MicroRNA-2366 Regulates Melanogenesis By Targeting Dopachrome Tautomerase In Melanocytes Of Xiang Pigs

As an important animal variety characteristic,hair color plays a crucial role in breeding.Recently,more and more studies have confirmed that miRNA plays an important role in the generation of mammalian hair color,but the regulatory mechanism of miRNAs on pig coat color has been less studied.The DCT gene is one of the tyrosine family members and is an important candidate gene in the melanin production process.The coat color phenotype of pigs is closely related to melanin,Jianbai Xiang pig has the coat color as head and tail black,the middle white,called "two-head-black" hair color phenotype,further study of melanin deposition mechanism is conducive to the breeding of local characteristics in Guizhou.In this study,through the previous transcriptome sequencing technology,the expressions of ssc-miR-2366 and DCT were different in the skin tissues of Xiang pigs with black and white coat,and it was speculated that it was associated with the skin melanin deposition of Xiang pigs,so a follow-up study was conducted.To determine the content of different kinds of melanin in different color hair and skin tissues,by analyzing the expression levels of ssc-miR-2366 and DCT and its upstream genes TYR,TYRP1 in different coat color by quantitative real-time PCR in skin tissues,combined analysis of the effects of ssc-miR-2366 and DCT on melanin deposition in the skin of fragrant pigs;The skin melanocytes were obtained and identified by Dispase Ⅱ enzyme and 0.25%trypsin/EDTA;Bioinformatics website was used to screen out DCT as one of the target genes of ssc-miR-2366,and the targeting relationship was verified by dual-luciferase detection technology;After transfection of ssc-miR-2366 mimic,inhibitor and NC groups,the m RNA and protein expression levels of DCT,TYR and TYRP1 were measured using quantitative real-time PCR and Western Blot techniques.The detection of total melanin in each transfection group were measured.This study aimed to resolve the molecular mechanism by which ssc-miR-2366 targeted DCT regulates melanogenesis in pig melanocytes.The test results are shown as follows:1.Correlation analysis of ssc-miR-2366 and DCT with skin melanin deposition in pigs.We found that ssc-miR-2366 expression was significantly lower in black skin tissue than in white skin tissue.However,the expression levels of DCT,TYR and TYRP1 in black skin tissue were significantly higher than that in white skin tissue;Compared to the white skin,the total melanin content in the black skin was significantly up-regulated;In the black hair,the content of total melanin,brown melanin and eumelanin is significantly higher than that of white hair;Pearson’s correlation analysis showed a negative correlation between ssc-miR-2366 expression and the content of various melanin classes;The DCT and TYR,TYRP1 expression and melanin content of various classes were positively correlated.To reveal the involvement of ssc-miR-2366,DCT and TYR and TYRP1 in regulating melanin deposition in Xiang pig skin and hair.2.In vitro isolation,culture and characterization of fragrant pig skin melanocytes.The skin melanocytes were successfully isolated and cultured in vitro by DispaseⅡenzyme and 0.25% trypsin/EDTA.The morphological structure,growth curves,specific gene,protein expression and dopa staining showed that the melanocytes maintained normal biological function.In vitro Xiang pig skin melanocyte culture system was successfully established,which provides an ideal cell model for the subsequent functional study of hair color gene.3.The DCT gene was targeted by ssc-miR-2366.Bioinformatic analysis revealed that DCT gene was one of the target genes of ssc-miR-2366 and the target binding site is in the DCT-3’UTR.The results of the dual luciferase assay showed that the fluorescence activity of the pmir GLO-DCT3’UTR-wt and ssc-miR-2366 mimic was compared with the pmir GLO-DCT 3’UTR-wt and NC,indicating that ssc-miR-2366 can be targeted to the 3’UTR of DCT gene.4.Targeted inhibition of DCT gene expression by ssc-miR-2366 subsequently affects melanin production in porcine melanocytes.Results were shown for ssc-miR-2366 mimic,inhibitor and NC transfection with pig melanocytes:the m RNA and protein expression of DCT,TYR and TYRP1 were significantly decreased in melanocytes overexpressing ssc-miR-2366,while the m RNA and protein expression of DCT,TYR and TYRP1 were significantly increased in the inhibitor group.Overexpression of miR-2366 reduced the production of total melanin in melanocytes by 36.0%,and the inhibition group by 20.0%.Showed that ssc-miR-2366 could target the DCT gene to affect melanin production in pig melanocytes.Overall,the results showed that ssc-miR-2366 could affect the melanin production in the Xiang pig melanocytes by targeting the inhibition of DCT gene expression.