。circRNAs则展现出在初、次级毛囊盛期差异最为活跃，胚胎90 与120天毛皮组织中差异的circRNA数目多达674个。GO和KEGG分析结果显示circRNA与lncRNA靶标在诸多与毛囊与皮肤发育密切相关的GO词条中显著富集，涉及到ECM−receptor，PDA，FA等途径。此外，互作网络图显示诸多miRNA与mRNA参与毛囊发育密切相关的MAPK、Notch和Wnt等信号通路。最终锁定circTGFβR3-bta-miR-205-KRT10、MSTRG.309130.11-bta-miR129-5p-TXNIP和MSTRG.158776.4-bta-miR129-5p-KRT5 ceRNA等调控网络进行后续研究。以上研究工作为进一步阐明中国美利奴细毛羊毛囊发育的分子调控机制奠定基础。