| Cashmere goat hair is composed of two different types of hair:short and fine unmedullated hair and long and thick medullated hair.Unmedullated hair,also known as cashinere,is produced by secondary hair follicles.The length and thickness of cashmere directly determine the economic value of cashmere.The formation of cashmere fiber is affected by many signal pathways,transcription factors,keratin and related proteins.Foxql is a member of the Forkhead Box family of proteins involved in DNA repair,embryonic development and the regulation of metabolic balance in organisms.Studies have shown that Foxql plays an important role in the hair follicle cycle of cashmere goats,but its regulatory mechanism has not been reported in detail.Thereforc,in this study,the differentially expressed genes Foxql and KAP8 were screened out based on the transcriptomic sequencing results of skin tissue in hair follicle induction and differentiation stages of Shanbei white cashmere goats,and the effect of Foxql on the proliferation of hair follicle stem cells wa studied through adenovirus overexpression system.The regulation mechanism of Foxql was studied by dual luciferase assay.RT-qPCR and immunofluorescence.The main results obtained are as follows:1.Differentially expressed Foxql and KAP8 genes were screened by transeriptome sequencing data from E65 d and E120 d of Shanbei White cashmere goat.RT-qPCR results showed that the expression levels of Foxql and KAP8 in skin tissues of cashmere goats were significantly higher than those in muscle and heart tissues.The expression level at E120 d was significantly higher than that at E65 d,which was consistent with the sequencing results.2.Foxql overexpressed adenovirus vector was constructed and successfully infected hair follicle stem cells.MTT results showed that compared with the control group,the activity of Foxq1 uverexpressed hair follicle stem cells was significantly enhanced.EdU results showed that the proportion of positive cells after Foxql overexpression was significantly higher than the control group.FCM results showed that the number of hair follicle stem cells in S and G2 phases increased after Foxql ovcrcxprcssion.The overexpression of Foxq1 could significantly promote the expression of antioxidant stress genes GSR and SOD2.and inhibit the expression of apoptosis related gene BCL2.These results suggested that Foxq1 could promote the proliferation of hair follicle stem cells.3.JASPAR database showed that KAP8 was the potential target gene of transcription factor Foxq1.Dual luciflucase assay showed that the core promoter sequence of KAP8 was KAP8-818,and the promoter activity of KAP8 gene decreased significantly after Foxq1 overexpression.RT-qPCR results showed that the expression of KAP8 decreased after Foxq1 uverexpression,and the above results indicated that Foxq1 targeted down-regulated the expression of KAP8 gene.4.The target genes of Foxq1 were predicted by TRRUST database and analyzed by GO and KEGG enrichment Foxq1 target genes were found to be enriched in Wnt/β-catenin signaling pathway.RT-qPCR results showed that Foxq1 overexpress ion could significantly promote the expression of key genes CTNNB1,TCF3,CCND1 and c-MYC in Wnt sign aling pathway.The results of immunofluoresccnce were consistent with those of RT-qPCR,suggesting that Foxq1 could enhance the activity of Wnt signaling pathway.This study clarified the effect of transcription factor Foxq1 on the proliferation of hair follicle stem cells in cashmere goats,and revealed the molecular regulatory mechanism between Foxql and KAP8 md Wnt signaling pathways,The results provided theoretical basis for clarifying the regulatory role of Foxq1 in hair follicle development in cashmere goats. |
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