Molecular mechanism of miR-2366 targeting DCT gene regulating melanogenesis in Xiang pig melanocytes

【Objective】To clarify the effect of miR-2366 on melanin production in melanocytes by targeting the dopa pigment isomerase gene(DCT), and to lay a theoretical foundation for the analysis of the formation mechanism of “twoblack-sheep” coat color in Jianbai Xiang pigs. 【Method】With the Jianbai Xiang pig, the extraction rate of total melanin in skin tissue and the amount of hair in different colors was determined by enzymatic hydrolysis. The content of different types of melanin in hair of different colors were determined by alkaline dissolution method. The expression levels of miR-2366 and DCT, TYR,TYRP1 were analyzed in different coat color skin tissues by real-time fluorescence quantitative PCR technology. Based on the targeting relationship of miR-2366 and the DCT gene, artificial dual luciferase reporter vector(pmirGL0-DCT-3'-UTR-wt and pmirGL0-DCT-3'-UTR-mut) and miRNA-2366 overexpression vector mimic, inhibitory vector inhibitor and negative control NC, after the transfection of the white pig melanocytes, respectively, the relative expression levels of DCT, TYR and TYRP1 genes and their protein expression levels were determined by real-time fluorescence quantitative PCR and Western blotting, and the total melanin content in the transfected melanocytes was determined by alkaline lysis. 【Result】The melanin extraction rate was extremely significantly higher in the Jianbai Xiang pig black skin tissue than the extraction rate in the white skin tissue(P<0.01,the same below). In the black hair,the total melanin,phaeomelanin and eumelanin contents were extremely significantly higher than those in the white hair;in black skin tissue, relative expression of miR-2366 were extremely significantly higher than in white skin tissue. In the transfection assay, miR-2366 mimic extremely significantly down-regulated the expression of DCT, TYR and TYRP1 genes, significantly down-regulated the expression of DCT, TYR and TYRP1 proteins(P<0.05). The content of melanin in melanocytes decreased extremely significantly by 36.0%. However, miR-2366 inhibitor extremely significantly up-regulated the expression of DCT, TYR and TYRP1 genes and their proteins, and extremely significantly increased the content of melanin in melanocytes by 20.0%. 【Conclusion】The miR-2366 regulates the melanin content in melanocytes by negatively regulating DCT gene expression, and then regulates the formation of Jianbai Xiang pig hair color, suggesting that miR-2366 regulates the production of melanin.