Screening Of Hub Genes Of Skin Hair Follicle Development And Its Regulatory Role In Inner Mongolia Cashmere Goats

Cashmere is the world's high-grade textile raw materials,cashmere goat industry is an important part of Inner Mongolia animal husbandry.The wool of cashmere goat is produced by primary hair follicle,and cashmere is produced by secondary hair follicle.This study WGCNA and analyzed the transcriptome data obtained from the research group and screened the WNT10A of hub genes of hair follicle development;screening and identified miRNA capable of regulating WNT10A through bioinformatics,real-time fluorescence quantitative PCR and double luciferase reporter system;Using real-time fluorescence quantitative PCR and Western blot to verify the regulation of miRNA on WNT10A in fetal skin fibroblasts and epithelial cells;CCK8 and Annexin V-FITC/PI were used to detect the effect of Wnt10A on the proliferation and apoptosis of fetal epithelial cells and fibroblasts.The main results were as follows:1.A total of ten co-expression modules(blue,black,green,turquoise,purple,red,brown,yellow,magenta and pink)were obtained though WGCNA analysis,by calculating the characteristics of each module,we can get the specific high expression module blue module in the development of skin and hair follicle 115-135 days in Inner Mongolia cashmere goats;584 candidate hub genes were obtained by calculating gene connectivity and the correlation between genes and module eigenvalues;Then,584 candidate hub genes were analysis by go and KEGG functional enrichment analysis,it was found that WNT10A is a hub gene in the skin and hair follicle development stage of Inner Mongolia cashmere goat2.Nine miRNAs that may regulate WNT10A were screened by bioinformatics and transcriptome data;real time fluorescence quantitative PCR analysis showed that the relative expression levels of chi-miR-130b-3p,chi-miR-301a-3p and chi-miR-532-3p in Inner Mongolia cashmere goats were negatively correlated with WNT10A.The Spearman correlation coefficients(RS)were-0.82,-0.86 and-0.84,respectively,It was preliminarily determined that there was a targeting relationship between chi-miR-130b-3p,chi-miR-301a-3p and chi-mir-532-3p with WNT10A.3.Double luciferase reporter system showed that chi-miR-301a-3p and chi-miR-532-3p could not down regulate the expression of WNT10A3'UTR wild type,indicating that chi-miR-301 a-3p and chi-miR-532-3p and WNT10A did not bind in this experiment;chi-miR-130b-3p successfully downregulated the expression of WNT10A3'UTR wild type,and the effect disappeared after mutation,indicating that WNT10A is the target gene of chi-miR-130b-3p.4.Real 'time fluorescent quantitative PCR and Western blot showed that chi-miR-130b-3p,chi-miR-301a-3p and chi-miR-532-3p could regulate WNT10A expression at mRNA and protein levels in fetal skin fibroblasts and epithelial cells.5.CCK8 and Annexin V-FITC/PI double staining showed that WNT10A may promote the proliferation of fetal epithelial cells and fibroblasts and inhibit the apoptosis of fetal epithelial cells.