Effects Of SOX5 On MITF-M Expression And Melanogenesis In Mouse Skin Melanocytes

SOX5 plays an important role in the expression of pigment gene and the promotion of melanogenesis in melanocyte.We discovered that the first SOX gene regulates the development of melanocytes ten years ago To date,we have successively discovered that SOX5,SOX9,SOX 10 and SOX 18 are all involved in the regulation and adjustment of key melanocyte genes as transcription factors.This article studies the important role that SOX5 plays in mouse skin color.In this study,in order to investigate the role of SOX5 in mouse skin color,three C57BL/6 strains of black,brown,and gray mice were each randomly selected from vigorous hair follicle growth at 12 days after birth The differences of SOX5 expression in the skin of black,brown and gray mice were analyzed by fluorescence quantitative PCR,Western blotting and immunohistochemistry.The SOX5 eukaryotic expression vector was constructed,the mouse melanocytes were transfected using in vitro transfection method,the expression levels of genes related to chromogenesis were detected and the melanin content was measured.The main research contents and results are as follows(1)The differences of SOX5 expression in mouse skin of black,brown and gray were analyzed by real-time quantitative PCR,Western blotting and immunohistochemistry.Immunohistochemistry showed that SOX5 had SOX5 protein expression in the mice skin of different coat colors,and its main expression site was the outer root sheath of hair follicles.The fluorescence intensity in black and gray mouse skin was significantly higher than in brown mouse skin.The results of qRT-PCR showed that the mRNA expression levels of SOX5 in black and gray mouse skin were 8.12 times(P<0.01)and 2.87 times(P<0.01)compared with brown mouse skin,respectively.Western blotting results showed that the total protein in the mice skin of different coat colors was immunologically positive by reacting with SOX5 antibody.By analyzing the protein bands,the protein levels of SOX5 in black and gray mouse skin were 2.46 times(P<0.01)and 1.91 times(P<0.01)compared with brown mouse skin,respectively.It can be seen that there was difference in the expression of SOX5 with different coat color mice skin,indicating that SOX5 has certain influence on the formation of coat color.(2)The mouse SOX5 and MITF-M eukaryotic expression vectors were constructed.Overexpressions of SOX5 and MITF-M in mouse melanocytes were used to demonstrate that SOX5 affected the expression of MITF-M in mouse skin melanocyte by using the reverse verification method.Overexpression of SOX5 results showed that the MITF-M mRNA was increased by 1.50 times(P<0.05)and the protein was increased by 1.95 times(P<0.01)compared with the empty vector group.Overexpression of MITF-M results showed that the mRNA of SOX5 was reduced by 1.34 times(P<0.05)and the protein was decreased by 2.05 times(P<0.01).Both showed that overexpression of SOX5 promoted the expression of MITF-M while overexpression of MITF-M inhibited the expression of SOX5(3)In order to further verify that SOX5 regulates melanocytes through MITF-M,we verified that mRNA expression differences,protein relative levels,and melanin contents were analyzed by SOX5 in the major pigment gene TYR,TYRP1,TYRP2,PMEL,and OA1 after overexpression of SOX5.The results showed that the mRNA,protein expression and melanin content of the corresponding genes were increased compared with the control group after overexpression of SOX5The above results indicate that overexpression of SOX5 gene in mouse skin melanocytes can promote the expression of MITF-M,TYR,TYRP1,TYRP2,PMEL and OA1,and thus increase the production of melanin.