MiR-370-5p Targets The Molecular Mechanism Of MAP3K8 To Inhibit Sheep Melanocytes Proliferation And Regulate Melanin Production

Sheep coat is the main raw material of the wool textile industry,because the natural coat color of sheep is relatively single,can not meet the current people’s pursuit of color,so the acquisition of natural high-quality sheep leather products,reduce the use of chemical dyes has become the focus of researchers.The color of an animal’s coat is determined by melanin produced by melanocytes in the papillae of hair follicles.MAPK kinase kinase 8(mitogen-activated protein kinase kinase 8;MAP3K8),which activates a large number of downstream molecules,including MEK,ERK and JNK,involved in melanin production.Previous studies have found that there are significant differences in the gene expression levels of miR-370-5p in the fur of alpacas of different coat colors,which is speculated to be involved in the production of animal coat color.Therefore,this study took white and brown sheep skin as the research object(n=3)to explore whether there is a regulatory relationship between the miR-370-5p-mediated MAP3K8 regulatory pathway and the animal coat color phenotype.The test methods and results are as follows:1 The expression of miR-370-5p in white melanocytes was significantly higher than that in brown melanocytes by RT-q PCR detection.miR-370-5p overexpression vector was constructed to transfect sheep melanocytes;The changes of melanin production indexes were detected by overexpression vector transfection into melanocytes,and the cell proliferation rate,melanin content,and tyrosinase activity in the treated group were significantly reduced compared with the control group.2 Mi Rbase software predicted that miR-370-5p and MAP3K8 have targeted binding site;The results of homology analysis showed that the targeted binding sites of MAP3K8 and miR-370-5p were highly conserved among sheep,cattle,mice and humans;The results ofdiluciferase experiment showed that there was a targeted adsorption relationship between miR-370-5p and MAP3K8;RT-q PCR and Western blot assays showed that overexpression of miR-370-5p significantly inhibited the expression of m RNA and protein of MAP3K8.3 In situ hybridization assays showed that MAP3K8 m RNA was located in hair follicles and cytoplasm.4 Three pairs of MAP3K8 interference vectors were constructed,and the RT-q PCR screening results showed that the interference effect of si RNA-710 was stable and in line with expectations,and it continued to be used in subsequent experiments.The test results of melanin production related indicators are displayed with the control group,the cell proliferation rate,melanin content and tyrosinase activity of the transfected si RNA-710 group were significantly reduced.5 Construction of MAP3K8 substitute,transfection of melanocytes,RT-q PCR and Western blot to detect the efficiency of MAP3K8 substitute,in line with expectations;detection results are displayed that the cell proliferation rate,melanin content,and tyrosinase activity of the MAP3K8 substitute group were significantly reduced compared with the control group.Based on the above results,it is concluded that miR-370-5p inhibits the proliferation of sheep melanocytes and inhibits melanin production by targeting MAP3K8.