西罗莫司诱导K562细胞γ珠蛋白基因表达机制探讨

Mechanisms of Sirolimus Inducing Expression of γ-Globin Gene in K562 Cells

目的探讨西罗莫司诱导K562细胞γ珠蛋白基因表达的作用机制。方法终浓度10 nmol·L-1西罗莫司处理K562细胞3 d,同时设立阳性药物(丁酸钠)对照、二甲基亚砜对照和空白对照。采用Western blot方法检测p38MAPK。采用基于Real time PCR的染色质免疫共沉淀方法分析γ珠蛋白基因启动子乙酰化组蛋白H3水平。结果经西罗莫司处理的K562细胞的磷酸化p38MAPK相对水平及γ珠蛋白基因启动子乙酰化组蛋白H3水平分别比空白对照细胞升高2.8和9.8倍、分别比二甲基亚砜处理组升高2.9和9.1倍,而与丁酸钠处理的细胞无显著性差别。SB203580预处理K562细胞可中止西罗莫司升高磷酸化p38MAPK及γ珠蛋白基因启动子乙酰化组蛋白H3的作用。结论西罗莫司诱导K562细胞γ珠蛋白基因表达的机制与其激活p38MAPK信号和上调启动子乙酰化组蛋白H3有关。

OBJECTIVE To investigate the mechanisms of sirolimus inducing 7-globin gene expression in K562 cells. METHODS K562 cells were cultured in the presence of 10 nmol · L-l sirolimus, butylate, or DMSO for 3 d. Western blot and real time PCR-based chromatin immunoprecipitation was employed to measure the levels of p38MAPK and acetylated histone H3 （acH3） at 7-globin gene promoter regions, respectively. RESULTS In K562 cells with l0 nmol · L-j sirolimus treatment, phospholylated p38MAPK （p-p38MAPK） was 2. 8-fold greater and acH3 was 9.8-fold greater than that in untreated K562 ceils, and there was a 2. 9- fold in p-p38MAPK and a 9. 1-fold in acH3 increase comparing with K562 cells treated with DMSO, no significant difference in p- p38MAPK and acH3 level was found between cells treated with sirolimus and with butylate. SB203580 completely abolished induction of p38MAPK activation and acH3 increase by sirolimus. CONCLUSION Our results indicate that sirolimus actives p38MAPK signal and increases acetylation of H3 at 7-globin gene promoter regions, which may be the mechanisms of induced expression of 7-globin genes by sirolimus in K562 cells.