MicroRNA-100-5p And FGF21 On The Regulation Of Melanogenesis In Alpaca Melanocytes

Recent studies have shown that miRNAs play important roles in the formation for mammalian coat color.Bioinformatics predictions(Targetscan 7.1,miRDB,mirRBase)indicated that there existed a target binding site between miR-100-5p and FGF21.However,studies on miR-100 and FGF21-involved in the regulation of melanogenesis in alpaca melanocytes have not been reported.To investigate the effect of miR-100 and FGF21 on melanogenesis in alpaca melanocytes,the experiments were as follows:PmirGLO-FGF21 3’UTR vector was constructed for double fluorescence detection.Subsequently,the eukaryotic expression vector encoding FGF21,FGF21-siRNA,eukaryotic expression vector encoding miR-100-5p were constructed and transfected into melanocytes,respectively.Finally,the varieties of melanin content were detected after transfection,and the coat color-related genes:P-ERK、MITF、TYR、TYRP2 in mRNA and protein levels were also detected.The results were as follows(1)We transfected the eukaryotic expression vector encoding FGF21into alpaca melanocytes and investigated the effect of FGF21 on melanogenesis.The results of melanin content assay showed that,the melanin contents decreased significantly in alpaca melanocytes which transfected with pMSCV-FGF21 compared to pMSCV control group.qRT-PCR results showed that,for the FGF21-transfected group,the relative mRNA levels of MITF、TYT、TYRP2 and ERK1/2 were decreaesd significantly when compared with the pMSCV-transfected group.Western blotting results showed that,the relative protein levels of MITF、TYR、TYRP2 in FGF21-transfected group were singnificantly lower than that in pMSCV-transfected group.However,the relative protein levels of P-ERK were singnificantly higher than that in pMSCV-transfected group.We transfected the FGF21-siRNA into alpaca melanocytes to further investigated the effect of FGF21 on melanogenesis.The results of melanin content assay showed that,the melanin contents decreased significantly in alpaca melanocytes which transfected with FGF21-siRNA compared to NC group.qRT-PCR results showed that,for the FGF21-siRNA-transfected group,the relative mRNA levels of MITF、TYR、TYRP2 and ERK1/2 were increased significantly when compared with the NC-transfected group.Western blotting results showed that,the relative protein levels of MITF、TYR、TYRP2 in FGF21-siRNA-transfected group were singnificantly higher than that in NC-transfected group.However,the relative protein levels of P-ERK were singnificantly lower than that in NC-transfected group(2)The pmirGLO-FGF21 3’UTR vector and the miR-100-5p expression vector were co-transfected into 293T cells for dual luciferase assay,which verified the targeted relationship between miR-100-5p and FGF21.The luciferase reporter assay showed that,luciferase activities in 293T cells which co-transfected with pmirGLO-FGF21 3’UTR plasmid and miR-100-5p plasmid were decreased by more than 20%.(3)We transfected the miR-100-5p overexpression plasmid into alpaca melanocytes and investigated the effect of miR-100-5p on melanogenesis.The results of melanin content assayt showed that,the melanin contents increased significantly in alpaca melanocytes which transfected with miR-100-5p overexpression plasmid compared to the corresponding control transfected group.qRT-PCR results showed that,for the miR-100-5p overexpression-transfected group,the relative mRNA levels of MITF、TYR、TYRP2 and ERK1/2 increased significantly when compared with the control-transfected group.Western blotting results showed that,the relative protein levels of MITF、TYR、TYRP2 in miR-100-5p overexpression-transfected group were singnificantly higher than that in control-transfected group.However,the relative protein levels of P-ERK were singnificantly lower than that incontrol-transfected group.Taken together,the results showed that FGF21 and miR-100-5p may involve in regulation of melanogenesis in alpaca melanocytes.There was a targeted relationship between the two,and the melanogenesis mechanism by FGF21 was conducted via the activating ERK signaling pathway.