MicroRNA-411a-3p靶向钙/钙调素依赖蛋白激酶4调控羊驼黑色素细胞中的黑色素生成

羊驼是重要的毛用型经济动物，具有22种天然色。毛色由皮肤中黑色素细胞产生的黑色素颗粒的分布和转运所决定的。然而，羊驼色素形成或毛色形成的分子机制复杂，由多个基因、miRNA 和lncRNA调控。但是，miR-411a-3p 对羊驼色素形成起调控作用未见报道。为研究miR-411a-3p在黑色素产生过程中的调控作用，本文将构建的miR-411a-3p载体转染到羊驼黑色素细胞中，并对毛色基因的表达进行研究。经生物信息学预测，钙/钙调素依赖蛋白激酶4(calcium-calmodulin dependent protein kinase 4, CaMK4)可能是miR-411a-3p的靶基因之一。双荧光素酶报告基因结果显示，与对照组相比，双荧光报告酶活性下降（34.78 ± 16.09）%(P<0.01)，其下降趋势明显，说明CaMK4可能是miR-411a-3p的靶基因之一。miR-411a-3p通过结合CaMK4的3′ untranslated region (3′-UTR)直接调控CaMK4的表达。在羊驼黑色素细胞中转染miR-411a-3p后，CaMK4、CDK5和TYRP1在转录水平的表达量与NC组相比具有显著下降趋势，其中TYRP1下降趋势尤为显著（53.66 ± 2.11）%（P<0.01）。Western印迹检测CaMK4、CDK5、TYRP1、p-CREB在蛋白质水平的表达与NC组相比下降趋势明显，尤其是CDK5和p-CREB基因下降极为显著，分别为（70.26 ± 4.84）%（P<0.01）和（70.11 ±9.05）%（P<0.01）。Masson-Fontana法检测黑色素颗粒，结果显示，miR-411a-3p抑制黑色素细胞产生黑色素颗粒。通过紫外分光度法检测真黑素（eumelanin，EM）和褐黑素（phenomelanin，PM）显示，EM和PM的含量均被下调。结果表明，miR-411a-3p靶向抑制CaMK4表达，从而改变CREB的表达以控制黑色素的产生，此研究结果对哺乳动物毛色形成机制有重要意义。

MicroRNA-411a-3p Modulates Melanin Production by Targeting CaMK4 in Alpaca Melanocytes

Alpaca is an important fiber-roducing species with 22 natural hair colors determined by the production and transport of melanin in the skin. The molecular mechanisms of melanogenesis and the formation of hair colors in alpaca are complex, which are regulated by genes, miRNAs and lncRNAs. However, miR-411a-3p regulates melanogenesis or hair colors formation has not been reported. To investigate the role of miR-411a-3p in melanogenesis, miR-411a-3p construction was transfected into alpaca melanocytes. miR-411a-3p was predicted to modulate the expression of gene encoding calcium-calmodulin dependent protein kinase 4 (CaMK4). The double fluorescent reporter assay showed that enzyme activity was significantly decreased (34.78±16.09)% (P<0.01) in experimental group as compared with the control, indicating that CaMK4 might be one of the target genes of miR-411a-3p to directly regulate the expression of CaMK4 by combining the 3′ untranslated region (3′-UTR) of CaMK4. After transfection of miR-411a-3p in alpaca melanocytes, the mRNA levels of TYRP1, CaMK4 and CDK5 were significantly decreased as compared with those of NC group, in which TYRP1 showed a particularly significant decline (53.66±2.11)% (P<0.01). The protein levels of CaMK4, CDK5, TYRP1 and phospho-cAMP-response element binding protein (p-CREB) were significantly decreased by Western blot analysis when compared with those of NC group. The expression of CDK5 and p-CREB were significantly decreased with (70.26± 4.84)% (P<0.01) and (70.11± 9.05)% (P <0.01), respectively. The detection of melanin particles by Masson-Fontana method showed that miR-411a-3p inhibited the production of melanin particles in melanocytes. The spectrophotometry assay showed that eumelanin (EM) and phenomelanin (PM) were decreased in melanocytes transfected with miR-411a-3p significantly. The results suggested that miR-411a-3p targeted CaMK4 and inhibited its expression to control melanin production via modulation of CREB, which provides important clues in understanding the mechanism of hair color formation in mammals.