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黑色素皮质素1受体(MC1R)是在黑色素细胞内表达的G蛋白耦合受体(G protein-coupled receptor,GPCR)家族成员,参与黑色素细胞中黑色素的生成。微RNAs(miRNAs)是一类非编码RNA,通过与靶基因3’-UTR结合抑制基因表达。已有研究证明,miR-338-3p在多种人类肿瘤细胞中(过)表达,可通过下调靶基因表达抑制肿瘤细胞的侵袭迁移能力。然而,有关miR-338-3p对羊驼皮肤黑色素细胞的黑色素合成影响却罕见报道。本研究证明,miRNA-338-3p通过靶向抑制MC1R基因表达,抑制羊驼黑色素细胞黑色素的生成。采用生物信息学预测MC1R基因是miRNA-338-3p的靶基因,其基因表达抑制羊驼黑色素细胞黑色素合成。随后构建miR-338-3p真核表达载体。其基因转染结合q PT-PCR和Western印迹结果揭示,与对照细胞比较,过表达miRNA-338-3p的羊驼黑色素细胞的MC1R基因,及其下游与黑色素生成相关的小眼相关性转录因子(MITF)、酪氨酸酶(TYR)、酪氨酸酶相关蛋白1(TYRP1)、酪氨酸酶相关蛋白2(TYRP2)编码基因mRNA及蛋白质表达水平明显下调。酶联免疫吸附分析显示,过表达miRNA-338-3p的羊驼皮肤黑色素细胞的黑色素产量,较对照细胞显著下降(P<0.01)。综上结果,miR-338-3p可通过抑制靶基因MC1R表达,下调其下游基因MITF、TYR、TYRP1和TYRP2基因的表达,从而抑制羊驼皮肤黑色素细胞黑色素的合成。miRNA-338-3p在羊驼生长发育过程中,是否参与调控体内皮肤黑色素细胞的黑色素生成尚待进一步研究。
The melanoma-1 receptor (MC1R) is a member of the G protein-coupled receptor (GPCR) family expressed in melanocytes and is involved in the production of melanin in melanocytes. MicroRNAs (miRNAs) are a class of non-coding RNA that inhibits gene expression by binding to the 3’-UTR of target genes. Studies have shown that miR-338-3p (overexpression) in a variety of human tumor cells can inhibit the invasion and migration of tumor cells by down-regulating target gene expression. However, the effects of miR-338-3p on the melanin synthesis of alpaca skin melanocytes are rarely reported. This study demonstrates that miRNA-338-3p inhibits the production of melanin in alpaca melanocytes by targeting the MC1R gene. Bioinformatics predicts that the MC1R gene is the target gene of miRNA-338-3p, and its gene expression inhibits melanin synthesis in alpaca melanocytes. Subsequently, a miR-338-3p eukaryotic expression vector was constructed. Its gene transfection combined with q-PT-PCR and Western blot revealed that compared with control cells, the MC1R gene of the alpaca melanoma cells overexpressing miRNA-338-3p and its downstream small eye associated transcription factor associated with melanin production The mRNA and protein expressions of MITF, TYR, TYRP1 and TYRP2 were significantly down-regulated. Enzyme linked immunosorbent assay showed that the melanin production of the alpaca skin melanocytes over-expressing miRNA-338-3p was significantly lower than that of the control cells (P <0.01). In conclusion, miR-338-3p can inhibit the melanoma melanin synthesis in the skin of the alpaca by inhibiting the expression of the target gene MC1R and down-regulating the expression of its downstream genes MITF, TYR, TYRP1 and TYRP2. Whether or not miRNA-338-3p is involved in the regulation of melanin production in vivo in the process of alpaca growth and development remains to be further studied.