5-AZA抑制人葡萄膜黑色素瘤细胞增殖的研究

The study on inhibition of 5-AZA on human uveal melanoma cell proliferation

目的 DNA甲基化是一种重要的表观修饰,由DNA甲基化转移酶介导完成,与癌症的发生密切相关。目前,已发现至少有50个与黑色素瘤发生发展相关的关键基因发生异常超甲基化修饰,而DNA甲基化酶抑制剂能抑制DNA甲基转移酶活性,具有调控甲基化状态的潜能。本研究旨在探讨DNA甲基化酶抑制剂5-AZA对人葡萄膜黑色素瘤细胞增殖的影响并初步研究其作用的分子机制。方法 4μM 5-AZA处理OCM-1和OCM-3细胞作为实验组,同时溶剂DMSO处理细胞作为对照组,通过MTS[3-(4,5-diethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-etrazolium,inner salt,MTS]实验和细胞克隆形成实验检测5-AZA对细胞增殖的作用;采用流式细胞术分析5-AZA作用于细胞后细胞周期的变化;通过细胞划痕实验评估5-AZA对细胞迁移的影响;分别应用Real-time PCR和Western blotting技术检测5-AZA对细胞周期相关蛋白基因表达水平;所得实验数据使用t检验判定实验组和对照组之间差异是否具有统计学意义。结果 5-AZA显著抑制人葡萄膜黑色素瘤细胞增殖(P<0.01)以及细胞克隆形成能力;5-AZA诱导人葡萄膜黑色素瘤细胞G_1期阻滞;5-AZA对人葡萄膜黑色素瘤细胞迁移影响不明显;5-AZA显著促进葡萄膜黑色素瘤细胞p21表达上调(P<0.01),抑制CDK2表达(P<0.01)。结论 5-AZA能通过上调p21和下调CDK2抑制人葡萄膜黑色素瘤细胞增殖,可作为治疗葡萄膜黑色素瘤的潜在药物。

Objective DNA methylation is an important epigenetic regulatory mechanism, which is catalyzed with DNMTs and closely related to tumorigenesis. Currently, it has been found that more than 50 key genes were hypermethylation during the process and development of melanoma. Our study is to investigate the effects of DNA methylase inhibitor 5-AZA on proliferation and migration of human uveal melanoma cells and explore its molecular mechanism. Methods OCM-1 and OCM-3 cells were treated with 4 μM 5-AZA as experimental group, and the cells were treated with DMSO as control group. Then, the effect of 5-AZA on cell proliferation was detected by MTS and cell clone formation assay;the cell cycle was analyzed by flow cytometry;and the effect of 5-AZA on cell migration was evaluated by cell scratch assay;Real-time PCR and Western blotting were used to determine the gene expression of 5-AZA on cell cycle. All data were analyzed using two-tailed t-test to determine statistical significance to assess the difference between the control group and experimental group. Results 5-AZA significantly inhibited the proliferation of human uveal melanoma cells(P<0.01) and the ability of cell clone formation. 5-AZA could induce G1 phase arrest of human uveal melanoma cells. 5-AZA had no significant effect on the migration of human uveal melanoma cells. 5-AZA significantly could upregulate p21 expression and downregulated CDK2 expression in uveal melanoma cells. Conclusion 5-AZA inhibits proliferation of human uveal melanoma cells by up regulating p21 and down regulating CDK2. And 5-AZA can be applied potentially into clinical treatment of uveal melanoma.