目的：探讨橙花叔醇通过 Wnt-β-catenin 通路抑制黑色素瘤 A-375 和 WM-115 细胞恶性生物学行为的分子机制。方法: 体外培养黑色素瘤细胞A-375 和WM-115，用不同浓度的橙花叔醇处理，采用SRB 法和克隆形成实验、FCM术、Transwell实验和细胞划痕实验、DCFH-DA 染色法、qPCR 和WB法分别检测橙花叔醇对A-375 和WM-115 细胞的增殖能力、细胞周期和凋亡、迁移能力、活性氧（ROS）水平和Wnt-β-catenin 通路及其下游相关基因和相关蛋白表达的影响。利用ULCAN和GEPIA2数据库分析黑色素瘤中Wnt-β-catenin 通路的激活与患者预后的关系。结果：与对照组比较，橙花叔醇处理组A-375 和WM-115 细胞的增殖能力受明显抑制（均P<0.01）、细胞周期阻滞于G2/M 期（P<0.05 或P<0.01）、细胞凋亡率增加（均P<0.01）、迁移能力降低（P<0.05或P<0.01）、ROS水平升高（均P<0.01）、Wnt-β-catenin 通路被抑制而其下游基因和蛋白表达明显上调（均P<0.01）。数据库数据分析显示，WNT1基因高表达患者OS低于低表达患者（P<0.01）。结论：橙花叔醇通过上调A-375 和WM-115细胞中ROS水平影响Wnt-β-catenin通路，从而抑制其恶性生物学行为；Wnt-β-catenin通路可能是黑色素瘤治疗的潜在靶点。

Objective: To investigate the molecular mechanism by which nerolidol inhibits the malignant biological behavior of melanoma A-375 and WM-115 cells through the Wnt-β-catenin pathway. Methods: Melanoma A-375 and WM-115 cells were cultured in vitro and then treated with different concentrations of nerolidol. The effects of nerolidol on the proliferation, cell cycle and apoptosis,and migration of A-375 and WM-115 cells were analyzed by SRB and clonogenic assays, FCM, Transwell, and cell scratch assays,respectively. The levels of reactive oxygen species (ROS) in the cells were examined with DCFH-DA staining. The Wnt- β -catenin pathway and the expression levels of its related downstream genes were determined by qPCR and WB. The relationship between patient prognosis and the activation of Wnt-β-catenin pathway in melanoma was analyzed using the ULCAN and GEPIA2 databases. Results:Compared with the control group, the proliferation, migration, and cell cycle of A-375 and WM-115 cells in the nerolidol-treated group were significantly inhibited (all P<0.01), while the apoptosis was significantly increased (all P<0.01); the ROS level was increased (P<0.01), and the Wnt-β-catenin pathway was inhibited, while its downstream gene expression was significantly up-regulated (P<0.01 or P<0.01). Analysis of database data showed that OS was lower in patients with high WNT1 gene expression than in patients with low expression (P<0.01). Conclusions: Nerolidol affects the Wnt-β-catenin pathway by upregulating ROS levels in A-375 andWM-115 cells, thereby inhibiting their malignant biological behaviors. The Wnt-β-catenin pathway may be a potential target for the treatment of melanoma.

甘肃省卫生行业科研项目（No. GSWSKY-2019-30）