Regulation And Molecular Mechanism Of Sox18 On The Induction Ability Of Dermal Papilla Cells In Hu Sheep

The Hu sheep is a unique sheep breed in China and is best known for its white lambskins with a wavy pattern.However,with the continuous development of the meat market in recent years,more attention has been paid to improving the meat performance of Hu sheep.The crossbreeding and breeding work has led to a gradual decline in the lamb skin quality of Hu sheep,and the excellent germplasm resources have been seriously threatened.Wool curvature is a key factor determining the quality of lamb skin in Hu sheep.Therefore,analyzing the molecular mechanism of wool curvature is of great significance of revealing the genetic mechanism of lamb skin pattern formation of Hu sheep and for protecting and utilizing lamb skin germplasm resources in Hu sheep.The curvature of wool is closely related to the growth,development,and quantity of hair follicles.As the signal center of hair follicles,dermal papilla cells(DPCs)can induce the formation of hair follicles and regulate their growth and development.Their induction ability is crucial for hair growth and morphology.Sox18 is a transcription factor,which has been proven to be a key factor affecting hair follicle growth and development,and plays an important role in the development and function of DPCs.Prelaboratory studies have found that Sox18 is differentially expressed in the DPCs of different pattern types of Hu sheep.We speculate that Sox18 also plays an important role in the DPCs of Hu sheep,thereby affecting wool bending,but the specific mechanism of action is still unknown.This study examined the effect of Sox18 on the induction ability of DPCs and protein secretion in exosomes of Hu sheep,and explored the molecular mechanism of its action through the Wnt/β-catenin signaling pathway.The main research results are as follows:1.Regulation of Sox18 on the induction ability of Hu sheep DPCsFirstly,Hu sheep DPCs were successfully isolated using a mechanical stripping method,and the expression of Sox18 in Hu sheep DPCs was detected by immunofluorescence staining,proving that Sox18 is specifically expressed in DPCs of Hu sheep and can be used as a marker for identifying genes in DPCs.In order to achieve overexpression and interference with Sox18 in Hu sheep DPCs,pcDNA3.1(+)-Sox18 was constructed and siRNA-Sox18 was synthesized.It was successfully transfected into Hu sheep DPCs,and the transfection effect was good.Then,alkaline phosphatase staining,cell agglutination observation,and RT-qPCR were used to detect the effect of Sox18 on the induction ability of DPCs.The results showed that Sox18 could enhance the alkaline phosphatase activity of DPCs,promote the aggregation of DPCs,promote the expression of Verican,HHIP,and FGFRI,and inhibit the expression of BMP4 and WIF1.The above results indicated that Sox 18 can enhance the induction ability of Hu sheep DPCs.2.Effect of Sox18 on protein secretion in exosomes of Hu sheep DPCsTo explore the effect of Sox18 on protein secretion in exosomes of Hu sheep DPCs,exosomes of Hu sheep DPCs were first extracted,and WB,TEMand NTA were used to characterize the extracted exosomes.Th e results showed that the extracted matters expressed exosome-specific marker proteins(CD9,CD63)and had a typical exosome structure with a diameter of about 128.4 nm,which was consistent with the particle size range distribution of exosomes.Then the Hu sheep DPCs transfected with pcDNA3.1(+)-Sox18 were set as the experimental group(Group G),and the Hu sheep DPCs transfected with pcDNA3.1(+)were set as the control group(Group PC),with three replicates in each group.We extracted exosomes and performed proteomic assays on the proteins in exosomes using Label free relative quantitative proteomic technology.A total of 1199 proteins were detected.Quantitative analysis of proteins identified a total of 40 significantly differentially expressed proteins,of which 38 proteins were up-regulated in the Sox18-overexpression experimental group and 2 proteins were down-regulated in the Sox18-overexpression experimental group.Based on the functional analysis of differential proteins and interaction network analysis,and combined with related literature,EZR,LAMA4 and TGFBI proteins were found to play important roles in the processes of cell growth,proliferation and migration,which can be used as candidate proteins for subsequent studies affecting hair follicle growth and development.The above results indicate that Sox18 can affect the expression of related proteins in DPCs exosomes of Hu sheep.3.Regulation of Sox18 on Wnt/β-catenin signaling pathway in Hu sheep DPCsThe Wnt/β-catenin pathway is the pathway most closely associated with hair follicle growth and development and is critical for the induction capacity of DPCs.To investigate the association between Wnt/β-catenin signaling pathway and Sox18 and its effect on the induction ability of Hu sheep DPCs,this study activated or inhibited the Wnt/β-catenin signaling pathway in Hu sheep DPCs,and then examined the induction ability of Hu sheep DPCs using alkaline phosphatase staining and observation of cell agglutination phenomenon.The results showed that activation of the Wnt/β-catenin signaling pathway enhanced the induction ability of Hu sheep DPCs with the same effect as overexpression of Sox18,while inhibition of the Wnt/β-catenin signaling pathway in Hu sheep DPCs overexpressing Sox18 weakened the induction ability of Hu sheep DPCs,which indicated that Sox18 through the Wnt/β-catenin signaling pathway affects the induction ability of Hu sheep DPCs and is able to activate the Wnt/β-catenin signaling pathway in Hu sheep DPCs.To verify this result,the expression of β-catenin,the core protein of Wnt/β-catenin signaling pathway,was firstly examined by RT-qPCR and WB,and the results showed that Sox18 could promote the expression of β-catenin in Hu sheep DPCs.Then,the expression of β-catenin in Hu sheep DPCs was identified by immunofluorescence staining,and the results showed that both the use of activator and overexpression of Sox18 could increase the expression amount of βcatenin in the nuclei of Hu sheep DPCs,which indicated that Sox18 could activate the Wnt/βcatenin signaling pathway in Hu sheep DPCs.This conclusion was further supported by the finding that overexpression of Sox18 could activate the already suppressed Wnt/β-catenin signaling pathway in Hu sheep DPCs.In conclusion,we found that Sox18 can enhance the induction ability of Hu sheep DPCs by regulating the Wnt/β-catenin signaling pathway and promote the expression of related proteins in the exosomes of Hu sheep DPCs.This study provides a theoretical basis for analyzing the molecular mechanism of the formation of lamb skin patterns in Hu sheep.