BIX-01294对Molt-4细胞增殖、凋亡及组蛋白甲基化的影响

Effect of BIX-01294 on cell proliferation,apoptosis and histone methylation in Molt-4 line

目的观察甲基化转移酶G9a抑制剂BIX-01294对急性T淋巴细胞白血病Molt-4细胞增殖、凋亡及组蛋白甲基化的影响。方法 MTT法绘制细胞生长曲线,流式细胞仪检测细胞凋亡的变化;Western blot检测凋亡相关蛋白Caspase-3、Bcl-2、Bax、抑癌蛋白P15、DNA去甲基化酶DNMT1、组蛋白H3乙酰化、组蛋白H3K9单、双、三甲基化水平,H3K27单甲基化、双甲基化水平的变化。结果 BIX-01294可下调Bcl-2蛋白、上调Bax蛋白的表达,Caspase-3表达上调,诱导细胞凋亡,浓度为0、1、2、4μmol·L-1的BIX-01294作用Molt-4细胞24 h后,凋亡率分别为(5.54±1.35)%、(10.24±2.26)%、(32.28±3.26)%、(47.52±4.37)%(P<0.05),差异有统计学意义。BIX-01294抑制DNMT1表达,上调P15蛋白表达,抑制细胞的增殖;BIX-01294下调组蛋白H3K9、H3K27单甲基化和二甲基化水平,而组蛋白H3乙酰化及H3K9三甲基化水平无明显改变。结论 BIX-01294通过抑制G9a的活性,下调H3K9、H3K27单甲基化、二甲基化水平,抑制DNMT1,使上调P15蛋白,最终抑制Molt-4细胞增殖,并诱导细胞凋亡。

Aim To investigate the effect of the specif-ic inhibitor BIX-01294 of G9a on the proliferation, ap-optosis,DNA methylation and histone modulation of a-cute leukemia cell line, Molt-4. Methods Cells were cultured with different concentrations of BIX-01294 . Cell growth was determined by MTT. Cell apoptosis was analyzed by flow cytometry. The expression of Caspase-3, Bcl-2, Bax, P15, acetylated H3, H3 K9 me1 , H3 K9 me2 , H3 K9 me3 and H3 K27 me1 , H3K27me2 methylation were detected by Western blot. Results BIX-01294 downregulated bcl-2 , and upreg-ulated Bax, Caspase-3 and induced apoptosis in （5. 54 ± 1. 35）%, （10. 24 ± 2. 26）%, （32. 28 ± 3. 26）%, （47. 52 ± 4. 37 ）% after 24 hours exposure to BIX-01294 in 0 , 1 , 2 , 4 μmol · L-1 . The difference be-tween them was statistically significant （ P 〈 0. 05 ） . BIX-01294 inhibited DMNT1 and promoted P15 , which resulted in cell proliferation inhibition. Further studies showed that BIX-01294 decreased H3 K9 me1 , H3 K9 me2 , and H3 K27 me1 , H3 K27 me2 , and didn′t change protein expression of acetylated H3 and H3 K9 me3. Conclusion BIX-01294 inhibits G9a, resulting in downregulation of methylation of H3 K9 me1 , H3 K9 me2 , H3 K27 me1 and H3 K27 me2 and DMNT1 and P15 denovo. It inhibits cell proliferation and in-duces cell apoptosis.