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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 mechanism of sirolimus inducing γ-globin gene expression in K562 cells. Methods K562 cells were treated with sirolimus at final concentration of 10 nmol·L-1 for 3 days. A positive control (sodium butyrate), dimethyl sulfoxide (DMSO) control and blank control were also established. Western blot was used to detect p38MAPK. The real-time PCR-based chromatin immunoprecipitation method was used to analyze the level of acetylated histone H3 in the promoter of γ-globin gene. Results The relative levels of phosphorylated p38MAPK and the levels of γ-globin gene promoter acetylated histone H3 in sirolimus-treated K562 cells were 2.8 and 9.8 times higher than those in blank control cells, respectively, compared with dimethylsulfoxide treatment group 2.9 and 9.1 times higher, respectively, but no significant difference with sodium butyrate-treated cells. SB203580 pretreatment K562 cells can abrogate the role of sirolimus in increasing phosphorylated p38MAPK and gamma-globin gene promoter acetylated histone H3. Conclusion The mechanism of sirolimus inducing γ-globin gene expression in K562 cells is related to the activation of p38MAPK signal and upregulation of acetylated histone H3.