Study On The Tissue Expression Of Goat MiR-29a-5p And Melanogenesis Function

Studies reported that more than 370 genes involved in coat color formation of tissues and organs,which is regulated by melanocortin receptor-1 / mouse gray gene / ?-melanocyte stimulating hormone signaling pathway,Wnt/?-catenin signaling pathway,stem cell factor/c-Kit signaling pathway,and melanocortin in the endothelin /PKC signaling pathway Gene 1,microphthalmia associated transcription factor,tyrosinase family,?-melanocyte stimulating hormone,Agouti signaling protein and so on.We recently conducted small RNA sequencing in the skin tissues of Youzhou dark goats and Yudong white goats revealed that miR-29a-5p was differentially expressed.In this study,the tissue expression profile of miR-29a-5p in goats was first established,analysis and compared of expression between different color skins tissues in Youzhou dark goat(black skin)and white goats;And then,the expression characteristics of miR-29a-5p and pigment related genes in melanoma cell line(B16f10)were detected.Moreover,the effects of miR-29a-5p mimic and inhibitor on the physiological analyse of melanocytes were detested by MTT,scratch and melanin content test.Finally,Asip and Kitl were verified by target gene prediction and dual luciferase.The main results as follows:1.miR-29a-5p was expressed in goatheart,liver,spleen,lung,kidney,leg muscle,skin and cerebrum,among which the expression level is highest in cerebrum and the lowest in skin and lung tissues,and the expression of miR-29a-5p in the skin of Youzhou dark goats was extremely significantly lower than Yudong white goats(P<0.01).2.The expression level of miR-29a-5p increased with the prolongation of B16f10 cell culture time.The analysis of differentiation status revealed that the expression of miR-29a-5p was not significantly different in cells on the 4th,6th and 8th day after differentiation(P>0.05),but it was significantly lower than that on the second day of differentiation(P<0.01).3.miR-29a-5p mimic and inhibitor have significant effects on cell viability(P<0.05),and the effect on cell melanin production is not significant(P> 0.05).Transfection of miR-29a-5p mimic promoted cell proliferation and migration,but there was no significant difference in the differentiation of melanocytes(P>0.05);and the expression of Pax3 mRNA in inhibitor miR-29a-5p expression was significantly increased(P<0.05).4.The dual luciferase reporter assay confirmed that there was no target relationship between miR-29a-5p and Asip gene,and Kitl was the target gene of miR-29a-5p.The results showed that the low expression of miR-29a-5p in melanin-deposited blackskin was negatively correlated with melanin deposition,indicating that inhibition of miR-29a-5p promoted melanin deposition.Overexpression of miR-29a-5p promotes cell proliferation and migration.In addition,miR-29a-5p can target the binding of a target site on the 3'UTR of Kitl to regulate expression of the gene.