褪黑激素调控绒山羊次级毛囊周期生长中关键lncRNAs的筛选及其分子机制

Long non-coding RNAs (lncRNAs) are non-protein-coding RNA with longer than 200 nucleotides that were widely existed in mammalian cells. It could be involved in biological processes in many ways, including transcriptional regulation and epigenetic regulation. Cashmere is a fine undercoat produced by the secondary hair follicle of the skin, which has important economic value. Previous studies from applicants found that exogenous melatonin could regulate the growth of secondary follicles, produce cashmere in telogen period and alter the expression of some genes and miRNA. However, it is unclear if the exogenous melatonin had an effect on lncRNAs..Some lncRNAs associated with hair follicle development were found to exist at skin tissue in the previous studies. Nevertheless, the regulation function of lncRNAs on the periodic growth of hair follicle is still vague. Therefore, on the basis of RNA-seq in the hair follicle，this study took the Inner Mongolia Cashmere goats as the research object and used melatonin as the mediating factor to screen some differentially expressed lncRNAs and target genes between vegetal and telogen. Additionally, the cell growth was detected through regulating expression of lncRNA in dermal papilla. Using qRT-PCR, immunohistochemistry, Western blot, RNA-RIP and luciferase technology to explore，the molecular mechanism of lncRNAs in the regulation of secondary hair follicle cycle by melatonin was explored. All of these will lay the foundation for Cashmere goats breeding.

lncRNAs是广泛存在于哺乳动物细胞中的一类蛋白非编码序列，长度超过200nt，可在多层面参与转录后调控和表观调控等生物学过程。羊绒是由皮肤次级毛囊产生的纤细底层毛，具有重要的经济价值。申请人在前期的研究中发现：外源褪黑激素可调控次级毛囊周期生长，诱发二次生绒，并改变部分基因和miRNA 的表达，是否对lncRNAs有影响未做研究。.据报道，在绒毛皮肤组织中存在一些lncRNAs与毛囊发育相关基因关联，但其对绒毛周期生长调控机理尚不清楚。本项目以内蒙古绒山羊为研究对象，褪黑激素为介导因子，拟在毛囊转录组测序的基础上，筛选绒毛生长期和休止期差异表达的关键lncRNAs，并调控其在毛乳头细胞中的表达，观测细胞生长，利用qRT-PCR、免疫组化、Western blot、RNA-RIP、荧光素酶等技术, 探讨lncRNAs 在褪黑激素调控次级毛囊周期生长中分子机制，为绒山羊育种奠定基础。

羊绒生长主要受季节性光周期调控，光照强度和日照时长对内蒙古绒山羊体内的褪黑激素分泌影响较大，体内血液中的褪黑激素含量在季节上也呈周期性变化规律，说明褪黑激素对毛囊发生发育起重要的调控作用，能够促进次级毛囊的生长发育，这对提高产绒量和绒质有着重要的意义。lncRNAs、miRNAs、circRNAs虽然已被证明在毛囊发育中发挥一定作用，但褪黑激索调控lncRNAs、miRNAs、circRNAs对毛囊发育分化的作用机制尚不清晰。因此，本课题利用转录组重测序技术检测生长早期和生长旺盛期绒山羊皮肤的lncRNAs、miRNAs及circRNAs、qRT-PCR、双荧光素酶基因检测、慢病毒介导过表达、RNAi、Western blot等技术探索lncRNAs、miRNAs、circRNAs及其靶基因在褪黑激素促进毛囊生长发育中的表达模式和分子功能。.主要研究内容和重要结果：.1、利用RNA-seq技术进行测序分析和生物信息学分析，筛选出差异表达的基因，其中，1106个编码基因、1963个lncRNAs、35个miRNAs和46个circRNA表达上调，1106个编码基因、1953个lncRNAs、31个miRNAs和50个circRNA表达下调。.2、通过GO和KEGG通路富集分析等发现一些lncRNAs的靶基因，如WNT11、TP63、XIAP、TRPM4、SHROOM3、KANK1、WNK1、LGR5、LOC102186320，WNT5b、PDGFA，Wnt10A、VEGFA、KRT35、COL17A1并富集于重要的毛囊发育通路，包括Wnt、NFKB、Notch、MAPK信号通路和调控上皮细胞发育的信号通路。.3、构建了lncRNA-miRNA-mRNA和circRNA-miRNA-mRNA的ceRNA网络。.4、双荧光素酶基因检测验证了关键miRNA与mRNA的靶向关系。.5、培养原代次级毛囊毛乳头细胞，细胞状态良好，呈现多角形，传代后的细胞可快速贴壁且生长均匀。.6、构建慢病毒介导的过表达载体、干扰载体，转染毛乳头细胞，转染效率良好。 .阐明关键lncRNAs、miRNAs、circRNAs在褪黑激素调控毛囊周期生长中的作用机制，为进一步揭示绒毛周期生长分子调控机理和提高产绒效应积累分子基础数据。