Identification And Functional Study Of MiRNAs Related To Proliferation And Differentiation Of Hair Follicle Stem Cell In Shaanbei White Cashmere Goats

The Shaanbei White cashmere goat,an important Chinese domestic goat breed,has great economic value because of its cashmere.Hair periodic regeneration depends onthe coorparative regulation of hair follicle stem cells(HFSCs)and dermal papilla cells(DPCs).In this study,we aimed at exploring the effects of the DPC derived exosomes(DPC-Exos)on the proliferation and differentiation of HFSC.First,we successfully separated HFSCs and obtained DPC-Exos from cashmere goats in vitro.Then mi RNA sequencing of DPCs and DPC-Exos were conducted,and precise quantitative transcriptome and mi RNA sequencing were used to analyze co-cultured HFSCs with DPCs(co-culture)and control HFSCs(NC).Analysis screen out the key genes or mi RNA regulating the proliferation and differentiation of HFSCs.We used the dual luciferase reporter assay to detect the targeting relationship between selected mi R-22-5p/let-7b-5p and predicted target genes,and the roles of mi R-22-5p/let-7b-5p on HFSCs prolifereation and differentiation were further explored.The main results are as follows:1.The HFSCs of Shaanbei White cashmere goat were successfully separated by tissue adherence,and further purified by using fast adhesion to collagen IV and fluorescence-activated cell sorting(FACS).Microscopy,transmission electron microscopy and HFSC markers(CD34,K15,and ITG?1)analysis demonstrated that the HFSCs were successfully separated.Meanwhile,the co-cultured HFSCs with DPCs system was successfully established to induce HFSCs differentiating into hair follicle(HF)cells.2.Six m RNA libraries and six small RNA libraries of co-culture and NC samples in Shaanbei White cashmere goat were constructed.There were 12,802 co-expressed genes and 656 differentially expressed genes in these two groups,of which 342 were up-regulated and 314 were down-regulated.The GO and KEGG enrichment analysis showed that these differentially expressed genes are associated with TNF signaling pathway,p53 signaling pathway and Apoptosis pathways et al.The mi RNA sequencing analysis showed that there were 13 differentially expressed genes,of which 6 were up-regulated and 7 were down-regulated.We found four candidate mi RNAs from these genes,which are mi R-1,mi R-151-3p,mi R-182,mi R-143-3p.The GO and KEGG enrichment analysis of target genes of these four genes indicated that they are associated with RNA transport and Legionellosis pathways.Conjoint analysis for m RNAs and mi RNAs showed that differential genes were enriched in TNF signaling pathway,FOXO signaling pathway,Amphetamine addiction pathways which are connected with cell proliferation,apoptosis and metabolism.3.The extracellular vesicle were separated successfully from Shaanbei White cashmere goat DPCs culture medium by using gradient centrifugation and ultracentrifugation.Exosomal marker proteins,particle size and transmission electron microscopy showed that the isolated extracellular vesicles were exosomes.HFSCs were co-cultured with Di I-labeled DPCs in vitro through Transwell chamber,and the HFSCs were incubated with Di I-labeled exosomes,demonstrating that DPC-Exos can be transported to HFSCs,and HFSCs can take up the exosomes derived from DPCs.4.Six small RNA libraries were established by mi RNA sequencing of Shaanbei White cashmere goat DPCs and DPC-Exos.There were 187 mi RNAs expressed in both DPCs and DPC-Exos,79 mi RNAs were differentially expressed,and 5 mi RNAs specifically expressed in DPC-Exos.There were 111 mi RNAs differentially expressed in both DPCs and DPC-Exos,of which 49 mi RNAs were up-regulated and 62 mi RNAs were down-regulated in DPC-Exos.The expression of mi R-21-5p,mi R-143-3p,let-7i-5p,mi R-148a-3p,and let-7b-5p were the top five of enriched mi RNAs.In the meantime,336 novel mi RNAs were predicted,114 novel mi RNAs were differentially expressed in both DPCs and DPC-Exos.5.The differentially expressed mi RNAs between DPCs and DPC-Exos were predicted to target 33,055 genes.GO analysis revealed that target genes mainly enriched in extracellular region of cell component and other functional subsets,enriched in binding and catalytic activity of molecular function and other functional subsets and enriched in macromolecule modification of biological processes and other functional subsets.KEGG analysis revealed that target genes were annotated in 271 pathways,which mainly participated in cell signaling transduction,endocytosis,regulation of protein translation,lipid and fatty acid metabolism.6.The IGF1 R,SOX9,MSI2,FOXC1,FOXP1,and LEF1 were selected for dual luciferase assay,the results showed mi R-22-5p mimics can inhibit the wild-type of FOXP1 and LEF1.However,by binding to a mutated sequence of these six genes,only the LEF1 luciferase activity can be restored.The results showed that LEF1 was a target gene of mi R-22-5p,and mi R-22-5p could target and regulate predicted target sites of the LEF1.Overexpression of mi R-22-5p suppress the proliferation of HFSCs through AKT signaling pathway,but with no significant effect on HFSCs differentiate to HF cells.7.The KDM6 A,FOXC1 and MSI2 were selected for dual luciferase assay,the results showed let-7b-5p mimics can inhibit the wild-type of FOXC1 and MSI2.However,by binding to a mutated sequence of these three genes,only the FOXC1 and MSI2 luciferase activity can be restored.The results indicated that FOXC1 and MSI2 are target genes of let-7b-5p,and let-7b-5p can target the predicted target sites of FOXC1 and MSI2.At the same time,the results showed that over-expression of let-7b-5p,let-7b-5p inhibited the proliferation of HFSCs via AKT signaling pathway and promoted the differentiation of HFSCs into HF cells by activating Notch signaling pathway.In summary,we successfully separated HFSCs and DPC-Exos of Shaanbei White cashmere goat.We used the transcriptomic research and mi RNA sequencing to explore the relationship between NC and co-culture,and also used the mi RNA sequencing of DPCs and DPC-Exos to seek the differentiation mechanism of HFSCs.Through these two ways,we further identified mi R-22-5p synergize with LEF1,let-7b-5p synergize with FOXC1,MSI2 to regulate the proliferation of HFSCs.There results will provide a foundation for further revealing the molecular mechanism of HFSCs proliferation and differentiation and the cycle of HF.