绒山羊次级毛囊lncRNA参与调控毛乳头细胞诱导功能的作用及分子机理研究

The inducing function of dermal papilla cell of secondary hair follicle is significantly important in improving the cashmere yield and quality in cashmere goat, however, its regulatory mechanism is unknown. Long chain noncoding RNA (lncRNA) was found to be widely involved in various physiological processes in cells. In our previous study, 1049 differentially expressed lncRNAs were identified in dermal papilla between anagen and telogen stages in Liaoning cashmere goat by RNA-seq technique. In present study, we will predict the potential function of the differentially expressed lncRNA, and construct lncRNA-miRNA-mRNA regulatory network through combining with mRNA and miRNA high-throughput sequencing data obtained in our provious work, and further determine the critical lncRNA which might regulate the inducing function of demal papilla cell. Through constructing overexpression and shRNA interference vectors of lncRNA and transfecting to dermal papilla cell, we will reveal the regulation effects of lncRNA on the inducing function of dermal papilla cell. Further by RNA pull down, CHIRP-Seq and methylation detection methods, we will reveal the target molecules (DNA, RNA or protein) and methylation regulation in order to clarify the melocular regulatory mechanisms of lncRNA on inducing function of dermal papilla cell of secondary hair follicle in cashmere goat. The results from the present study will provide a novel scientific evidence to further improve the cashmere yield and quality in cashmere goat.

绒山羊次级毛囊毛乳头细胞诱导功能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。长链非编码RNA（lncRNA）被发现广泛参与细胞多种生理过程。前期研究中，我们从辽宁绒山羊次级毛囊生长期和休止期毛乳头中，通过RNA-seq筛选到1049个差异表达的lncRNA。本项目对这些lncRNA进行功能预测，结合前期mRNA和miRNA高通量测序数据，构建lncRNA-miRNA-mRNA 调控网络，确定出调控毛乳头细胞诱导功能的关键lncRNA，构建过表达和shRNA干扰载体，转染毛乳头细胞，揭示lncRNA 对毛乳头细胞诱导功能的调控作用；进一步通过RNA pull down、CHIRP-Seq 和甲基化检测等，分析与lncRNA结合的靶分子（DNA、RNA或蛋白质）及甲基化调控，阐明lncRNA调控绒山羊次级毛囊毛乳头细胞诱导功能的分子机理，为提高山羊绒产量及改善绒品质提供新的科学依据。

① 项目的背景：绒山羊次级毛囊毛乳头细胞诱导功能对山羊绒产量及品质有重要意义。项目“绒山羊次级毛囊lncRNA参与调控毛乳头细胞诱导功能的作用及分子机理研究”（31672388），起止日期：2017年1月1日-2020年12月31。.② 主要研究内容：从次级毛囊筛选差异表达lncRNAs，构建lncRNA-miRNA-mRNA网络，确定关键lncRNAs，功能获得/缺失试验揭示lncRNAs对毛乳头细胞诱导功能的调控作用；RNA pull down等解析与lncRNA结合靶分子阐明其分子调控机理。.③ 重要结果：共筛选出lncRNA-599547、lncRNA-599554、lncRNA-000133、lncRNA-H19和lncRNA-HOTAIR等22个候选lncRNAs；功能获得/缺失试验发现lncRNA-599547、lncRNA-599554和lncRNA-000133正向调控毛乳头细胞诱导功能；lncRNA-599547通过miR-15b-5p/Wnt10b 轴，lncRNA-599554吸附miR-15a-5p，上调Wnt3a表达实现其对毛乳头细胞诱导功能的调控。.④ 关键数据：lncRNA-599547、lncRNA-599554、lncRNA-000133、lncRNA-H19和lncRNA-HOTAIR等5个 lncRNA在生长期显著上调；lncRNA-H19、lncRNA-HOTAIR和lncRNA-000133启动子甲基化参与其转录。lncRNA-000133、lncRNA-599547和lncRNA-599554的功能获得/缺失试验表明均显著改变ALP等标志基因表达。RNA Pull-down 等技术分析表明与lncRNA-599547和lncRNA-599554互作的靶分子分别是miR-15b-5p和miR-15a-5p；甲基化和双荧光素酶报告试验表明lncRNA-599547和lncRNA-599554不是通过甲基化途径发挥作用，而是通过miR-15b-5p和miR-15a-5p分别靶向结合毛乳头细胞诱导功能关键基因Wnt10b和Wnt3a的mRNA 3’-UTR发挥作用。.⑤ 科学意义：项目研究结果为阐明绒山羊次级毛囊发育和生绒的分子调控机理提供新线索，也为人工调节山羊绒生长、提高山羊绒产量及改善绒品质提供新的思路和科学依据。