miR-380调控羊驼黑色素细胞MAPK/ERK信号通路

miR-380 Regulates MAPK/ERK Signal Pathway in Alpaca Melanocytes

miR-380是不同羊驼毛色中差异表达的基因之一,但是否与黑色素生成有关未见报道。为了丰富调控黑色素生成的机制,挖掘黑色素生成路径中所涉及到的更多新的基因并揭示miR-380在黑色素细胞中的功能,本实验通过生物信息学方法预测出MAPK信号通路的成员MAP3K6是miR-380的靶基因之一。在293T细胞中共转染miR-380和MAP3K6后,与对照组相比双荧光报告酶活性下降(28.92±25.63)%(P<0.01),下降趋势明显,说明MAP3K6可能是miR-380的靶基因之一;在羊驼黑色素细胞中转染miR-380后,MAP3K6、MEK1、ERK1/2、CREB和MITF在转录水平的表达量与NC组相比具有显著下降趋势,其中CREB下降趋势尤为显著(64.20±54.30)%(P<0.01),Western印迹检测MAP3K6、p-MEK1、p-ERK1/2、CREB和MITF在蛋白质水平的表达与NC组相比下降趋势明显且p-MEK1和CREB基因下降极为显著,分别为(29.09±10.68)%(P<0.001)和(47.12±6.70)%(P<0.001),抑制组则反之。通过Masson-Fontana黑色素颗粒染色法检测miR-380抑制黑色素细胞产生黑色素颗粒,用紫外分光度法检测真黑素(eumelanin,EM)和褐黑素(pheomelanin,PM),含量结果提示EM与PM含量分别下降为(38.63±2.00)%(P<0.01),(54.10±5.73)%(P<0.001)且PM含量下降极为显著。综上所述miR-380通过靶向抑制MAP3K6等基因的表达,从而对MAPK/ERK信号通路起调控作用,最终影响黑色素生成生物学功能,此研究对哺乳动物毛色形成机制和防止皮肤受紫外辐射有重要意义。

miR-380 is one of the genes differentially expressed in alpaca with different hair colors,which suggested that miR-380 might be related to the melanogenesis. Basing on the bioinformatics analysis,MAP3 K6,the member of MAPK signal,is one of the target genes of miR-380. miR-380 and MAP3 K6 were co-transfected with 293 T cells to verify the binding relationship by luciferase assay. The results showed that the luciferase activity was significantly decreased（28. 92% ± 25. 63%）（P 0. 01）,and MAP3 K6 was one of the target genes of miR-380. qRT-PCR and Western blotting were used to measure the levels of mRNA and protein of MAP3 K6,MEK,ERK1/2,CREB and MITF in alpaca melanocytes which were transfected by miR-380 or miR-380 inhibitor. After miR-380 was overexpressed in alpaca melanocytes, the expression of CREB protein was significantly decreased（47. 12% ±6. 70%）（P 〈0. 001） and the expression of downstream genes MEK,ERK1/2,MITF were downregulated. The effect of miR-380 on melanogenesis was detected by Masson-Fontana melanin staining and the result indicated that the number of melanin particles was significantly decreased after miR-380 was overexpressed. The spectrophotometry assay showed that eumelanin（EM） and pheomelanin（PM） were decreased in melanocytes transfected with miR-380 significantly,or vise verse with miR-380 inhibitor.These results indicated that miR-380 could regulate the MAPK/ERK signal pathway by targeting MAP3 K6 gene and subsequently affect the biological function of melanocytes for melanin production. This study is of great interest for mammalian coat color formation and UV radiation prevention.