Study Of The Function And Mechanism Of MiR-27a-3p On Melanogenesis In Mouse And Sheep

Coat color is determined mainly by the synthesis and transport of melanin, which is synthesized in melanosomes of melanocytes. Melanogenesis is regulated by hundreds of genes. Tyrp2 is the third member of tyrosinase family which is key enzyme for melanogenesis and can catalyze dopachrome into 5, 6-Dihydroxy indole carboxylic acid (DHICA) to promote melanogenesis. Compared with genes, there are a few of studies about miRNAs regulating melanin synthesis. MiRNAs participate in cytogenesis, proliferation, differentiation, apotosis, tumor matastasis and melanogenesis by inhibiting target gene expression in post-transcription level. Target gene of miRNAs is mainly predicted through bioinformatics software and verified by experiments. Aim To confirm the function and mechanism of miR-27a-3p to melanogenesis in mouse and sheep and to find melanocyte specific promoter. Target gene Wnt3a of miR-27a-3p was predicted in three databases. Wnt3a 3’UTR dual luciferase reporter vector and miR-27a overexpression vector were constructed. Then co-transfect HEK 293T cells with the two vector to verify relationship of and Wnt3a. Expression levels of miR-27a-3p and Wnt3a in gray & brown mouse skin were quantifed using QRT-PCR. MiR-27a-3p mimic, inhibitor and their negative control were transfected into mouse melanocytes. MiR-27a-3p, Wnt3a mRNA and protein and melanin content were detected at 48 h after transfection. Promoter region of sheep Tyrp2 gene was predicted based sequence from NCBI and UCSC. Eukaryotic expression vectors of sheep Tyrp2 gene promoter with various fragments were constructed, sequenced and analyzed its regulation elements and binding sites of transcription factor. Sheep melanocytes were transfected with Tyrp2 promoter expression vectors with different fragments to determine their activities. Sheep pri-miR-27a was fused together with Tyrp2 promoter expression vectors and transfected melanocytes. Expression of miR-27a-3p and melanin content were detected in pri-miR-27a and negative control group.1. Target genes of miR-27a-3p were predicted in three databases. Prediction shows that Wnt3a is a target gene of miR-27a-3p. Luciferase assay show that relative luciferase activity of co-transfected pri-miR-27a and Wnt3a 3’UTR decreases 41% than others.2. Expression of miR-27-3p is significantly high in mouse melanocytes transfected with miR-27-3p mimic than others. Expressions of Wnt3a mRNA among different groups are no significant difference, while Wnt3a protein amount in miR-27-3p mimic group is significantly low and in miR-27-3p inhibitor group is siginificantly high. Trend of melanin content is similar to Wnt3a protein amount.3.834 bp (-807～+27 bp) promoter region of sheep Tyrp2 has the highest activity.4. Expression of miR-27-3p is significantly high in sheep melanocytes transfected with miR-27-3p than NC.5. Compared to NC, melanin content in sheep melanocytes trasfected with miR-27-3p is siginificantly high.In conclusion, Wnt3a is a target gene of miR-27a-3p. MiR-27a-3p inhibits Wnt3a expression by post-transcriptional regulation. MiR-27a-3p inhibits melanogenesis both in mouse and sheep.