The Study Of Regulation On The Key Genes Of Pigementation By MicroRNA-182-5p And MicroRNA-96-5p In Alpaca Melanocytes

The economic value of mammalian coat is more and more higher,and the researchers pay attention to its traits.The color of coat is one of the important features.The melanin content is the key factor of different coat colors.Through researching the regulation mechanism that about melanin synthesis-related genes,it can provide a theoretical basis for breeding high-quality and rich-bred animals in animal genetic breeding.In order to study the regulatory mechanisms of microRNA-182-5p(miR-182-5p)and microRNA-96-5p(miR-96-5p)on pigment-associated genes in alpaca melanocytes,first,the assay optimized the digestion conditions that cultured alpaca melanocytes in vitro.,iR-182-5p and miR-96-5p were differently expressed in cultured alpaca melanocytes by transfection of mature miR-182-5p and miR-96-5p series.Using the methods of qRT-PCR and immunohistochemistry.The expression levels of TYR,TYRP1,and TYRP2 genes associated with MITF and its downstream hair coloring genes were analyzed.The melanin content was determined by microplate reader.The results showed:the best time for trypsin-digestion of alpaca melanoma cells in vitro was 7 min,the viable cell rate was the highest.The qRT-PCR analysis showed that in alpaca melanocytes,the transfected miR-182-5p mimic group compared to the blank group,the expression of was 72.29 times higher than endogenous miR-182-5p(P<0.001),the expression of MITF mRNA was unchanged(P>0.05),and the expression of TYR,TYRP1 and TYRP2 mRNA was decreased,The difference was extremely significant(P<0.01):The transfected miR-182-5p inhibitor group compared to the blank group,the expression of TYR,TYRP1,and TYRP2 mRNAs were increased,and the difference was extremely significant(P<0.01).The transfected miR-96-5p mimic group compared to the blank group,the expression of miR-96-5p was 55.65 times higher than endogenous miR-96-5p(P<0.001),the expression of MITF mRNA was not changed(P>0.05),the mRNA expression of TYR,TYRP1 and TYRP2 were decreased,the difference was extremely significant(P<0.01);The transfected miR-96-5p inhibitor group compared to the blank group,the expression of MITF mRNA was not changed(P>0.05),the relative expression of TYR,TYRP1,and TYRP2 were increased,and the difference was extremely significant(P<0.01).Immunohistochemical analysis results showed that the transfected miR-182-5p mimic group compared to the blank group,the relative expression levels of MITF,TYR,TYRP1 and TYRP2 protein were decreased,The difference was extremely significant(P<0.01):The transfected miR-182-5p inhibitor group compared to the blank group,the relative expression levels of MITF,TYR,TYRP1,and TYRP2 were were increased,the difference was extremely significant(P<0.01);The transfected miR-96-5p mimic group compared to the blank group,the relative expression levels of MITF,TYR,TYRP1 and TYRP2 protein were decreased,the differences were extremely significant(P<0.01);The transfected miR-96-5p inhibitor group compared to the blank group,the relative expression levels of MITF,TYR,TYRP1 and TYRP2 protein were increased,and the difference was extremely significant(P<0.01).The melanin content in each group was measured using a microplate reader.The results showed that the transfected miR-182-5p and miR-96-5p mimic group compared to the blank group,the melanin content were decreased,the difference was extremely significant(P<0.001);the transfected miR-182-5p and miR-96-5p inhibitor group compared to the blank group,the melanin content were increased,the difference was extremely significant(P<0.001).In summary,the optimal digestion time for trypsin is 7 min;miR-182-5p and miR-96-5p can both targeted the mRNA of the transcription factor MITF,thereby downregulating its downstream genes TYR,TYRP1,and TYRP2,and inhibited melanin synthesis.