绒山羊次级毛囊周期性发育的microRNA调控机理研究

The secondary follicles of cashmere goat are accurate renewable organs and show periodic changes. This kind of periodic changes control the growth and apoptosis of follicular cells. By elucidating the molecular regulation mechanisms of development and cycle of cashmere goat secondary follicles, it would provide theoretical basis for the realization of artificial regulation on cashmere growth, speed up the progress of genetic breeding of cashmere goats. All differentially expressed microRNAs and differentially expressed genes will be screened in anagen, catagen and telogen of cashmere goat secondary follicle using high-throughput sequencing approach. The integrative analysis of microRNA and mRNA expression is made to explore related microRNAs and their possible target genes during secondary follicle development and cycling. In order to confirm related microRNAs of the secondary follicle development, the microRNA expression levels at different development stages will be evaluated by real time PCR method. Moreover, the candidate target genes of microRNA will be verified with the luciferase reporter assay. The recombinant adenovirus over-expressing vectors will be constructed and identified. The dermal papilla cells of the secondary follicles will be transfected and cultured in vitro, to detect the impact of the microRNA on candidate target genes and signal pathway genes regulating hair follicle development and cycle. In summary, this project will reveal the important regulatory roles of microRNAs in cashmere goat secondary follicle development and cycle.

绒山羊次级毛囊是精确的可再生器官，呈周期性变化，控制着绒毛生长与脱落。阐明绒山羊次级毛囊周期性发育的分子调控机理，能够为实现人工调控绒毛生长、加快遗传育种进展奠定理论基础。本研究运用高通量测序技术，在筛选绒山羊次级毛囊生长期、退行期和休止期差异表达microRNA以及差异表达基因的基础上，通过microRNA与mRNA联合信息分析，寻找次级毛囊周期性发育相关的重要microRNA及其作用靶基因；运用实时定量PCR检测microRNA在次级毛囊不同发育时期表达量，确认次级毛囊发育相关microRNA；构建荧光素酶报告基因系统，验证次级毛囊发育相关microRNA的候选靶基因；构建microRNA过表达腺病毒载体，转染体外培养的绒山羊次级毛囊毛乳头细胞，探索microRNA与其靶基因以及毛囊周期性发育相关信号通路基因间的作用关系。揭示microRNA在绒山羊次级毛囊周期性发育中的重要调控作用。

绒山羊次级毛囊是精确的可再生器官，呈周期性变化，控制着绒毛生长与脱落。阐明绒山羊次级毛囊周期性发育的分子调控机理，能够为实现人工调控绒毛生长、加快遗传育种进展奠定理论基础。根据项目研究任务，主要研究内容包括：（1）通过绒山羊皮肤次级毛囊不同发育阶段（生长期、退行期、休止期）miRNA与mRNA测序数据的联合分析，鉴定到与次级毛囊周期性发育密切相关 的miRNA及其靶基因；（2）通过qPCR等实验室方法，对差异miRNA进行了实验室验证；选择3个miRNA(mir125-b、mir206、miR-144-3p)，构建荧光素酶报告基因系统，验证次级毛囊发育相关microRNA 的候选靶基因；（3）对mir125b构建过表达腺病毒载体Ad-pri-miR-125b，转染体外培养的绒山羊次级毛囊毛乳头细胞，从从中预测的靶基因中，发现对FGF5，TNF-α具有显著的调控作用，阐明了其与毛囊周期性发育相关的作用机制。此外，本项目还完成相关拓展性研究任务：（1）构建了绒山羊次级毛囊毛乳头细胞的体外培养体系体外培养体系，并对体系进行了优化。我们发现在毛乳头细胞培养液中β2、ITS和EGF的单独添加浓度分别为0.3 μg/mL、5.0 μg/mL 和7.0 μg/mL时，次级毛乳头的贴壁及迁出的时间和效率最佳；（2）鉴定到173生长期和退行期差异表达lncRNAs，72个差异表达miRNAs和3500个差异表达的转录本，并对生长期和休止期的lncRNA-miRNA-mRNA关系进行了的深度分析；（3）利用CRISPR/Cas9技术，创制了FGF5基因敲除的基因编辑绒山羊，用于 FGF5基因的功能研究。本项目的研究结果有助于揭示microRNA及mRNA和lncRNA在绒山羊皮肤次级毛囊周期性发育中的重要调控作用，尤其是诱发次级毛囊从生长期到退行期这一个过程的转变。