Study On The Function And Mechanism Of MiR-186-5p On Sheep Melanogenesis

In animals,Melanogenesis is a complex process,and which is regulated by hundreds of genes.such as TYR、TYRP1、TYRP2/DCT、MITF、α-MSH、MC1R、KIT、ASIP and so on,of which TYR、TYRP1、TYRP2/DCT are key enzymes in melanogenesis;a-MSH/MC1R is an important signal pathway in melanogenesis;MITF is an important transcription factor that regulates key enzymes in the formation of melanin.microRNAs(miRNAs)are non-coding single-stranded small RNAs molecule,and are consisting of 19-25 nucleotides.First microRNAs are binding to the 3’untranslated region(3’UTR)of target gene,and then to regulate target gene expression in post-transcription level.MiRNAS is involved in the biological processes of cell occurrence,proliferation,differentiation and apoptosis.Recent years,microRNAs have been found to have an important regulatory role in the production of melanin.In this study,through analysis of the melanin pathway,using the bioinformatics software to predict and screen target genes and miRNAs;To construct eukaryotic expression vectors;The expression vectors and cytokines were transfected to and added to the sheep melanocytes,respectively;Through variety of biological detection means to reveal the role of miRNA in the formation of melanin.The experiment contents and results are as follows:1.Through analysis of the melanin pathway,MITF gene was selected.The miRbase and TargetScan database predict that MITF is the target gene of miR-186-5p.The expression difference of miR-186 and MITF in different sheep coat color skin were detected by QRT-PCR;Constructed the double fluorescent reporter vector of the MITF 3 ’UTR and the miR-186-5p overexpression vector;The target relationship between miR-186 and MITF was verified by double fluorescence reporter assay.2.The expressing vector of miR-186-5p,inhibitor and negative control were transfected into sheep melanocytes.The melanin content of the transfected melanocytes was detected by spectrophotometric method;The mRNA expression level of miR-186-5p、MITF、TYR、TYRP1、TYRP2 were tested by QRT-PCR;The protein expression level of MITF、TYR、TYRP1、TYRP2 were tested by western blot;The expression in situ of MITF in the sheep melanocytes was detected by immunohistochemistry;The migration and proliferation of the sheep melanocytes were detected by cell scratches.3.The localization and distribution of miR-186-5p in sheep melanocytes were detected by cell culture and in situ hybridization.4.miR-186-5p and a-MSH have regulatory function in the synthesis of melanin.The expressing vector of miR-18 6-5p was transfected to sheep melanocytes,at the same time a certain concentration of a-MSH also added to the sheep melanocytes.Study the synergistic effects of miR-186-5p and a-MSH on melanogenesis in sheep melanocytes.The melanin content of the melanocytes was detected by spectrophotometric method;The mRNA expression level of MITF、TYR、TYRP1、TYRP2 were tested by QRT-PCR;The protein expression level of MITF、TYR、TYRP1、TYRP2 were tested by western blot;The expression in situ of MITF in the sheep melanocytes was detected by immunohistochemistry;The migration and proliferation of the sheep melanocytes were detected by cell scratches.The experiment results are as follows:1.The database of miRNAs predicted that MITF is the target gene of miR-186-5p;QRT-PCR show that the expression of miR-186-5p in sheep black skin was significantly lower than that of in white skin,while MITF was significantly higher in sheep black skin.Luciferase assay show that relative luciferase activity of co-transfected miR-186-5p and MITF 3’ UTR decreases 41%than others.2.The situ hybridization show that miR-186-5p was distributed in the nucleus and cytoplasm of sheep melanocytes3.After transfected,the result of QRT-PCR,western blot and immunohistochemistry show that the over expression of miR-186-5p suppressed the expression of MITF both in mRNA and protein level.QRT-PCR and western blot show that the over expression of miR-186-5p suppressed the expression of TYR,TYRP1 and TYRP2 in mRNA and protein level.The spectrophotometric method result show that the miR-186-5p overexpression reduced the melanin content in melanocytes;The scratch test show that miR-186-5p overexpression inhibited the proliferation and migration of melanocytes4.The result of QRT-PCR,western blot and immunohistochemistry show that the inhibitory effect of miR-186-5p on the target gene MITF can be alleviated by the synergistic effect of miR-186-5p and a-MSH on the sheep melanocytes;QRT-PCR and western blot show that the synergistic effect of miR-186-5p andα-MSH also alleviated the inhibitory effect of miR-186-5p on TYR,TYRP1 and TYRP2;The results of spectrophotometry show that the synergistic effect of miR-186-5p and a-MSH alleviated the decrease of melanin content by miR-186-5p;The results of scratch test show that the synergistic effect of miR-186-5p and a-MSH did not alleviated the inhibitory effect of miR-186-5p on the proliferation and migration of melanocytes.