Differential Expression Of MiRNA-203 In Hair Follicle Development Cycle Of Cashmere Goats And Identification Of Target Gene Function

Liaoning Cashmere goats rank among the top in the world in terms of villus quality and cashmere yield.The cashmere produced is a rare special animal fiber and precious textile raw material.Recently,the domestic cashmere and goat data was released by the national bureau of statistics show that domestic raw cashmere output has been declining year by year since 2014.The self-produced cashmere raw materials are far from meeting the production and export needs of local enterprises and foreign-invested enterprises.As an important part of hair growth and development,the hair follicle has the characteristics of self-renewal and periodic growth both in embryonic stage and after birth,and the whole process involves multiple genes.Therefore,it has become a research goal in recent years to improve the quality and yield of cashmere by studying the mechanism of regulating hair follicle development cycle.miRNAs are a kind of endogenous single-strand non-coding small RNA that inhibit its translation or cause degradation by acting on non-coding regions of target gene mRNA,thereby participating in the basic biological processes such as the development,cell proliferation,differentiation and apoptosis of organisms.In recent years,the research team has carried out systematic studies on miRNA in hair follicle development of Cashmere goats and fine wool sheep.Through high-throughput sequencing,it was found that miRNA-203,which is specifically expressed of epithelial tissues,is involved in the development of hair follicles,but how it plays a regulatory role in hair follicle development of Cashmere goats.There are no relevant reports on the regulation.In this study,miRNA-203 was selected as the research target.Target genes of miRNA-203 were predicted using online biological software such as miRBase,microRNA.org,TargetScanHuman 7.1,and microRNA target prediction,and target genes related to hair follicle development were screened out.At the mRNA level,protein level and cell level,the expression differences of miRNA-203 and candidate target genes in the hair follicle development of Cashmere goats were verified,and the binding sites of miRNA-203 to the target genes were determined.The regulation mechanism of miRNA-203 on the hair follicle development cycle of Cashmere goats was clarified,and the role of miRNA in the hair follicle development cycle was perfected.It provides a new idea for the breeding of high-quality Cashmere goats and improving the yield and wool quality of Cashmere goats.The specific research results are as follows:1.The target genes of miRNA-203 were predicted using online bioinformatics software miRBase,microRNA.org,TargetScanHuman 7.1 and microRNA target prediction,and the target genes related to hair follicle development were screened out: DDOST,NAE1 and GLYATL2.2.Homology analysis: DDOST,NAE1 and GLYATL2 sequencing showed that the homology with sheep,cattle and other species was up to 95%,indicating that the target gene sequences were genetically conserved.3.At the mRNA level,miRNA-203,DDOST,NAE1 and GLYATL2 were identified,and the results showed that miRNA-203 was expressed in anagen and telogen of hair follicle development in Cashmere goats,and the expression level of miRNA-203 in telogen was significantly higher than that in anagen.On the contrary,the expression levels of DDOST,NAE1 and GLYATL2 in anagen of Cashmere goats were higher than those in telogen.The opposite was true for candidate target genes.4.The protein identification of target genes DDOST and NAE1 showed that DDOST and NAE1 were up-regulated in anagen of Cashmere goats,and the results were consistent with the fluorescence quantitative results.The results of in vivo experiments showed that miRNA-203 was involved in the hair follicle cycle development of Cashmere goats through DDOST and NAE1.5.The Dual-luciferase reporter gene system identified the specific binding sites of miRNA-203 to target genes DDOST and NAE1,and the results showed that there are miRNA-203 target sites on the 3'UTR of DDOST and NAE1.6.In vitro experiments were conducted to transfect cells.And miRNA-203,DDOST and NAE1 after the over-expression of miRNA-203 were verified at mRNA and protein levels.The results showed that the expression of miRNA-203 in mimics group was significantly higher than that in the negative control group,while the expression of target genes was contrary.miRNA-203 significantly down-regulated the expression of DDOST and NAE1.In summary,miRNA-203 negatively regulates the involvement of target genes(DDOST and NAE1)in hair follicle cycle development,which can be used as a method to further select high-quality sheep varieties.