绒山羊circRNA参与调控毛乳头细胞诱导的次级毛囊干细胞向毛囊细胞分化的作用及分子机制

The differentiation of secondary hair follicle stem cell to hair follicle cell is significantly important in improving the cashmere yield and quality in cashmere goats, however, its regulatory mechanism is unknown. Circular RNA (circRNA) was found to regulate various biological processes in cells. In our previous study, 1219 differentially expressed circRNAs were identified in bulge of secondary hair follicle between anagen and telogen stages in cashmere goats by circRNA-seq technique. In the present study, we will predict the potential functions of the differentially expressed circRNAs, and construct circRNA-miRNA-mRNA network through combining with the mRNA-seq and miRNA-seq sequencing data of bulge obtained in our previous work, and further determine the critical circRNAs. We will construct overexpression and shRNA interference vectors of the circRNA and transfect into hair follicle stem cell. And then, the transfected hair follicle stem cells will be induced by dermal papilla cells to differentiate to hair follicle cells, in order to reveal the regulatory effects of circRNA on the differentiation of secondary hair follicle stem cell to hair follicle cell. Further, through circRNA pull-down, CHIRP-Seq/MS and methylation analyses, we will reveal the target molecules that interact with circRNA, and methylation regulation, in order to clarify the molecular regulatory mechanisms of circRNA on the differentiation of secondary hair follicle stem cell to hair follicle cell. The results from the present study will provide novel scientific evidences for further improving the cashmere yield and quality in cashmere goats.

绒山羊次级毛囊干细胞向毛囊细胞分化对提高山羊绒产量及品质有重要意义，但其调控机制不清楚。环状RNA(circRNA)被发现调控细胞多种生物过程。前期中我们从绒山羊次级毛囊生长期和休止期隆突部，经circRNA-seq筛选到1219个差异表达circRNAs。本项目对这些circRNAs进行功能预测，结合前期隆突部mRNA-seq和miRNA-seq数据，构建circRNA-miRNA-mRNA 网络，确定出关键circRNAs，构建其过表达和shRNA干扰载体，转染毛囊干细胞，并经毛乳头细胞诱导向毛囊细胞分化，揭示circRNA对毛囊干细胞向毛囊细胞分化的调控作用；进一步通过circRNA pull-down、ChIRP-seq/MS和甲基化分析，揭示与circRNA互作的靶分子及甲基化调控，阐明circRNA调控毛囊干细胞向毛囊细胞分化的分子机制，为提高山羊绒产量及品质提供新的科学依据。

①项目的背景：绒山羊次级毛囊干细胞向毛囊细胞分化对提高山羊绒产量及品质有重要意义。项目“绒山羊circRNA参与调控毛乳头细胞诱导的次级毛囊干细胞向毛囊细胞分化的作用及分子机制”（31872325），起止日期：2019年1月1日-2022年12月31。.②主要研究内容：从次级毛囊隆突部筛选差异表达circRNAs，构建circRNA-miRNA-mRNA网络，确定关键circRNAs，功能获得/缺失试验揭示circRNAs对毛囊干细胞向毛囊细胞分化的调控作用；RNA pull down等解析与circRNA结合靶分子阐明其分子调控机理。.③重要结果: 共筛选出circRNA-1926、circRNA-1967和circRNA-0100等17个候选circRNAs；功能获得/缺失试验发现circRNA-1926、circRNA-1967和circRNA-0100正向调控毛囊干细胞向毛囊细胞分化；circRNA-1926通过 miR-148a/b-3p/ CDK19轴，circRNA-1967吸附miR-93-3p以上调LEF1表达，circRNA-0100作为miR-153-3p的分子海绵上调KLF5表达,分别实现对毛囊干细胞向毛囊细胞分化的调控。.④关键数据：circRNA-1926, circRNA-1967, 和circRNA-0100等17个circRNA在生长期显著上调；circRNA-1926,circRNA-1967和circRNA-0100的功能试验表明均显著改变Keratin 7等标志基因表达。RNA Pull-down等分析表明与circRNA-1926,circRNA-1967和circRNA-0100互作的靶分子分别是miR-148a/b-3p，miR-93-3p和miR-153-3p；试验表明circRNA-1926,circRNA-1967和circRNA-0100均不是通过甲基化途径发挥作用，而是分别通过miR-148a/b-3p，miR-93-3p和miR-153-3p靶向结合CDK19,LEF1和KLF5基因的mRNA3’-UTR发挥作用。.⑤ 科学意义：研究结果为阐明绒山羊次级毛囊重建、发育和绒纤维形态发生及生长的分子机理提供新的科学依据。