The Function Of Lpa-miR-nov-66on The Melanogenesis In Melanocytes Of Alpaca(Lama Pacos)

In mammals, both hair and skin colors depend on the type, quantity and distribution of the melanins in melanocytes. MicroRNAs (miRNAs) are a class of small single-stranded RNA length of about22nt, involving in the multiple physiological and biochemical process of plants and animals through the transcription regulation mechanism. miRNAs play important roles in the regulation of mammalian melanogenesis and color, and more and more miRNAs are being found to elucidate the mechanism. The object of this study is to get the function of lpa-miR-nov-66, a noval miRNA expressed in alpaca skin. sGC was one of the targets of lpa-miR-nov-66, which was verified by the luciference reporter vector. The over-expression plasmid of lpa-miR-nov-66was constructed and transfected into alpaca melanocyte to explore the function of lpa-miR-nov-66in melanogenesis by detecting the expression level of mRNA and protein in sGC, MITF, TYR, TYRP1, the melanin content, the content of cAMP and cGMP in melanocytes by ELASA assay. All those results suggested that the mechanism of lpa-miR-nov-66in the melanogenesis. The results were as following:1The real-time PCR showed that the expression level of sGC, MITF, TYR, TYRP1mRNA was5.83,0.98,0.75,0.53fold changes, respectively, compared with the control group (P<0.05).2Western blotting results showed the protein expression level of sGC, MITF, TYR, TYRP1was3.15,0.50,0.187,0.621fold changes in melanocytes transfected lpa-miR-nov-66plasmid, respectively, compared with the control group (P<0.05).3The melanins quatity test results showed the melanins quatity transfected by lpa-miR-nov-66plasmid was0.7fold changes (P<0.05), compared with the control group.4The quatity of cAMP and cGMP was measured by ELISA assay. The results showed the production of cGMP was increased by2.05fold and the production of cAMP was decreased0.57fold in melanocytes transfected by lpa-miR-nov-66plasmid, compared with the control group (P<0.05).5Dual luciferase reporter assays showed that sGC is one of the target genes of lpa-miR-nov-66. The result showed the activity of reporter in transfected group was0.69fold changes compared with the control group (P<0.05).All those results showed that lpa-miR-nov-66might regulate the mechanism of melanogenesis by cAMP pathway in alpaca melanocytes. It will supply the molecular mechanism of melanogenesis or coat color formation of alpaca.