The Influence Of Oct-1 On Melanogensis In Mice Melanocytes

Objective:A Study shows that ethanol activate induction of Slc7a11 gene expression via inhibition binding activity of transcriptional reprseeor octamer-binding transcription factor 1(Oct-1).The formation of brown pigment was regulated by Slc7a11 gene in sheep.According to these results,we speculate that Oct-1 could play an important role in process of melanin.However,whether it can regulate the production of melanin is not certified.The aim of the study was to clone the octamer-binding transcription factor 1 and detect the expression of genes and proteins,to investigate whether over-expression of Oct-1 regulated the major genes of coat color in melanocytes of mouse at the transcriptional levels and to explore its influence on the formation of melanin.Methods:The mouse Oct-1 gene sequence was analyzed by bioinformatics method.The CDS region in Oct-1 gene were cloned from melanocytes of mouse by primers and PCR to build a eukaryotic expression vector.After over-expression of Oct-1,the contents of melanin in melanocytes was detected by spectrophotometer.The level of major genes were detected using real-time PCR and the proteins of MITF,TYR,TYRP-1 and TYRP-2 were detected using western blot.Results:(1)The length of mouse Oct-1 gene was 2 313 bp analyzed by bioinformatics method.Oct 1 was found can regulate the gene of coat color by Transfec promoter analysis software.Which regulate the expression of 13 colour genes in mouse melanocytes.There were binding sites of Oct-1 on the promoters of MITF,TYR,TYRP1,TYRP2.(2)The cDNA sequence of Oct-1 gene was obtained.Eukaryotic expression vector was successfully constructed with specific TYRP-2 gene promote of mouse and a startup report gene of green fluorescent protein.Its sequence been verified by sequencing.(3)Melanin cells in mouse were cultivate and were transfected by Oct-1.Under the fluorescence microscope,green fluorescence could be identified in melanocytes of mouse.Real-time PCR results showed that Oct-1 mRNA was significantly increased,witch indicated Oct-1 high transfection efficiency.The contents of melanin in melanocytes were reduced.These results show Oct-1 could Inhibit melanin synthesis.(4)The results of Real-time PCR showed that MITF and TCF mRNA were significantly reduced.The expressions of Ras,Frizzled,ERK2 and TYRP-2 mRNA did not change.TYR,a-MSH,AC,c-kit and ETB-R mRNA were significantly increased.TYRP-1?ET-1 and CAM mRNA were increased.The results of western blot showed that MITF protein expression was significantly reduced;TYR and TYRP-1 protein expression were were increased;TYRP-2 protein expression did not change.Conclusion:After over-expression of Oct-1,the expression of MITF and TCF was reduced,while that of TYR,TYRP-1,a-MSH,AC,c-kit,ET1,ETB-R and CAM was increased.This study indicates that Oct-1 may could regulate the production of melanin by mediating the expression of major genes of coat color through regulating genes involved in the formation process of coat color.