辽宁绒山羊绒相关LncRNA生物学功能和作用机制的研究

MT can promote the increase of secondary follicle quantity and concentrate and enlarge follicle groups, and then stimulate cashmere growth and increase cashmere yield. FGF5 have remarkable effect on follicle growth and clothing hair length during different period. Thus, further research on the molecular mechanism that MT and FGF5 affect follicle growth and development and cashmere quality has important theoretical and practical significance. In this project, transcriptome sequencing is implemented on the skin cells of Liaoning cashmere goat treated with MT and FGF5 respectively are sequenced by RNA-seq technology, Expression specificity and expressive sites of the target lncRNAs are studied using Real Time PCR, fluorescence in situ hybridization；after the overexpression or silence of the target lncRNAs by lentiviral vector, expressive change of the genes and proteins which are related to follicle development are conducted，and then we can further research biological function of the target lncRNAs；transcription regulation mechanism of the upstream and downstream areas of the target lncRNAs is studied by CHIRP-Seq and RNA Pull Down, RIP-Seq. The project revealed the mechanism that MT and FGF5 regulate follicle development in a whole new perspective, it will lay a strong theoretical foundation for molecular genetic breeding of Liaoning cashmere goat.

褪黑激素(MT)能促使次级毛囊数量增加并使毛囊群集中变大，进而刺激羊绒生长并增加产绒量。成纤维细胞因子5 (FGF5)对不同时期毛囊的生长及被毛的长度有显著的影响。因此，深入研究MT及FGF5对毛囊生长发育及绒毛品质影响的分子作用机制具有重要的理论和实践意义。本项目通过RNA-seq技术对分别经MT和FGF5处理的辽宁绒山羊皮肤细胞进行转录组测序；利用Real Time PCR、荧光原位杂交技术对目标lncRNA进行表达特异性及表达位点研究；用慢病毒载体使目标lncRNA过表达和干扰，通过毛囊发育相关基因和蛋白的表达变化，从而对目标lncRNA的生物学功能进行研究；利用CHIRP-Seq及RNA Pull Down、RIP-Seq技术对目标lncRNA上下游区域的转录调控机制进行研究。以全新的视角揭示MT和FGF5影响毛囊发育调控的作用机制，为辽宁绒山羊分子遗传育种奠定坚实的理论基础。

深入了解辽宁绒山羊产绒机理和调控机制对培育高产优质辽宁绒山羊品种具有重要科学意义。本项目对不同浓度褪黑素（MT）/成纤维细胞生长因子5（FGF5）处理的皮肤成纤维细胞进行转录组测序，筛选出一条MT介导的LncRNA：LncRNA MTC和FGF5调控绒毛生长相关的LncRNAs：XLOC_011424、XLOC_011157。通路抑制剂分析表明LncRNA MTC通过NF-κB信号通路促进皮肤成纤维细胞增殖；LncRNA MTC过表达组中TNF-α、NF-κB基因的表达量显著上调，而IκBα基因的表达量显著下调；WB结果显示LncRNA MTC过表达组中TNF-α、NF-κB-P-p65蛋白表达量明显上调；蛋白质核质分离后，检测到细胞核内NF-κB-p65表达上调，胞浆中IκBα表达下调、P-IκBα表达上调。FISH结果显示LncRNA MTC和p65蛋白在细胞内共定位；RNApull-down结果表明LncRNAMTC可能通过与靶基因（TNFα、TNFAIP3(A20）、NFKBIA（IкBα）、NFKB2、IL8）、p65蛋白结合而参与NF-ΚB通路对皮肤细胞生长的调节。过表达LncRNA MTC前后iTRAQ蛋白质组学筛选出74个差异表达的蛋白，细胞衰老相关的蛋白在LncRNAMTC过表达条件显著下调；结合生物信息学分析筛选出靶蛋白半乳糖凝集素-3(galectin-3)通过RIP证明与LncRNA MTC确实结合。接下来利用Co-IP鉴定到天然状态下与galectin-3结合的蛋白有15个，包括4个角蛋白、细胞增殖正调控的TenascincC蛋白、参与毛囊调控的Piezo1和Gal-3BP蛋白，说明LncRNA MTC主要通过结合靶蛋白调控皮肤成纤维细胞的增殖来影响绒毛生长发育。项目为辽宁绒山羊分子遗传育种奠定坚实的理论基础，为其他毛皮动物产绒的分子机理研究提供参照。