基于miRNA测序数据解析苏博美利奴羊毛囊发育分子机制

Analyzing the Molecular Mechanism of Hair Follicle Development in Subo Merino Based on miRNA Sequencing Data

【背景】羊毛由毛囊(hair follicle,HF)产生并控制,HF结构功能和形态发生是一个复杂的生物过程。细毛羊胚胎期HF发育过程决定了羊毛的产量和品质。苏博美利奴羊是我国自主培育的羊毛细度达17—19μm的精纺用超细毛羊新品种。超细毛羊HF形态发生过程中miRNA及其调控机制有待更为深入的研究。【目的】解析超细毛羊早期HF发育中miRNA的分子调控机制对于更好地理解HF形态发生过程以及对细毛羊的育种工作具有重要的指导意义,为解析超细毛羊HF发育分子调控机制和选育优质超细毛羊提供可参考的分子标记。【方法】对相同饲养条件下的苏博美利奴羊进行同期发情和人工授精,授精日被指定为胚胎第0天(E0)。在胚胎期第65天(E65)、85天(E85)、105天(E105)和135天(E135)将妊娠母羊安乐死后进行剖宫产收集胚胎的皮肤组织;在羔羊出生后第7天(D7)和30天(D30)采集左侧肩胛部皮肤组织,每个时期采3个样本。运用miRNA-Seq鉴定毛囊发育不同时期的保守miRNA和Novel miRNA,并构建苏博美利奴羊HF发育不同时期的皮肤组织miRNA文库。对差异表达miRNA(DE-miRNA)进行靶基因预测及生物信息学分析,筛选参与超细毛羊HF发育的关键miRNA和候选基因,并构建miRNA-靶基因调控网络。运用RT-qPCR和双荧光素酶报告基因检测方法验证miR-433-3p与NOTCH1的靶向关系。【结果】构建了苏博美利奴羊HF发育不同时期18个皮肤组织miRNA文库,并筛选出87个DE-miRNA和446个Novel DE-miRNA。对DE-miRNA聚类分析可知毛囊诱导分化阶段(E65、E85、E105)有21个DE-miRNA,其中有5个(oar-miR-23b、oar-miR-133等)是关键候选miRNA;毛囊成熟阶段(E135、D7、D30)有28个DE-miRNA。SOM分析结果表明DE-miRNA可聚为10个Cluster,每个Cluster中miRNA有相似功能。对DE-miRNA和Novel DE-miRNA进行混合预测靶基因并进行功能富集发现靶基因主要富集通路有AMPK、Notch、hedgehog。将DE-mRNA与靶基因结合生物信息学分析构建了与HF发育相关的miRNA-靶基因调控网络。在293T细胞中将NOTCH1野生型和突变型载体与miR-433-3p mimics和mimics-NC共转染,结果表明NOTCH1为miR-433-3p的靶基因。【结论】构建了苏博美利奴羊毛囊发育不同时期miRNA表达谱,分析了miRNA及其靶基因的表达调控关系,并构建了miRNA-靶基因调控网络,进一步了解了miRNA在毛囊发育不同时期的作用及分子机制;为解析超细毛羊毛囊发育分子调控机制和选育优质超细毛羊提供可参考的分子标记。

【Background】Wool is produced and controlled by hair follicles(HFs).The structure,function and morphogenesis of HF is a complex biological process.The morphogenesis of HF in the embryonic stage of fine wool sheep determines the wool yield and quality after sheep adulthood.Subo Merino sheep is a new breed of ultra-fine wool sheep for worsted spinning with wool fineness up to 17-19μm independently bred in China.The miRNAs and their regulatory mechanisms during the morphogenesis of the HF in superfine wool sheep need to be studied in depth.【Objective】Analyzing the molecular regulatory mechanisms of miRNAs in the early development of HF in superfine wool sheep was of great significance for a better understanding of the morphogenesis of HF as well as for the breeding of ultrafine wool sheep,and it could provide the reference molecular markers for the analyzing of molecular mechanisms of the molecular regulatory mechanisms of the development of HF in ultrafine wool sheep and for the selection and breeding of high-quality ultrafine wool sheep.【Method】In this study,simultaneous estrus treatments and artificial insemination were performed on Subo Merino sheep under the same feeding conditions,with insemination day designated as embryonic day 0(E0).Skin tissues from embryos were collected by cesarean section after euthanasia of pregnant ewes at embryonic days 65(E65),85(E85),105(E105),and 135(E135),respectively;skin tissues from the left scapular region were collected at 7(D7)and 30(D30)days after the birth of the lambs,and three samples were taken at each period.miRNA-Seq were used to identify conserved miRNAs and Novel miRNAs at different periods of hair follicle development,and constructed skin tissue miRNA libraries at different periods of HF development in Super Fine Wool Merino sheep.The target gene prediction and bioinformatics analysis of differentially expressed miRNAs(DE-miRNAs)were also performed,the key miRNAs and candidate genes involved in HF development in Superfine wool sheep were screened,and constructed miRNA-target gene regulatory networks.The targeting of miR-433-3p with NOTCH1 was validated by using RT-qPCR and dual luciferase reporter gene assays.【Result】In this study,18 skin tissue miRNA libraries were constructed at different periods of HF development in Subo Merino sheep,and 87 DE-miRNAs and 446 novel DE-miRNAs were screened.DE-miRNA clustering analysis showed that there were 21 DE-miRNAs in the HF-induced differentiation stage(E65,E85,and E105),in which 5 DE-miRNAs(oar-miR-23b,oar-miR-133,etc.)were key candidate miRNAs;there were 28 DE-miRNAs in the HF-maturation stage(E135,D7,and D30).SOM analysis showed that DE-miRNAs could be clustered into 10 clusters,and miRNAs in each cluster had similar functions.Mixed prediction of target genes and functional enrichment of DE-miRNAs and Novel DE-miRNAs revealed that the main enriched pathways of target genes were AMPK,Notch,and hedgehog pathways.The miRNA-target gene regulatory network associated with HF development was constructed by combining DE-mRNA and target gene with bioinformatics analysis.NOTCH1 wild-type and mutant vectors were cotransfected with miR-433-3p mimics and mimics-NC in 293T cells,and the results showed that NOTCH1 was a target gene of miR-433-3p.【Conclusion】In summary,this study constructed miRNA expression profiles in different periods of hair follicle development in Subo Merino sheep,analyzed the relationship between miRNAs and their target genes,and built a miRNA-target gene regulatory network,to further understand the roles of miRNAs in different periods of hair follicle development and molecular mechanisms.This study also provided a reference molecular marker for analyzing the molecular regulation mechanism of hair follicle development,as well as selecting and breeding high-quality ultra-fine wool sheep.