The Regulation Of Alpaca Melanocortin-1 By MiR-338-3p

MicroRNAs(miRNAs)are small non-coding RNAs,which can suppress the gene expression by targeting with 3 ’UTR of target genes.It has been reported that many miRNAs targeted regulate the production of melanin in melanocytes.However,the effects of miR-338-3p on the melanogenesis have not been reported.In this study,several biological methods was used to search the relationship between miR-338-3p and MC1R gene,then research the change of the melanin production by miR-338-3p.The main results of the research are as follows:1.The TargetScan and miRBase bioinformatics were employed to predict that the target gene of miRNA-338-3p is MC1R,which can regulate the production of melanin.And the study predicted the miRNA-338-3p target binding site with target gene MC1R.2.The study constructed the dual luciferase reporter construction and eukaryotic expression construction of miR-338-3p for the dual luciferase reporter gene detection system,the experimental group with pmirGLO-MC1R-3’UTR and miR-338-3p,the control group with pmirGLO-MC1R-3’UTR and miR-NC.Then transfected 293T cells.The results show that the relative luciferase activity of experimental group is 0.65 times than that of the control group.we demonstrated that the MC1R gene was a target for miR-338-3p.3.Two groups were as follows:miR group(Transfected the eukaryotic expression construction of miR-338-3p),NC group(Transfected the empty construction of miR-NC).Transfected the constructions to alpaca melanocytes,qRT-PCR were used to measure the mRNA levels of miRNA-338-3p、MC1R、Mitf、Tyr、Tyrp1 and Tyrp2 in miR group and NC group.The results show:(1)The expressive quantities at the mRNA levels of miRNA-338-3p in miR group was 2.23 times than NC group,which was significantly higher than NC group.(2)The expressive quantities at the mRNA levels of MC1R、Mitf、Tyrp1 and Tyrp2 genes in miR group were decreased 0.85、0.702、0.746 and 0.51 times compared with NC group,which were significantly different with NC group(P<0.01);The expressive quantities at the mRNA levels of Tyr genes in miR group was decreased 0.531 times compared with NC group,which was markedly different with NC group(P<0.05).4.48 h after transfected,extracted melanin cell protein,western blotting was used to measure the expressive quantities at protein levels of MC1R、Mitf、Tyr、Tyrp2 in miR group and NC group.The results show:The experessive quantities at protein levels of MC1R、Mitf and Tyrp2 were 0.509、0.289 and 0.486 times compared with NC group,which were significantly different with NC group(P<0.01);The expressive quantities at protein levels of Tyr was 0.696 times compared with NC group,which was observably different with NC group(P<0.05).5.56 h after transfected,The production of melanin in groups,were detected by Microplate Reader.The results show that the rproduction of melanin in miR group is 0.4143048±0.0104589,the production of melanin in NC group is 1.000000±0.0200072,the relative melanin production in miR group was decreased 59%than NC group,which were significantly different with NC group(P<0.01)In conclusion:miR-338-3p can target the MC1R gene and subsequently,down-regulates the Mitf,Tyr,Tyrpl and Tyrp2 genes,thereby inhibits the melanogenesis in alpaca skin melanocytes.The significance of the study is offering for reliable basis on the Regulation of MC1R by miR-338-3p.