Integrating Transcriptome And MiRNA Data To Analyze The Molecular Mechanism Of Hair Follicle Development In Subo Merino Sheep

As a natural textile raw material,wool has high economic value and is increasingly favored by modern textile enterprises and consumers.However,the output of domestic fine wool is far from meeting the processing needs of the textile market,especially high-quality superfine wool.Therefore,improving wool yield and quality has always been the research focus of fine wool sheep breeding.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 adulthood.The regulation mechanism of HF morphogenesis in superfine wool sheep need further study.Therefore,analyzing the molecular regulation mechanism of HF development in the embryonic stage of super fine wool sheep can better clarify the morphogenesis of HF,and has important guiding significance of the breeding of super fine wool sheep.Subo merino sheep is a new breed of worst super fine wool sheep,which is independently bred in China,with wool fineness of 17-19μm.In this study,the skin tissue of scapular region at different stages of HF development was collected from Subo merino.RNA-Seq sequence,bioinformatics analysis,RT-qPCR,WB,flow cytometry and other methods were used to screen candidate miRNAs and genes involved in HF development of ultra-fine wool sheep,and construct miRNAs-mRNAs regulatory network.This study provides a reference molecular marker for understanding the molecular regulation mechanism of HF development in ultra-fine wool sheep.1.Screening and bioinformatics analysis of miRNA in different stages of HF development.In this study,we constructed a miRNA library of 18 skin tissues at different stages of HF development in Subo merino,and screened 87 DE-miRNAs and 446 novel DEmiRNAs.Cluster analysis of DE-miRNA showed that there were 21 DE-miRNAs in HF induced differentiation stage(E65,E85,E105),including 5 candidate miRNAs(oar miR-23 b,oar miR-133,etc.).There were 28 DE-miRNAs in HF mature stage(E135,D7,D30).SOM analysis showed that DE-miRNA could be clustered into 10 clusters,and miRNA in each cluster had similar functions.The mixed prediction targeted genes of DE-miRNAs and Novel DE-miRNAs were performed.The main enrichment pathways to target genes are AMPK,Notch and hedgehog.2.Gene network analysis reveals candidate genes related to the HF development.In this study,7879 DE mRNA were screened by RNA-seq sequence.Enrichment analysis of DE-mRNA showed that E65 vs.E85 gene is mainly enriched in the negative regulation of the canonical Wnt signaling pathway,HF development,negative regulation of the BMP signaling pathway.The main pathways to DE-mRNA enrichment are PI3K-Akt,Hippo,PPAR,MAPK and ECM-receptor interactions.The network by Cytoscape software results showed that biological processes such as HF development,hair cycle process,skin development,epidermal epithelial morphogenesis and TGF-β,Wnt,Notch,Hippo,MAPK and Hedgehog signal pathways have 17 candidate genes(ACVR1B,NOTCH1,TGFβ2.WNT10 A,etc.).In order to further to analyze the function of differential genes,nine gene expression modules were obtained by WGCNA method.The analysis of the Hub gene in the module showed that 36 key genes(ACVR1B,WNT5 A,TGFβ2,NOTCH1,TGFβ3,WNT10 A,etc.)in yellow,green,red,brown and turquoise modules were involved in HF morphogenesis.3.Construction of gene regulatory network by joint analysis of mRNA and miRNA.In this study integrated miRNA and mRNA data,and divided mRNA and miRNA into HF induced differentiation stage(Stage A)and HF maturation stage(Stage B)according to the clustering results of gene expression.In order to understand the function of specific expressed genes in each stage,the functional enrichment analysis of specific DE-mRNA in Stage A and Stage B was carried out.The results showed that Stage A enrichment was in the negative regulation of canonical Wnt signaling pathway,establishment of skin barrier,skin development,cellular response to transforming growth factor beta stimulus,negative regulation of BMP signaling pathway,negative regulation of epithelial cell proliferation,epithelial to mesenchymal transition,positive regulation of epidermal cell differentiation,HF morphogenesis.Also enriched in Hippo,PI3K-Akt,TGF-β and Hedgehog signal pathways.Furthermore,the specific DEmRNA of Stage A was combined with the target gene of DE-miRNA to construct a miRNA-mRNA regulatory network related to HF development.4.Double luciferase reporter gene validation of target genes of oar-miR-23 b and oar-miR-133.We selected oar-miR-23 b to target WNT10 A,NOTCH1,TGFβ2,ACVR1 B,and oar-miR-133 to target NOTCH1,TGFβ2,ACVR1 B to verify the targeting relationship using RT-qPCR and double luciferase gene detection methods.RT-qPCR results showed that oar-miR-23 b,oar-miR-133 and their target genes were differentially expressed in the skin tissues at six stages of HF development.293 T cells were cotransfected with vector and miR-23 b mimic,miR-133 mimic and mimic-NC.The results showed that WNT10 A,NOTCH1,TGFβ2 were the target genes of oar-miR-23 b,and ACVR1B、NOTCH1、TGFβ2 were the target genes of oar-miR-133.5.Functional study of oar-miR-23 b and oar-miR-133 in SDFsIn this study,oar-miR-23 b and oar-miR-133 were overexpressed and inhibited in SDFs.RT-qPCR and WB detection showed that overexpression of miRNA in SDFs could inhibit the expression of target genes at mRNA level and protein level.CCK-8assay showed that overexpression of oar-miR-23 b and oar-miR-133 could inhibit the proliferation of SDFs.Annexin V-FITC method combined with flow cytometry showed that overexpression of oar-miR-23 b and oar-miR-133 could significantly promote the apoptosis of SDFs.Transwell results showed that overexpression of oar-miR-23 b and oar-miR-133 inhibited the cell cycle progression of SDFs.The results showed that overexpression of oar-miR-23 b and oar-miR-133 inhibited gene expression in WNT,TGF-β and Hippo signal pathways.In conclusion,five important miRNAs(oar-miR-23b、oar-miR-133、oar-miR-381-5p、oar-miR-381-3p和 oar-miR-655-3p)and 17 candidate genes(LAMA5,KRT25,ACVR1 B,SOSTDC1,ZDHHC21,FZD1,BMP7,LRP4,TGFβ2,NOTCH1,WNT10 A,TMEM79,SOX10,ITGB4,KRT14,ITGA6,GLI2)screened by transcriptome sequencing combined with relevant molecular biology methods are closely related to the biological process of hair follicle morphogenesis.We further integrated miRNA and mRNA data to construct a regulatory network of 9 miRNAs and 17 key genes at the stage of hair follicle induction and differentiation.At the same time,it was identified that oar miR-23 b inhibited the proliferation and migration of SDFs by targeting NOTCH1,WNT10 A,TGFβ2and oar miR-133,and promoted cell apoptosis by targeting ACVR1 B,NOTCH1 and TGFβ2,thereby affecting hair follicle morphogenesis.The results revealed the molecular mechanism of miRNA and mRNA regulation in HF development of Subo merino,which provided a reference molecular marker and theoretical basis for super fine wool sheep breeding.