Identification of microRNAs in Wool Follicles during Anagen,Catagen, and Telogen Phases in Tibetan Sheep

Background

Wool quality is one of the most important economic traits in sheep. The wool fiber is derived from specialized skin cells that are referred to as wool follicles. To understand the roles of microRNAs (miRNAs) in wool fiber growth, we detected the expression patterns of miRNAs in wool follicles at the anagen, catagen, and telogen stages from Tibetan sheep through Solexa sequencing.

Results

A total of 244 mature miRNAs were identified. Of these, only five miRNAs are listed in the database of sheep miRNAs (miRBase Database V19), and the other 239 miRNAs have not been previously described in this species. Further analyses indicated that 204 miRNAs are evolutionarily conserved among mammal species, whereas 35 of the identified miRNAs were first found specifically in sheep. The expression pattern analyses showed that the expression levels of 39, 34, and 20 of the miRNAs significantly change between anagen and catagen, between anagen and telogen, and between catagen and telogen, respectively. The results of the bioinformatics analysis show that these differentially expressed miRNAs might regulate wool follicle development by targeting genes in many different pathways, such as the MAPK and Wnt pathways, as well as the pathways that regulate the actin cytoskeleton, focal adhesion, and tight junctions. Furthermore, we identified six differentially expressed miRNAs (oar-miR-103-3P, oar-miR-148b-3P, oar-miR-320-3P, oar-miR-31-5P, oar-novel-1-5P, and oar-novel-2-3P) that might target the key genes of the Wnt pathway. It has been reported that the Wnt pathway is critical for wool follicle development. Therefore, these miRNAs may regulate wool development through the Wnt pathway.

Conclusions

Our results provide new information on the identification and expression pattern of miRNAs in wool follicles. Our data might therefore aid in the understanding of the mechanisms of wool follicle development in sheep.     

蛋白质组学技术对不同性别中国美利奴细毛羊(军垦型) 皮肤差异蛋白的筛选

旨在了解性别因素对绵羊毛性状的影响。以周岁雄性和雌性中国美利奴羊（军垦型）为研究对象,利用液相色谱-串联质谱联用技术和数据非依赖性采集策略的定量蛋白质组学技术筛选皮肤组织差异表达蛋白,并对筛选获得的差异蛋白进行基因本体（gene ontology,GO）功能注释、京都基因与基因组百科全书（Kyoto encyclopedia of genes and genomes,KEGG）代谢通路和蛋白互作分析。结果显示,共计筛选获得差异表达蛋白674种,其中,280种蛋白表达上调,394种蛋白表达下调;通过进一步分析发现,与皮肤毛囊发育及羊毛表型相关的差异蛋白有43种,上调差异蛋白30种,下调差异蛋白13种。GO注释结果显示,在分子功能方面,差异蛋白在氧结合、硫酸软骨素结合、亚铁血红素结合、谷胱甘肽过氧化物酶活性和转运活性等37个过程显著富集;在生物过程方面,差异蛋白在细胞氧化解毒作用、肌肉收缩调节、Notch信号通路、钙离子跨膜转运和谷胱甘肽新陈代谢等120个过程显著富集;在细胞组分方面,主要富集在肥大细胞颗粒、细胞核、肌质网状组织和内质网等31个过程。KEGG通路结果表明,这些差异蛋白涉及16条信号通路,其中,MAPK、P53信号通路和羊毛生长发育密切相关。蛋白质网络互作结果显示,COL1A1蛋白与MMP2、SPARC、THBS1等差异表达蛋白联系较为紧密,其可能在羊毛生长发育过程中发挥着关键作用。本研究为揭示不同性别绵羊毛性状的分子机制积累了基础数据。     

Expression profile analysis to identify circular RNA expression signatures in hair follicle of Hu sheep lambskin

CircRNAs are involved in the regulation of various cellular and biological processes, but none of the studies have focused on hair follicle in sheep. In this study, the expression profile of circRNAs between small waves (SM) and straight wool (ST) groups was investigated using RNA-Seq. The results showed that a total of 5,527 circRNAs were identified and 114 of them were differentially expressed between two groups. Enrichment analysis revealed that the host genes with DE circRNAs were mainly enriched in TGF-beta pathway, Notch pathway. Miranda software was used to found that 129 miRNAs might be binding to 114 DE circRNAs, including miR-10a, miR-143, miR-let-7a, miR-199a-3p, miR-200a, which also had important influence on hair follicle morphogenesis. Furthermore, the coding potential of circRNAs was predicted, and 11 circRNAs were simultaneously identified with coding potential. In summary, circRNAs have important effects on hair follicle growth and development, and these results will provide a basis for molecular mechanism of pattern formation.     

