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目的哺乳动物细胞中H3K9的甲基化是引起基因表达抑制的主要修饰标记之一,H3K9的二甲基化在维持肿瘤细胞自我复制方面所起的作用仍不清楚。本研究将探讨组蛋白甲基化转移酶G9a抑制剂BIX-01294对肝癌细胞增殖的影响及其作用机制。方法将肝癌细胞HepG2和Huh7分为BIX-01294处理组和空白对照组,用5μmol/L的BIX-01294分别处理24、48和72h。CCK-8法检测BIX-01294对肝癌细胞增殖的影响;流式细胞术检测对细胞周期的影响;蛋白质印迹法检测细胞周期相关蛋白CyclinB1、细胞周期蛋白依赖激酶抑制因子p27、组蛋白H3及组蛋白H3K9二甲基化(H3K9me2)水平的变化。结果随着作用时间的延长,5μmol/L的BIX-01294对肝癌细胞有抑制增殖作用,且具有时间依赖性;在处理肝癌细胞24、48和72h后,HepG2细胞的存活率分别为95.27%、92.19%和53.94%,F=76.058,P<0.001;Huh7细胞的存活率分别为93.11%、86.12%和47.72%,F=99.832,P<0.001。流式细胞术检测结果表明,BIX-01294处理组肝癌细胞发生G0/G1阻滞,且阻滞呈时间依赖性。分别处理24、48和72h时,HepG2细胞G0/G1细胞比例分别为(70.55±1.38)%、(72.27±0.59)%和(79.63±3.40)%,F=19.937,P<0.001;Huh7细胞G0/G1细胞比例分别为(72.34±0.60)%、(80.36±0.78)%和(79.77±1.01)%,F=256.060,P<0.001。蛋白质印迹法检测结果显示,与对照组相比,组蛋白H3表达无显著改变,G9a、组蛋白H3K9二甲基化(H3K9me2)水平及细胞周期相关蛋白CyclinB1表达显著降低,细胞周期依赖激酶抑制因子p27的表达显著上调。结论 BIX-01294通过抑制G9a的活性,下调H3K9me2水平,从而抑制肝癌细胞HepG2及Huh7的增殖,促进G0/G1的周期阻滞,其机制可能与上调细胞周期依赖激酶抑制剂p27,以及下调细胞周期相关蛋白CyclinB1表达有关。
Purpose Methylation of H3K9 in mammalian cells is one of the major modified markers that cause inhibition of gene expression. The role of dimerization of H3K9 in maintaining tumor cell self-replication remains unclear. This study will investigate the histone methyltransferase G9a inhibitor BIX-01294 on the proliferation of hepatocellular carcinoma cells and its mechanism of action. Methods Liver cancer cells HepG2 and Huh7 were divided into BIX-01294 treatment group and blank control group, and treated with 5 micromol / L BIX-01294 for 24, 48 and 72 hours respectively. The effect of BIX-01294 on the proliferation of hepatoma cells was detected by CCK-8 assay. The effect of BIX-01294 on cell cycle was detected by flow cytometry. The expressions of cyclinB1, p27, histone H3 and histone H3 Changes in protein H3K9 dimethylation (H3K9me2) levels. Results With the prolongation of action time, 5μmol / L BIX-01294 inhibited the proliferation of HepG2 cells in a time-dependent manner. The survival rates of HepG2 cells after treatment with HepG2 cells for 24, 48 and 72 h were 95.27% 92.19% and 53.94% respectively, F = 76.058, P <0.001. The survival rates of Huh7 cells were 93.11%, 86.12% and 47.72% respectively, F = 99.832, P <0.001. The results of flow cytometry showed that G0 / G1 arrest occurred in BIX-01294-treated hepatocarcinoma cells in a time-dependent manner. The percentages of G0 / G1 cells in HepG2 cells were (70.55 ± 1.38)%, (72.27 ± 0.59)% and (79.63 ± 3.40)%, F = 19.937, P <0.001 for 24, 48 and 72 h / G1 cells were (72.34 ± 0.60)%, (80.36 ± 0.78)% and (79.77 ± 1.01)% respectively, F = 256.060, P <0.001. The results of Western blotting showed that there was no significant change of histone H3 expression compared with the control group. The levels of G9a, H3K9me2 and the expression of CyclinB1 were significantly decreased. The cell cycle dependent kinase inhibitor The expression of p27 was significantly up-regulated. Conclusions BIX-01294 can inhibit the proliferation of HepG2 and Huh7 cells and promote the arrest of G0 / G1 by inhibiting the activity of G9a and down-regulating the level of H3K9me2. The mechanism may be related to up-regulation of p27 and down-regulation of cell cycle Related protein CyclinB1 expression.