Effect Of Silencing MicroRNA-508 By STTM On Melanogenesis In Alpaca(Vicugna Pacos)

MicroRNAs(miRNAs)have been shown to play important roles in the regulation of melanogenesis and hair color formation in mammals.Short tandem target simulation(STTM)has been used to effectively block micro RNA function in plants and animals.Our previous studies have shown that miR508-3p plays a functional role in regulating melanogenesis in alpaca melanocytes by directly targeting the small eye deformity-associated transcription factor(MITF).To verify the effect of miR508-3p function on melanogenesis in alpaca melanocytes,miR508-3p was blocked using STTM technology here.In this study,the effects of miR508-3p function on melanogenesis in alpaca melanocytes were verified by bioinformatics analysis,dual luciferase reporter assay,quantitative real-time PCR assay,Western blotting,Fontana-Masson staining,cellular immunochemical assays and melanins contents assay.The results are as follows:1.Using miRBase and Target Scan target gene bioinformatics analysis,it was found that one of the target genes of miR508-3p is SOX6,which is co-expressed in 293 T cells by SOX6 3’UTR double fluorescent reporter vector(PGL0-SOX6)and STTM-miR508-3p(PGL0-STTM-miR508)overexpression vector.The activity of luciferase was 1.38 folds change compared to the control group.Therefore,it is speculated that SOX6 was one of target genes of miR508-3p.2.In the alpaca melanocytes,after overexpressing STTM-miR508-3p,the m RNA of miR508-3p was detected by quantitative real-time PCR.The results showed that STTM-miR508-3p overexpression down-regulated the expression of miR508-3p m RNA.Therefore,it is speculated that STTM-miR508 might regulate in inhibiting the express melanogenesis in melanocytes.3.After overexpressing STTM-miR508-3p in alpaca melanocytes,the target gene SOX6,the downstream gene Cyclin D1,CREB,the main transcript factor MITF,were detected by quantitative real-time PCR,western blotting and cellular immunochemical assays.Also,m RNA and protein expression of TYR,TYRP1,TYRP2 and the localization of target gene SOX6 in alpaca melanocytes were examined.The results showed that STTM-miR508-3p overexpression up-regulated the m RNA expression and protein levels of SOX6,Cyclin D1,CREB,MITF,TYR,TYRP1 and TYRP2 genes.The target gene SOX6 positivereaction in alpaca melanocytes was mainly distributed in the cytoplasm.4.In the alpaca melanocytes,after overexpressing STTM-miR508-3p,the alki-total melanins contents,phenomelanin contents and eumelanin contents of melanocytes were detected.The experimental results showed that overexpression of STTM-miR508-3p caused a significant increase of total melanins content,phenomelanins content and eumelanins content.Fontana-Masson staining revealed a significant increase in melanins granues produced in melanocytes.Conclusion: STTM-miR508-3p regulate the expression of hair color related genes CREB,MITF,TYR,TYRP1 and TYRP2 by targeting SOX6 to regulate the downstream gene Cyclin D1,which affected the change of melanins content.