MicroRNA-5110 Regulates The Pigmentation By Co-targeting The Wnt1 And MLPH In Melanocytes

Pigmentation involves two processes including melanocytes produce melanin and melanin transport to surrounding keratinocytes.The process of pigmentation is complex and multiple genes and microRNAs have been found to regulate this process.The analysis of miRNA expression profiles of different coats of alpaca skin revealed that microRNA-5110 was differentially expressed and may be associated with hair color formation.In this study,we have revealed microRNA-5110 regulates pigmentation mechanism by using bioinformatics analysis,target gene verification,dual fluorescent reporter experiments,fluorescence quantitative PCR experiments,western blottingting experiments,tissue immunochemistry and cell immunochemistry experiments,in situ hybridization experiments,and deep sequencing.The experiment contents and results are as followed:1 miRBase and Targetscan predict that Wntl and MLPH are two target genes of microRNA-5110.Dual luciferase reporter assays showed that MLPH and Wntl are two of the target genes of microRNA-5110.The result showed the activity of reporter in transfected group was 0.42 fold changes and 0.53 fold changes compared with the control group,respectively(P<0.05).2 The expressing vector of microRNA-5110 and negative control were transfected into alpaca melanocytes.Overexpression of microRNA-5110 in melanocytes resulted in the reduce in the levels of mRNAs encoding MLPH,Wntl,Rab27a,myosin Va,MITF,tyrosinase(TYR),and tyrosinase-related protein 1(TYRP1),while the level of tyrosinase-related protein 2(TYRP2)mRNA was significantly increased.western blotting analysis showed that the expression of MLPH,Wntl,Rab27a,myosin Va,MITF,TYR and TYRP1 proteins was significantly reduced,while the expression of TYRP2 protein was significantly increased in melanocytes overexpressed by microRNA-5110.3 Establishment of microRNA-5110 transfected co-cultured melanocytes and keratinocytes,the melanin in the melanocytes and keratinocytes were significantly reduced by Fontana-Masson staining;after subcutaneous injection of the microRNA-5110 overexpression vector,the mouse hair color phenotype was changed.Overexpression of microRNA-5110 in mouse resulted in the reduce levels of MITF,TYR,and TYRP1 mRNA,while the level of TYRP2 mRNA was significantly increased.western blotting analysis showed that the expression of MLPH,Wntl,Rab27a,myosin Va,MITF,TYR and TYRP1 proteins was significantly reduced,while the expression of TYRP2 protein was significantly increased in mouse overexpressed by microRNA-5110.4 In order to find more melanin synthesis and transport mechanism,melanocytes overexpressing microRNA-5110 were deeply sequenced.The results showed that there are four molecular pathways:melanogenesis pathway,MAPK pathway,Wnt signaling pathway and cAMP signaling pathway.Of these,48 were up-regulated genes and 212 were down-regulated genes.qRT-PCR shown the different expression of DKK1,gp100,IGF1R,CDK5,Ednrb,Kitl,Myc and sGC mRNA and MAPK signaling pathway mRNA,the results are consistent with RNA sequencing results.5 Quantitative real time PCR analysis of sGC showed that the expression of sGC mRNA is higher in white sheep skin relative to black sheep skin with significant difference(P<0.01).Using a rabbit polyclonal anti-sGC antibody,an immune reactive band corresponding to sheepsGC protein was detected in the skin samples by western blottingting analysis,and the expression of sGCprotein was significantly higher in white sheep skin compared to black sheep skin(P<0.01).The immunocytochemical analysis revealed that sGC was expressed in melanocytes in vitro of sheep skin.Over expression of sGC in melanocytes resulted in decreased expression of key melanogenic genes including microphthalmia transcription factor(MITF),tyrosinase(TYR),tyrosinase related protein 1(TYRP1),and tyrosinase related protein 2(TYRP2)both at mRNA and protein level.