Knockdown Of MicroRNA-143-5p By STTM Technology Affects Melanin Production And Transport In Melanocytes

MicroRNAs(miRNAs)play an important role in regulating the production of melanins in mammals melanocytes and the formation of coat colors.Short tandem target mimic(STTM)is a technique that can effectively silence miRNAs in plants and animals.Mi R-143-5p regulates the migration,proliferation and melanin synthesis of alpaca melanocytes by targeting TGF-β-activated kinase 1(TAK1).It was predicted that myosin Va gene(MYO5A)was one of the target genes of miR-143-5p by biological analysis and dual fluorescent reporter vector assay.To investigate the function of miR-143-5p in melanocytes,the expression of miR-143-5p(STTM-miR-143-5p)was blocked using STTM technology.The mRNA and protein levels of related genes in mice melanocytes transfected with STTM-miR-143-5p were detected by real-time PCR,Western Blotting and Fontana-Masson staining.The main experimental results as follows:1.The dual fluorescent reporter vector of MYO5 A and the STTM-miR-143-5p overexpression vector were co-transfected into 293 T cells.The results showed that the activity of the dual luciferase of the reporter gene was 1.334 fold changes,compared to that in the control group(P<0.001).2.Quantitative real-time PCR(qPCR)was used to detect the difference of mRNA expression in mice melanocytes transfected with STTM-miR-143-5p.The results showed that the mRNA expression levels of MYO5 A,MLPH,Rab27 a,MITF,TYR and TYRP1 genes in melanocytes in group were significantly higher than that in the control group,while the mRNA level of TYRP2 gene in the transfected group was 0.583 fold changes,compared to that in the control group(P<0.05,P<0.01,P<0.001).3.Western Blotting was used to detect the difference of protein expression in mice melanocytes transfected with STTM-miR-143-5p.The results showed that STTM-miR-143-5p overexpression up-regulated the expression of MYO5 A,MLPH,Rab27 a,MITF,TYR and TYRP1,while the protein level of TYRP2 gene in the transfected group was 0.550 fold changes,compared to that in the control group(P<0.01,P<0.001).4.Using NaOH pyrolysis method,the contents of total melanin,eumelanin and phoemelanin were determined.The results showed that the content of total melanin and phoemelanin in the transfection group was significantly higher than that in the control group,while the content of eumelanin in the transfection group was 0.919 fold changes,compared to that in the control group(P<0.05,P<0.01).5.In the co-culture of mouse melanocytes and keratinocytes,the content and distribution of melanins particles in the transfected group were significantly increased by Fontana-Masson staining.The melanin particles in the keratinocytes were counted,and the analysis showed that the melanin particles in the keratinocytes transfected group were far more than the control group(P<0.001).Conclusion: STTM-miR-143-5p regulates the expression of MLPH,Rab27 a,MITF,TYR,TYRP1 and TYRP2 genes by targeting MYO5 A,which thereby regulated the production and transport of melanins.