【摘要】 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。更多还原

【Abstract】 Melanocortin-1 receptor(MC1R) as a member of G protein-coupled receptor(GPCR) family is involved in melanogenesis by melanocytes.MicroRNAs(miRNAs),a small non-coding RNA(ncRNA) class,can suppress gene expression by targeting the 3’-untranslated region(3’-UTR) of target genes.It has been showed that over-expression of miR-338-3p may inhibit invasion and migration in many human cancer cells.However,the effect of miR-338-3p on the melanogenesis in alpaca skin melanocytes is rarely reported.In this study,we demonstrated that miR-338-3p inhibited themelanogenesis in alpaca skin melanocytes through targeting the MC1 R gene.The bioinformatics was employed to predict that the human MC1 R gene was a target for miR-338-3p.The eukaryotic expression vector was constructed.The combination of gene transfection with quantitative real-time PCR(qRTPCR) and Western blotting revealed that compared with the control cells,the mRNA and protein expression of the MC1 R gene and their down-stream genes encoding for microphthalmia-associtated transcription factor(MITF),tyrosinase(TYR),tyrosinase-related protein 1(TYRP1) and tyrosinase related protein 2(TYRP2) that participate in melanogenesis were significantly down-regulated.Moreover,ELISA showed that the production of melanin in miR-338-3p-overexpressed alpaca melanocytes was greatly decreased(P < 0.01).Taken together,miR-338-3p can target the MC1 R gene and subsequently,down-regulate the MITF,TYR,TYRP1 and TYRP2 genes,thereby inhibiting the melanogenesis in alpaca skin melanocytes.The miR-338-3p participates in the regulation of melanogenesis in skin melanocytes during alpaca growth and development would to be studied.更多还原