miR-146a抑制酪氨酸酶基因家族在小鼠黑色素细胞中表达

miR-146a Inhibits the Expression of Tyrosinase Gene Family in Mouse Melanocytes Cells

miRNA是在许多生物过程中都起着至关重要作用的一类内源性非编码的小RNA,与癌症、肿瘤的发生有关。现发现很多miRNA在黑色素生成中都有重要的调控作用,但miR-146a是否对黑色素的生成具有影响未见报道。本研究发现miR-146a通过靶向抑制酪氨酸酶相关蛋白1(tyrosinase related protein 1,TYRP1)的表达而使黑色素生成降低。在小鼠黑色素细胞中分别转染miR-146a mimic和miR-146a抑制剂,通过qRT-PCR与Western印迹分析比较各实验组中TYRP1基因与酪氨酸家族相关基因酪氨酸酶(tyrosinase,TYR)、酪氨酸酶相关蛋白2(tyrosinase related protein 2,TYRP2)的表达差异。双荧光报告实验验证TYRP1与miR-146a的靶向关系,双荧光酶活性结果显示,实验组相比对照组,荧光素酶活性明显降低,说明TYRP1是miR-146a的靶基因之一;qRT-PCR和Western印迹结果显示实验组TYR、TYRP1及TYRP2在mRNA水平和蛋白质水平表达均显著降低;紫外分光光度法检测黑色素含量,结果显示miR-146a mimic转染组黑色素含量明显下降,而抑制组的黑色素含量呈上升趋势。综上所述,miR-146a通过靶向抑制TYRP1基因的表达,而影响TYR家族成员的表达,调控黑色素的生物合成。

MicroRNAs（ miRNAs） are endogenous small non-coding RNAs,which play crucial roles in numerous biological processes and are related to the occurrence of tumor. It has been reported that many miRNAs regulate the production of melanin in melanocytes. However,the effects of miR-146 a on the melanogenesis have not been reported. In this study,miR-146 a mimic was over-expressed in mouse melanocytes in vitro. PCR method was used to amplify TYRP1-3＇ UTR,which was ligated into PGLO vector to construct pmir GLO-TYRP1-3＇ UTR. Following pmir GLO-TYRP1-3＇ UTR and miR-146 a mimic were co-transfected into 293 T cells,the luciferase activity was measured. qRT-PCR and Western blottingwere used to measure the mRNA and protein levels of TYR,TYRP1 and TYRP2 in mouse melanocytes which were transfected by miR-146 a mimic or miR-146 a inhibitor. The results showed that compared to the negative control,luciferase activity was lower in miR-146 a mimic group,which suggested that TYRP1 was one of the target genes of miRNA-146 a. Furthermore,the mRNA and protein levels of TYR,TYRP1 and TYRP2 were decreased significantly in miR-146 a mimic group. Over-expression of miR-146 a mimic in mouse melanocytes decreased production of melanin. In summary,miR-146 a regulates the expression of TYR and TYRP2 by targeting TYRP1 to modulate melanin production.