Objective: To explore the effect and mechanism of Rafkinase inhibitor protein (RKIP) on proliferation and migration of malignant melanoma cells in vitro. Methods: The RKIP overexpression and down-regulated stable transfected strains of mouse malignant melanoma cell line B16 were constructed by recombinant lentiviral transfection technique and established as RKIP overexpression group and RKIP down-regulation group, the mouse malignant melanoma B16 cells without any treatment were used as a blank control group, and the proliferation activity and migration ability of each group were detected by cell counting kit-8 (CCK-8) and cell scratch test. The relative expression levels of CyclinD1, Calcium-dependent cell adhesin, Ki-67, Matrix metalloproteinase (MMP)-9, MMP-13, MMP-2 and Phosphatidylethanolamine binding protein (PEBP-1) were detected by quantitative real-time polymerase chain reaction (qPCR). Western blot was used to detect the difference of RKIP expression and the protein expression level of nuclear factor-kappa B (NF-κB) signaling pathway in each group. Results: Comparison of RKIP overexpression group and blank control group shown cell proliferation and migration were significantly inhibited in RKIP overexpression group (0.794±0.038 vs 1.200±0.081) (P<0.001). However, there was no significant difference in cell proliferation between RKIP down-regulation group and blank control group (1.077±0.084 vs 1.200±0.081) (P>0.05), and the cell migration ability of RKIP down-regulation group was significantly higher than that of the blank control group (P<0.001). In addition, there was no significant difference between the RKIP down-regulation group and the blank control group in PEBP-1 expression (P>0.05), while the expression levels of the remaining genes in the RKIP overexpression group were significantly lower than those in the blank control group, and the expression levels in the RKIP down-regulation group were significantly higher than those in the blank control group (P<0.001). Furthermore, the protein level of phosphorylated P65 (p-P65) in RKIP overexpression group was significantly lower than that in blank control group (0.080±0.000 vs 0.236±0.000), and RKIP down-regulation group was significantly higher than that in blank control group (1.139±0.001 vs 0.236±0.000) (both P<0.001). Conclusion: RKIP overexpression can inhibit the proliferation and migration of malignant melanoma cells, which may be related to the regulation of NF-κB signaling pathway-related protein p-P65.
目的： 探讨Raf激酶抑制蛋白（RKIP）在体外对恶性黑色素瘤细胞增殖、迁移能力的影响及其机制。 方法： 利用重组慢病毒转染技术构建小鼠恶性黑色素瘤细胞株B16的RKIP过表达及调低的稳定转染株，并将其设立为RKIP过表达组和RKIP调低组，以未经任何处理的小鼠恶性黑色素瘤B16细胞为空白对照组；通过细胞增殖/毒性检测试剂盒（CCK-8）法与细胞划痕实验检测各组细胞的增殖活性及迁移能力，利用实时荧光定量聚合酶链反应（qPCR）检测细胞周期蛋白D1（CyclinD1）、钙离子依赖的细胞黏附素、Ki-67、基质金属蛋白酶（MMP）-9、MMP-13、MMP-2及磷脂酰乙醇胺结合蛋白1（PEBP-1）的相对表达量；Western印迹检测各组细胞间RKIP表达差异及其在核因子（NF）-κB信号通路的蛋白相对表达量。 结果： RKIP过表达组细胞增殖率显著低于空白对照组（0.794±0.038比1.200±0.081）（P<0.001），而RKIP调低组细胞的增殖率与空白对照组差异无统计学意义（1.077±0.084比1.200±0.081）（P>0.05）；RKIP过表达组细胞的迁移能力显著低于空白对照组（P<0.001），而RKIP调低组细胞的迁移能力显著高于空白对照组（P<0.001）；除RKIP调低组与空白对照组PEBP-1相对表达量差异无统计学意义（P>0.05）外，其余检测基因相对表达量在RKIP过表达组均显著低于空白对照组，RKIP调低组均显著高于空白对照组（均P<0.001）。RKIP过表达组中磷酸化的P65（p-P65）蛋白相对表达量显著低于空白对照组（0.080±0.000比0.236±0.000），而RKIP调低组中p-P65蛋白相对表达量则显著高于空白对照组（1.139±0.001比0.236±0.000）（均P<0.001）。 结论： RKIP过表达可以抑制恶性黑色素瘤细胞的增殖及迁移，其可能与调控NF-κB信号通路相关蛋白P65的磷酸化有关。.
Keywords: Cell movement; Cell proliferation; Melanoma; NF-kappa B; Raf kinases.