雌激素促乳腺癌细胞MCF-7增殖中组蛋白乙酰化的作用及其意义

Role and significance of histone acetylation in proliferation of breast cancer cell line MCF-7 induced by estrogen

摘要:

目的：研究组蛋白乙酰化水平改变在雌激素促乳腺癌细胞增殖中的作用及其意义。方法：以人乳腺癌细胞MCF-7为研究对象，应用CCK-8法检测17-β雌二醇（17β-estradiol，１７β-E2）对MCF-7细胞增殖影响，根据增殖率确定后续实验处理浓度；应用CCK-8法检测Garcinol 对17β-E2促MCF-7细胞增殖作用的影响，根据抑制率求取其中效抑制浓度；流式细胞术检测细胞周期、细胞凋亡情况；Western blot检测乙酰化H３（acetylated histone 3，ac-H3）、乙酰化H4（acetylated histone 4，ac-H4）、细胞周期蛋白１、B细胞淋巴瘤/白血病-２（B cell lymphoma/leukemia-2，Bcl-2）、B细胞淋巴瘤/白血病-xl（B cell lymphoma/leukemia-xl，Bcl-xl）蛋白水平表达；RT-PCR检测CyclinD1 mRNA、Bcl-2 mRNA、Bcl-xl mRAN水平。结果：17β-E2的促MCF-7细胞增殖作用受到乙酰化酶抑制剂Garcinol抑制，使细胞周期转化阻滞于G0/G1期，细胞凋亡率增加（P=0.000）；17β-E2促进MCF-7细胞ac-H3、ac-H4的表达（P=0.007、P=0.013），促进CyclinD1、Bcl-2、Bcl-xl 蛋白表达（P=0.000、P=0.002、P=0.000），Garcinol能抑制17β-E2对ac-H3的促进表达（P=0.006），对ac-H4无显著影响（P=0.347），17β-E2的促CyclinD1、Bcl-2、Bcl-xl表达亦受到Garcinol抑制（P=0.000、P=0.001、P=0.001）；相应可见，17β-E2促进CyclinD1、Bcl-2、Bcl-xl mRNA转录（P=0.007、P=0.044、P=0.007），该作用受到Garcinol抑制（P=0.001、P=0.017、P=0.002）。结论：17β-E2促MCF-7细胞增殖、凋亡抑制作用与组蛋白乙酰化水平增高有关，Garcinol 对雌激素促乳腺癌细胞增殖具有抑制作用。

Abstract:

Objective：To investigate the role and significance of histone acetylation in proliferation of breast cancer cell line MCF-7 induced by estrogen. Methods：Human breast cancer MCF-7 cells were used as the research object. Effect of 17β-estradiol（１７β-E2）on proliferation of MCF-7 cells was investigated with CCK-8 assay and the optimal concentration of 17β-E2 in the following experi－ment was determined according to the proliferation rate. Effect of Garcinol on proliferation of MCF-7 cells by 17β-E2 was investigat－ed with CCK-8 assay and the intermediate concentration of Garcinol in the following experiment was determined according to the in－hibition rate. Cell cycle and apoptosis were analyzed by flow cytometry. Expression of acetylated histone 3（ac-H3），acetylated histone ４（ac-H4） protein was determined by Western blot. CyclinD1 mRNA，B cell lymphoma/leukemia-2（Bcl-2） mRNA，B cell lymphoma/leukemia-xl（Bcl-xl） mRAN expression in MCF -7 cells was measured by RT-PCR and expression of CyclinD1，Bcl-2，Bcl-xl protein was determined by Western blot. Results：Proliferation of MCF-7 cells treated with 17β-E2 was inhibited by acetyltransferase inhibitor Garcinol and transformation of MCF-7 cell cycle was arrested at G0/G1 phase and rate of apoptosis was increased（P=0.000）. Expres－sion level of ac-H3，ac-H4 protein in MCF-7 cells treated with 17β-E2 was increased（P=0.007，P=0.013），in which ac-H3 was inhib－ited by Garcinol（P=0.006） but no ac-H4（P=0.347）. Correspondingly，17β-E2 promoted the mRNA transcription（P=0.007，P=0.044，P=0.007） and protein expression（P=0.000，P=0.002，P=0.000） levels of CyclinD1，Bcl-2，Bcl-xl in MCF-7 cells，which were also in－hibited by Garcinol（P=0.001，P=0.017，P=0.002，P=0.000，P=0.001，P=0.001）. Conclusions：17β-E2 promoting proliferation and inhibiting apoptosis of human breast cancer MCF-7 cells is related with elevated acetylation level of histone. Garcinol plays an inhibitive role in 17β-E2 promoting proliferation of MCF-7 cells.