Effects Of MiR-1 On Proliferation And Differentiation Of Hair Follicle Stem Cellsin Shaanbei White Cashmere Goat

The Shaanbei White Cashmere Goat is a local breed in northern Shaanxi province.The cashmere grown by the secondary hair follicle(HFs)of the skin tissues has high economic value due to its excellent quality.MiRNAs are a class of non-coding small molecules that,in most cases,bind to the 3'-UTR region of a target gene to cause degradation of the target gene or inhibit transcriptional regulation of mRNA translation.Studies have shown that a variety of miRNAs regulate the cyclical development of HFs.However,it is unclear how miRNAs regulate the growth and development of cashmere goats.The miRNA sequencing analysis of dermal papilla cells and dermal papilla-derived exosomes have found that miR-1 was highly expressed in exosomes.In order to analyze the effect of miR-1 on the growth and development of hair follicle stem cells(HFSCs),we used molecular techniques to explore the mechanism of miR-1 in the cyclical development of HFs.The main results are as follows:1)After overexpression of miR-1,the regulation of miR-1 on the proliferation and differentiation of HFSCs in Shaanbei White cashmere goats was investigated by CCK8,EdU and flow cytometry.The results showed that the proliferative capacity of HFSCs was significantly weakened(p < 0.01),and the proportion of cells in the S phase was significantly decreased(p < 0.01),while the proportion of cells in the G0-G1 phase was significantly increased,indicating that the cells were arrested in the G0-G1 phase.These results indicate that overexpression of miR-1 could inhibit the proliferation HFSCs.2)qPCR and Western blot were used to detect the effect of signaling pathway of miR-1 on the proliferation and differentiation of HFSCs.The results showed that the mRNA and protein expression levels of cell proliferation marker KI67,PCNA,and AKT were significantly lower than those of the NC group after overexpression of miR-1(P < 0.05),indicating that overexpression of miR-1 inhibited cell proliferation in HFSCs by AKT pathway.The mRNA and protein expression levels of K6,S100A3,and Notch1 in mimics group were significantly higher than those in NC group(P < 0.05),which indicating that overexpression of miR-1 in HFSCs could promotes the expression of the cell differentiation marker K6 and S100A3,and further promotes the differentiation of HFSCs by activating the Notch pathway.3)The RNAhybrid and TargetScan 7.1 software were used to predict target genes of miR-1,the 3'-UTR region of the candidate target genes were successfully cloned according to the predicted results.The luciferase reporter vectors of wild-type(WT)and mutant of the miR-1 candidate target genes FOXP1,FOXC1,IGF1 R,SOX9,MSI2,and LEF1 were successfully constructed.The miR-1 mimics and WT/mutant vector plasmids were co-transfected into 293 T cells.The dual fluorescein reporter gene assay showed that miR-1 can inhibit the targets of wild-type IGF1 R and LEF1.The luciferase activity of IGF1 R and LEF1 was recovered after mutated the target gene binding site,while FOXP1,FOXC1,SOX9,and MSI2 were still significantly inhibited.There results showed that miR-1 has a targeting relationship with the 3'UTR of IGF1 R and LEF1.In summary,miR-1 can regulate the proliferation and differentiation process of HFSCs,it is provides a theoretical basis for further study on the periodic development of HFs in cashmere goats.