胚胎外胚层发育蛋白抑制剂抑制肝癌细胞增殖活性及其分子机制

Anticancer activity of embryonic ectoderm development inhibitor in hepatocellular carcinoma cells and molecular mechanism

目的研究应用胚胎外胚层发育(EED226)抑制组蛋白甲基化过度修饰是否具有抗癌活性并研究相关分子机制。方法体外培养肝癌细胞株BEL-7402和SMMC7721,用不同浓度的EED226处理肝癌细胞4、6、8 d,细胞计数试剂盒(CCK-8)检测细胞活性;5μmol/L的EED226或者二甲基亚砜(DMSO)处理肝癌BEL-7402和SMMC7721细胞株48 h,分别采用蛋白质印迹(Western blot)检测EED226对肝癌细胞组蛋白3上的第27位赖氨酸的三甲基化(H3K27me3)的表达水平的影响和定量反转录-聚合酶链反应(qRT-PCR)、Western blot检测EED226对肝癌细胞株的Bcl-2相互作用细胞死亡介导因子(Bim)和细胞周期依赖性蛋白激酶抑制因子1A(p21)的表达水平的影响。组间比较采用t检验。结果EED226能强效抑制肝癌细胞株BEL-7402和SMMC7721的增殖,抑制效应呈现明显的时间、浓度依赖作用。处理8 d后,EED226在两个细胞株中的半数细胞活性抑制率(IC50)分别为0.8μmol/L和0.9μmol/L。分子机制研究结果显示,2个肝癌细胞株分别经EED226处理与DMSO处理后H3K27me3的表达相比较显著下降。在BEL-7402和SMMC7721细胞中,EED226处理组H3K27me3的下游基因Bim和p21的mRNA相对表达水平高于DMSO组[1.00±0.15比5.67±1.53(t=-5.266,P<0.01),1.00±0.05比6.67±1.53(t=-6.422,P<0.05)和1.00±0.25比6.30±1.50(t=-5.968,P<0.05),1.00±0.10比6.00±1.00(t=-8.617,P<0.05)],差异有统计学意义;相应的Bim和p21蛋白质水平均显著升高。结论表观遗传调控新型抗癌制剂EED226能够抑制肝癌细胞中组蛋白三价甲基化过度修饰,进而解除对Bim和p21表达的抑制,达到抑制肝癌细胞增殖,表明EED抑制剂具有一定的抗肝癌作用。

Objective Embryonic ectoderm development(EED)226 is a newly developed anticancer agent through targeting trimethylation of trimethylation of lysine 27 on histone 3(H3K27me3).H3K27me3 is associated with malignancy characteristics of hepatocellular carcinoma(HCC).However,the anticancer activity of EED226 in HCC remains to be investigated.Methods Liver cancer cell lines BEL-7402 and SMMC7721 were treated with different concentrations of EED226 for 4,6,and 8 days,and cell counting kit-8(CCK-8)assay was used to examine cell growth.Liver cancer lines BEL-7402 and SMMC7721 were treated with 5μmol/L EED226 or dimethyl sulfoxide(DMSO)for 48 h.Western blotting was used to examine the expression level of H3K27me3,bcl2-interacting mediator of cell death(Bim)and cyclin-dependent kinase inhibitor 1A(P21).Reverse transcription-polymerase chain reaction(RT-PCR)was used to examine the mRNA level of Bim and p21.Results EED226 time-and dose-dependently inhibited cell growth in both HCC BEL-7402 and SMMC7721 cell lines.EED226 at 8th day achieved half maximal inhibitory concentration(IC50)values of 0.8 and 0.9μmol/L,respectively,in BEL-7402 and SMMC7721 cell lines.In both HCC cell lines,EED226 at 2nd day effectively inhibited the level of H3K27me3.Moreover,EED226 at 2nd day also substantially increased the expression of Bim and p21 at both mRNA level(1.00±0.15 vs.5.67±1.53,t=-5.266,P<0.01;1.00±0.05 vs.6.67±1.53,t=-6.422,P<0.05 and 1.00±0.25 vs.6.30±1.50,t=-5.968,P<0.05;1.00±0.10 vs.6.00±1.00,t=-8.617,P<0.05),respectively,and the protein level of Bim and p21 increased correspondingly.Conclusion Inhibition of H3K27me3 by targeting EED results in derepression of Bim and p21,subsequently leading to strong anticancer activity in HCC cells.EED inhibitors may represent a promising therapeutic in HCC.