DZNep靶向EZH2抑制葡萄膜黑色素瘤细胞增殖

DZNep Targeting EZH2 Inhibits Uveal Melanoma Cell Proliferation

目的:探讨EZH2抑制剂3-deazaneplanocin A(DZNep)对人葡萄膜黑色素瘤细胞增殖的影响。方法:实验研究。先采用Western blot法检测EZH2在正常葡萄膜黑色素细胞(UM95、UM96)和葡萄膜黑色素瘤细胞(M23、SP6.5)中的表达,然后在M23和SP6.5细胞的培养基中加入DZNep进行处理,作为实验组;以溶剂DMSO处理作为阴性对照组。采用MTS、平板克隆形成、EdU实验以及流式细胞术分别检测细胞存活、克隆形成能力、细胞增殖以及细胞周期。Western blot技术检测DZNep对细胞中H3K27me3修饰水平,细胞周期相关蛋白以及ERK信号通路的影响。组间数据均采用独立样本t检验进行比较。结果:EZH2在M23和SP6.5细胞中的表达水平与在UM95细胞中的表达相比显著升高(t=25.050,P=0.002;t=14.220,P=0.005)。MTS结果显示,DZNep显著抑制M23和SP6.5细胞的活性,呈浓度和作用时间依赖性。与阴性对照组相比,实验组中M23和SP6.5细胞的克隆形成数量显著降低(t=3.364,P=0.015;t=6.997,P<0.001),并且反映细胞增殖的EdU标记细胞比例明显减少(t=5.117,P=0.002;t=3.399,P=0.015)。流式细胞术检测结果显示,与阴性对照组相比,实验组中M23和SP6.5细胞处于G1期的细胞比例均显著升高(t=6.199,P=0.003;t=3.729,P=0.020)。Western blot检测结果显示,与阴性对照组相比,实验组M23和SP6.5细胞中的EZH2(t=5.214,P=0.035;t=13.530,P=0.005)、p-Rb(t=4.551,P=0.045;t=4.655,P=0.043)、E2F1(t=8.090,P=0.015;t=9.313,P=0.011)以及p-ERK(t=4.819,P=0.040;t=8.951,P=0.012)表达均有降低,H3K27me3修饰水平显著下降(t=5.257,P=0.034;t=5.697,P=0.030)。结论:DZNep能够抑制葡萄膜黑色素瘤细胞的增殖,具有治疗葡萄膜黑色素瘤的应用前景。

Objective:To investigate the effect of the EZH2 inhibitor DZNep on the proliferation of uveal melanoma cells.Methods:In this experimental study,the expression of EZH2 in uveal melanocytes(UM95,UM96)and uveal melanoma cells(M23,SP6.5)was detected by Western blot analysis.The effect of DZNep on cell viability and clonal growth was evaluated by MTS and colony formation assay.Furthermore,cell proliferation was detected by EdU assay,whereas cell cycle was determined by flow cytometry.Then,Western blotting was carried out to test the protein levels of EZH2,H3K27me3,and cell cycle-related proteins as well as p-ERK.Data were analyzed by an independent t test.Results:Compared with UM95,the expression of EZH2 increasedsignificantly in M23 and SP6.5 cells(t=25.050,P=0.002;t=14.220,P=0.005).MTS assay showed that the viability of M23 and SP6.5 cells was inhibited by DZNep ina doseor time-dependent manner.When compared with the DMSO-treated group,the clone numbers of M23 and SP6.5 cells in the DZNep group were obviously decreased(t=3.364,P=0.015;t=6.997,P<0.001),and the ratio ofthe proliferative marker EdU labelling cells was lower(t=5.117,P=0.002;t=3.399,P=0.015).Also,flow cytometry revealed that the percentage of cells in the G1 phase in the DZNep group was markedly higher than that in the DMSO group(t=6.199,P=0.003;t=3.729,P=0.020).Furthermore,Western blot analysis showed that compared with the DMSO-treated group,some proteins were reduced after exposureto DZNep,including EZH2(t=5.214,P=0.035;t=13.530,P=0.005),p-Rb(t=4.551,P=0.045;t=4.655,P=0.043),E2F1(t=8.090,P=0.015;t=9.313,P=0.011),p-ERK(t=4.819,P=0.040;t=8.951,P=0.012).as well as the level of H3K27me3(t=5.257,P=0.034;t=5.697,P=0.030).Conclusions:DZNep can inhibit the proliferation of uveal melanoma cells,and is a promising epigenetic drug for the treatment of this malignancy.