miR-186-5p与α-MSH互作对绵羊黑素细胞黑色素生成的影响

黑色素合成受多种基因和miRNAs的调控，为了研究miR-186-5p与α-MSH协同作用对绵羊黑素细胞黑色素生成的影响。本研究在绵羊黑素细胞中转染miR-186-5p表达载体，同时添加α-MSH，通过实时荧光PCR（quantitative real time polymerase chain reaction，qRT-PCR）和蛋白质免疫印迹（Western blotting）检测MITF、TYR、TYRP1和TYRP2 4种可能与之相关的基因表达情况；利用免疫组化检测MITF蛋白在黑素细胞中的表达和亚细胞定位；通过分光光度法测定黑色素含量变化；利用划痕试验测定细胞迁移情况，试验分为miR-186-5p转染组、miR-186-5p+α-MSH互作组和对照组，每组3个重复。结果显示，miR-186-5p能够抑制绵羊黑素细胞中MITF、TYR、TYRP1和TYRP2的表达，并最终抑制黑色素的生成。而添加α-MSH能缓解miR-186-5p对MITF、TYR、TYRP1和TYRP2表达的抑制作用，进而在一定程度上恢复黑色素的含量。另外，miR-186-5p还能抑制细胞分裂，并阻碍细胞的迁移，而α-MSH无法抵消miR-186-5p产生的这种负面影响。上述结果表明，在绵羊黑素细胞中，miR-186-5p和α-MSH均参与调控黑色素合成途径，其中，miR-186-5p主要起负调控作用，而α-MSH主要参与相关位点的正调控，并能部分恢复miR-186-5p导致的黑色素含量下降。值得注意的是，miR-186-5p还参与调控细胞的增殖和迁移，而α-MSH不参与相关调控。

Effect of Interaction between miR-186-5p and α-MSH on Melanogenesis in Sheep Melanocytes

Melanogenesis is regulated by many genes and miRNAs. This study aimed to explore the effect of interaction between miR-186-5p and α-MSH on melanogenesis in sheep melanocytes. The miR-186-5p expression vector was transfected into sheep melanocytes and α-MSH was also added to the cells at the same time. Expression patterns of associated genes(MITF, TYR, TYRP1, TYRP2) were detected by qRT-PCR and Western blotting. The expression and localization of MITF protein in sheep melanocytes were further detected by immunohistochemistry. The melanin contents and the migration capability of melanocytes were measured by spectrophotometry and scratch test, respectively. Three experiment groups were set up, containing miR-186-5p transfected group, miR-186-5p+α-MSH interaction group and negative control group, and there were 3 replicates in each group. The results showed that the expression of MITF, TYR, TYRP1 and TYRP2 were down-regulated, the production of melanin was inhibited in miR-186-5p transfected group, while the above effects of miR-186-5p were alleviated by adding α-MSH to the cells. In addition, miR-186-5p also could inhibit cell division and block cell migration, but α-MSH could not affect the negative effects of miR-186-5p. In summary, the results indicated that in sheep melanocytes, both miR-186-5p and α-MSH were involved in regulating melanogenesis.Between them, miR-186-5p mainly played a negative regulatory role, while α-MSH was involved in the positive regulation of related genes and relieved the inhibitory effect of miR-186-5p. It was worth noting that miR-186-5p played an important role in regulating cell proliferation and migration, while α-MSH did not participate in the related regulation.