Differential Expression Of MiR-27a In The Skin Of White And Brown Cashmere Goats And Identification Of Its Target Genes

China is a country with large output of cashmere and has abundant resources of Cashmere goat breeds,Among which Liaoning Cashmere goat is a native breed in northeast China.This breed has stable genetic traits,high cashmere yield and good cashmere quality,and has the reputation of "soft gold".The highest records of cashmere production were 1800 g for adult males and 845 g for adult females.Cashmere is also an important textile raw material,but the natural color of cashmere is relatively single and cannot meet people's daily needs.Therefore,white wool,which can be dyed into different colors for commercial products,holds the highest value in the textile industry.However,the application of chemical dyes harms human health and pollutes the environment.Therefore,it is an urgent problem to change animal coat color through gene regulation technology and breed more animal varieties with natural coat color.MicroRNAs(MiRNAs)are the length of 19-23 nt single non-coding small RNA,which down-regulate the expression of target genes at the post-transcriptional level and can fine-tune to target genes in almost all animal organs.In recent years,many reports have suggested that miRNAs play a key role in controlling skin morphogenesis,hair follicle development,and coat color forming.Using highthroughput sequencing technology,the researchers found that the expression of miR-27 a was significantly different in white and brown Alpaca skin,which indicated that miR-27 a may be involved in the formation of animal coat color.Therefore,miR-27 a was chosen as the research target in this study to explore its role in coat color formation of Cashmere goats.In this study,the expression differences of miR-27 a in the skin of white and brown cashmere goats were verified by fluorescence quantitative PCR.Then,TargetScan was used to predict the target genes of miR-27 a,and GO and KEGG pathway analysis was used to screen out the target genes related to hair color formation.The expression of mRNA and protein levels of target genes were detected by fluorescence quantitative PCR and Western blot,and the expression sites of target proteins in white and brown Cashmere goats skin were detected by immunohistochemistry.In addtion,the targeting relationship between miR-27 a and its candidate target genes was detected using dual luciferase reporter assay.After transfection of miR-27 a mimics into HEK293 T cells,the mRNA and protein expression levels of target genes were detected.Through the above studies,the role of miR-27 a in the coat color formation of Cashmere goats was clarified,providing a theoretical basis for the subsequent artificial modification of animal coat color.The results are as follows:1.The expression of miR-27 a in skin of white and brown cashmere goats was detected by fluorescence quantitative PCR.The results showed that the expression of miR-27 a in skin of white cashmere goats was significantly higher than that of brown cashmere goats.2.Target genes of miR-27 a were predicted by TargetScan and target genes WNT3 A and KITLG related to coat color formation were screened through GO and KEGG pathway analysis.3.The expression of WNT3 A and KITLG in white and brown Cashmere goats skin was detected by fluorescence quantitative PCR.The results showed that the expressions of WNT3 A and KITTL in white skin were significantly lower than that in brown skin.4.Western blot results showed that WNT3 A and KITLG protein expressions in white Cashmere goats skin were significantly lower than brown Cashmere goats skin.5.Immunohistochemical results showed that WNT3 A and KITLG protein in white and brown Cashmere goats skin tissue were widely expressed in epidermis(basal layer),inner and outer root sheath,but the expression level in white skin was significantly lower than that in brown skin,and WNT3 A KITLG protein was also expressed in the dermal papilla of brown skin.6.The melanin content in the skin of white and brown Cashmere goats was detected,and it was found that the melanin content in the skin of white Cashmere goats was significantly lower than that of brown Cashmere goats.7.The dual luciferase reporter gene verified the targeting relationship between miR-27 a and target genes(WNT3A and KITLG).The results showed that the luciferase activity of the experimental group(co-transfection of psiCHECKWNT3A/KITLG-WT and miR-27 a mimics)was significantly lower than that of the control group(co-transfection of psiCHECK2-WNT3A/KITLG-WT and negative control),indicating that WNT3 A and KITLG were the target genes of miR-27 a.8.In vitro experiments,miR-27 a mimics(mi)and negative control(nc)were transfected into HEK293 T respectively,and the expressions of miR-27 a and target genes were detected at the mRNA level.It was found that the expression of miR-27 a in the over-expressed group was significantly higher than that in negative control group,while the expression of target genes was opposite.The detection of protein level showed that the expression level of target protein was consistent with mRNA level.In conclusion,miR-27 a is involved in the coat color formation of Cashmere goats by down-regulating the expression of WNT3 A and KITLG,which provides a theoretical basis for gene regulation to change the coat color of animals and cultivate more natural color wool-producing varieties.