关键词: 毛囊发育, 苏博美利奴羊, miRNA, 靶基因, 调控网络

Abstract:

【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 17-19 μm 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.

Key words: hair follicle development, Subo Merino, miRNA, target gene, regulatory network