Comparative study on seasonal hair follicle cycling by analysis of the transcriptomes from cashmere and milk goats

Guard hair and cashmere undercoat are developed from primary and secondary hair follicle, respectively. Little is known about the gene expression differences between primary and secondary hair follicle cycling. In this study, we obtained RNA-seq data from cashmere and milk goats grown at four different seasons. We studied the differentially expressed genes (DEGs) during the yearly hair follicle cycling, and between cashmere and milk goats. WNT, NOTCH, MAPK, BMP, TGFβ and Hedgehog signaling pathways were involved in hair follicle cycling in both cashmere and milk goat. However, Milk goat DEGs between different months were significantly more than cashmere goat DEGs, with the largest difference being identified in December. Some expression dynamics were confirmed by quantitative PCR and western blot, and immunohistochemistry. This study offers new information sources related to hair follicle cycling in milk and cashmere goats, which could be applicable to improve the wool production and quality.     

绵羊肌肉中FAS基因和HSL基因的发育性变化及其对肌内脂肪含量的影响

选取不同日龄的雄性哈萨克羊和新疆细毛羊共54只,屠宰后取背最长肌,用索氏抽提法检测肌内脂肪(intramuscular fat,IMF)含量,用荧光实时定量PCR法检测肌肉脂肪酸合成酶(fatty acid synthase,FAS)和激素敏感脂肪酶(hormone-sensitive lipase,HSL)基因表达的发育性变化,并分析基因表达对肌内脂肪沉积的影响。结果表明:1)随着日龄的增加,雄性哈萨克羊的IMF含量持续上升,各生长时期差异显著(P<0.05),而新疆细毛羊的IMF含量在各生长时期无显著差异(P>0.05)。雄性哈萨克羊的IMF含量30~90日龄期间极显著高于新疆细毛羊(P<0.01)。2)FAS基因mRNA水平在哈萨克羊肌肉中初生时最高(P<0.05),然后随日龄的增加呈下降趋势;在新疆细毛羊肌肉中,FAS mRNA水平表现出"下降-上升-下降-上升"的发育模式,其中60日龄显著高于90日龄(P<0.05),其余日龄之间差异不显著。HSL基因在2品种绵羊肌肉中的表达模式基本类似,在哈萨克羊肌肉中随年龄的增加而下降,初生时的水平显著高于60~90日龄(P<0.05);在新疆细毛羊中30日龄时达到最高(P<0.01),到60日龄时下降到最低(P<0.05),随后保持这种低表达水平。3)FAS和HSL基因mRNA的表达量均与哈萨克羊IMF含量呈负相关,相关系数分别为:r=-0.485(P=0.02),r=-0.423(P=0.05);在哈萨克羊中两基因表达量水平比值(FAS:HSL)与IMF呈极显著负相关r=-0.552(P=0.01)。在新疆细毛羊中两基因的表达水平及比值均与IMF无显著相关性(P>0.05)。     

绵羊ILK基因的克隆及其在毛囊生长期的表达

整合素连接激酶(ILK)是一种支架蛋白,在毛囊发育过程中发挥重要作用。文章利用PCR技术,首次获得绵羊ILK基因的编码区全长序列,并进行了生物信息学分析;同时对该基因的组织表达谱及其在不同绵羊品种毛囊生长期皮肤组织中的表达变化进行了研究。结果表明,绵羊ILK基因ORF全长1 359 bp,编码452个氨基酸。ILK蛋白结构经预测含有3个锚定重复序列和1个激酶结构域,并存在多个磷酸化位点和蛋白激酶C的磷酸化位点。半定量RT-PCR结果显示该基因在绵羊心脏、肝脏、脾脏、肺脏、肾脏、骨骼肌、皮肤和小肠组织中均有表达,皮肤、脾脏和肝脏中表达量较高;实时荧光定量PCR结果表明在3~5月份(毛囊生长起始期),ILK基因在中国美利奴超细型和哈萨克羊皮肤组织中的表达水平较高并均呈逐月上升趋势;在6~10月份(毛囊生长期),中国美利奴超细型皮肤ILK基因表达水平高于同期的哈萨克羊。分析认为ILK可能在调控绵羊次级毛囊生长发育过程中起一定作用。     

