绒山羊生后毛囊周期中let-7基因家族的表达和作用机制研究

Cashmere goat is a high quality fiber-producing animal of commercial importance. The guard hair and down hair locate in similar skin niches are different in both growth and cycling. microRNAs regulate temporal transition in gene expression associated with cell fate progression and differentiation throughout animal development. It was recently recognized that let-7 expressed differentially in postnatal cashmere goat skin. Genetic analysis of developmental timing identified let-7 which performing analogous developmental timing function in many animal species. As core components of the heterochronic pathway, let-7 act in genetic switches that regulate progression through stage-specific developmental events. Investigating the let-7 microRNA family expression and function in postnatal hair follicle cycling of cashmere goat will help to orchestrate developmental events involve in tissue homeostasis roles important for hair follicle. To understand how let-7 are temporally regulated in particular cell lineages, and how they engage specific targets in specific cell types in the context of developmental progression, much work needs to be done, employing quantitative real time PCR, in situ hybridization, laser capture microdissection, two-dimensional gel electrophoresis-Mass spectrometry, RNA-seq, MicroRNA Target Finger Print (MTFP) pipeline and RNAi strategies. The project will postulate gene regulation network of let-7 family and their target genes in postnatal hair follicle and benefit the hair follicle research and molecular breeding of cashmere goat in the near future. As a handy model for mammal hair follicle, cashmere goat hair follicle offers a powerful system for employing genetic screens to identify cofactors that regulate let-7 family and their targets activity.

绒山羊是生产高档纺织原料的绒毛用家畜，其被毛外层的粗毛和内层的绒毛共生于相似的皮肤微环境，但在周期生长方面存在着差异。微小RNA 通过不同的时序表达在动物发育过程中调节细胞的增生与分化。最近研究表明，let-7基因家族在出生后不同时期的绒山羊皮肤上有差异表达。遗传分析发育时序发现let-7基因家族在不同物种中具有类似的功能。作为差异时序调控途径的核心成员，let-7是调节特异发育阶段转换的遗传开关。研究let-7基因家族在绒山羊生后毛囊周期中的表达和作用机制将有助于理解毛囊发育中的分子事件及其协调作用方式。本课题采用实时定量PCR、原位杂交、激光显微捕获、二维蛋白质电泳和质谱、RNA-Seq、微小RNA靶基因指纹图谱和RNAi等技术研究let-7基因家族在时序性调控细胞谱系发育中的作用。该课题构建的绒山羊皮肤毛囊let-7基因家族及其靶基因调节网络，将促进毛囊生物学研究和绒山羊分子育种。

本研究利用皮肤、初级毛囊和次级毛囊三种不同组织类型,分别比较了不同生长发育阶段和不同试验处理方式下毛囊相关miRNA和mRNA的表达模式，系统分析了let-7及其靶基因在山羊皮肤和毛囊中的作用机制。结果表明，let-7的11个家族成员在生绒季节和非生绒季节具有两类不同的网络调控模块，即相对恒定的调控模式和相对变动的调控模式关系。（Let-7a-5p）-（let-7b）-（let-7c）和（miR-98）-（let-7i）-（let-7g）分别形成稳定的“静三角”关系，在不同月份、不同处理模式下均保持相对恒定。而（let-7a-5p）-（let-7b）-（let-7f）和（let-7a-5p）-（let-7b）-（let-7d）分别形成可变的“动三角”关系，在不同月份和不同处理下表现为动态变化。此外，let-7a-3p和miR-3596则表现为明显的“游离”，在整个毛囊周期中均表现出相对独立的作用特点。值得指出的是，这些家族成员中miR-3596在次级毛囊中表现为“组成型”表达，其表达水平明显高于其他家族成员。Let-7家族成员内部关系的紧密程度显著与毛囊周期有关，并且其靶基因也形成了以“静三角”为轴心的基因调控网络关系。Let-7基因家族成员表达的整体上调与生绒有关，而且其内部成员集聚程度与生绒和褪黑激素埋植明显相关。Let-7家族与绒毛生长分子机理的复杂性决定了以let-7家族成员为核心开展各种生理性状之间的关联，特别是将特定组织特异表达的靶基因进行网络构建具有重要的理论价值和科学意义。