SIRT1对葡萄膜黑色素瘤细胞增殖的表观遗传调控

The Epigenetic Regulation of SIRT1 on the Proliferation of Uveal Melanoma Cells

目的:探讨沉默信息调节蛋白1(SIRT1)对人葡萄膜黑色素瘤细胞增殖的影响。方法:实验研究。通过定量PCR和Western blot法检测SIRT1在葡萄膜黑色素瘤细胞(M23和SP6.5)和正常人葡萄膜黑色素细胞(DM78、UM95和UM96)中的表达。在葡萄膜黑色素瘤细胞(M23、SP6.5)中,用EX527抑制SIRT1活性,以溶剂DMSO作为阴性对照组;用SIRT1小干扰RNA(siSIRT1)转染细胞抑制SIRT1表达,以无义小干扰RNA(NC)转染作为阴性对照组。细胞增殖实验(MTS)、流式细胞术、平板克隆形成实验分别检测细胞活性、细胞周期以及细胞克隆形成能力。裸鼠眼内成瘤实验检测葡萄膜黑色素瘤细胞眼内成瘤能力。Western blot法检测增殖相关蛋白。组间数据均采用独立样本t检验进行比较。结果:与葡萄膜黑色素细胞相比,葡萄膜黑色素瘤细胞(M23、SP6.5)中SIRT1 mRNA(t=17.08,P<0.001;t=13.24,P<0.001)和蛋白(t=6.26,P=0.008)表达水平显著升高。MTS结果显示,EX527抑制M23和SP6.5细胞活性,并呈药物浓度依赖性;与NC转染组相比,siSIRT1转染组细胞活性显著减弱(t=5.94,P<0.001;t=10.73,P<0.001)。与溶剂DMSO组相比,EX527处理组(t=5.10,P=0.047;t=7.57,P=0.002)和SIRT1 siRNA转染组(t=6.41,P=0.003;t=6.10,P=0.004)中处于G1期的细胞比例均显著升高。另外,M23和SP6.5细胞EX527处理组(t=5.04,P=0.001;t=5.93,P<0.001)和siSIRT1转染组(t=11.44,P<0.001;t=8.24,P<0.001)克隆形成数量显著降低。在裸鼠眼内成瘤实验中,与NC组相比,siSIRT1转染组眼内瘤体大小显著变小(t=5.50,P<0.001)。Western blot结果显示,与DMSO处理组相比,EX527处理组中P21(t=4.63,P=0.010;t=7.90,P=0.001)表达升高,E2F1(t=12.10,P=0.003;t=5.96,P=0.004)、CYCLIND2(t=36.28,P<0.001;t=16.58,P<0.001)、p-RB(t=17.55,P<0.001;t=21.34,P<0.001)表达降低,p-AKT表达下调。与NC转染组相比,siSIRT1转染组中,P21(t=5.88,P=0.004;t=10.51,P<0.001)表达升高,E2F1(t=4.60,P=0.01;t=4.89,P=0.008)、CYCLIND2(t=5.13,P=0.007;t=5.63,P=0.005)、p-RB(t=4.45,P=0.011;t=6.53,P=0.003)表达水平降低,p-AKT(t=9.61,P<0.001;t=6.44,P=0.003)表达下调。结论:抑制SIRT1活性或表达可以明显抑制葡萄膜黑色素瘤细胞的增殖,提示SIRT1可以作为治疗葡萄膜黑色素瘤的一个新靶标。

Objective:To investigate the effects of the silent information regulator protein1(SIRT1)on the proliferation of uveal melanoma cells.Methods:In this experimental research,the expression of SIRT1 was detected in uveal melanoma cells(M23,SP6.5)and uveal melanocytes through Western blot and real-time quantitative PCR analysis.M23 and SP6.5 were treated with EX527 to inhibit SIRT1 activity and SIRT1 siRNA transfection to inhibit SIRT1 expression.DMSO treatment and siNC transfection were used in the control group.MTS,flow cytometry and colony formation assays were used to evaluate cell viability,cell cycle and colony formation ability.A mouse tumor-genesis experiment tested the ability of tumor formation in vivo.Proliferation-associated proteins were detected by Western blot analysis.Data were analyzed by independent t test.Results:SIRT1 was highly expressed in M23 and SP6.5 at mRNA(t=17.08,P<0.001;t=13.24,P<0.001)and protein(t=6.26,P=0.008)levels.MTS assay showed that the viability of M23 and SP6.5 was inhibited by EX527 and depended on the concentrations;siSIRT1 transfection also inhibited viability significantly(t=5.94,P<0.001;t=10.73,P<0.001).Flow cytometry revealed that EX527 treatment resulted in G1 phase arrest in M23 and SP6.5(t=5.10,P=0.047;t=7.57,P=0.002);the percentage of cells in the G1 stage were markedly high in the siSIRT1 transfection group compared to the NC group(t=6.41,P=0.003;t=6.10,P=0.004).Compared to the control groups,the clone numbers of M23 and SP6.5 cells in the EX527 group(t=5.04,P=0.001;t=5.93,P<0.001)and siSIRT1 transfected group(t=11.44,P<0.001;t=8.24,P<0.001)were obviously decreased.In addition,siSIRT1 transfection markedly suppressed the tumor-genesis in vivo(t=5.50,P<0.001).Western blot analysis showed that the expression levels of E2F1(t=12.10,P=0.003;t=5.96,P=0.004),CYCLIND2(t=36.28,P<0.001;t=16.58,P<0.001),p-RB(t=17.55,P<0.001;t=21.34,P<0.001),as well as p-AKT were significantly decreased while P21(t=4.63,P=0.010;t=7.90,P=0.001)was increased in EX527-treated cells.In siSIRT1 transfected cells,E2F1(t=4.60,P=0.01;t=4.89,P=0.008),CYCLIND2(t=5.13,P=0.007;t=5.63,P=0.005),p-RB(t=4.45,P=0.011;t=6.53,P=0.003),and p-AKT(t=9.61,P<0.001;t=6.44,P=0.003)decreased but P21(t=5.88,P=0.004;t=10.51,P<0.001)increased significantly.Conclusions:Inhibition of SIRT1 activity or expression suppresses UM cell proliferation,suggesting SIRT1 is a promising target for the treatment of this malignancy.