Expression of bacterial cysteine biosynthesis genes in transgenic mice and sheep: toward a newin vivo amino acid biosynthesis pathway and improved wool growth

It is possible to improve wool growth through increasing the supply of cysteine available for protein synthesis and cell division in the wool follicle. As mammals can only synthesis cysteine indirectly from methionine via trans-sulphuration, expression of transgenes encoding microbial cysteine biosynthesis enzymes could provide a more efficient pathway to cysteine synthesis in the sheep. If expressed in the rumen epithelium, the abundant sulphide, produced by ruminal microorganisms and normally excreted, could be captured for conversion to cysteine. This paper describes the characterisation of expression of the cysteine biosynthesis genes ofSalmonella typhimurium, cysE,cysM andcysK, and linkedcysEM,cysME andcysKE genes as transgenes in mice and sheep. The linked transgenes were constructed with each gene driven by a separate promoter, either with the Rous sarcoma virus long terminal repeat (RSVLTR) promoter or the mouse phosphoglycerate kinase-1 (mPgk-1) promoter, and with human growth hormone (hGH) polyadenylation sequences. Transgenesis of mice with the RSVLTR-cysE gene afforded tissue-specific, heritable expression of the gene. Despite high levels of expression in a number of tissues, extremely low levels of expression occurred in the stomach and small intestine. Results of a concurrent sheep transgenesis experiment using the RSVLTR-cysEM and-cysME linked transgenes revealed that the RSVLTR promoter was inadequate for expression in the rumen. Moreover, instability of transgenes containing the RSVLTR sequence was observed. Expression of mPgk-cysME and-cysKE linked transgenes in most tissues of the mice examined, including the stomach and small intestine, suggested this promoter to be a better candidate for expression of these transgenes in the analogous tissues of sheep. However, a subsequent sheep transgenesis experiment indicated that use of the mPgk-1 promoter, active ubiquitously and early in development, may be inappropriate for expression of the cysteine biosynthesis transgenes. In summary, these results indicate that enzymically active bacterial cysteine biosynthesis gene products can be coexpressed in mammalian cellsin vivo but that expression of the genes should be spatio-temporally restricted to the adult sheep rumen epithelium.     

Analysis of gene expression in granulosa cells of ovine antral growing follicles using suppressive subtractive hybridization

Follicular growth, development and ovulation are highly ordered processes that involve the expression of many genes under precise temporal and spatial regulation. However, information on stage-specific gene expression during the antral follicle phase in sheep is not well understood. In the present study, suppressive subtractive hybridization (SSH) was performed to screen genes that were differentially expressed in the granulosa cells between large follicles (LF, >5 mm) and small follicles (SF, 3–5 mm), and subtractive cDNA library was constructed. Furthermore, with dot-blot analysis, a total of 90 clones randomly selected from the library were proven to be differentially expressed in the granulosa cells. Among these, 38 exhibited high homology to known genes, 14 sequences were corresponding to novel expressed sequence tags (ESTs). Four ESTs, LAPTM4A, SERPINE2, GSTA1, and INHBA, were further examined the reproducibility of the SSH data by the real-time quantitative PCR. Results confirmed an increase expression of respective mRNA in granulosa cells of large follicles compared with that of small follicles. It is concluded that we have identified several genes (known or unknown) that may effect follicular growth, dominance or ovulation in ewes.     

Hoxc13在毛囊发育中的作用

Hoxc13属于Hox(Homobox)基因家族Abd-B类成员之一, 与毛囊形成和毛发生长密切相关。毛发结构蛋白KP(角蛋白)和KAP(角蛋白关联蛋白)的表达都受Hoxc13的严格调控, Hoxc13表达水平会直接影响毛发的特性, 对维持毛囊的正常形态也至关重要。文章就Hoxc13的表达水平对毛囊发育和毛发生长的影响及Hoxc13与相关基因的调控进行了综述。