表观遗传学机制对白血病细胞维甲酸受体β基因表达的调控作用及意义

Epigenetic regulation of expression of retinoic acid receptor beta gene in leukemia cells

目的探索表观遗传学机制对白血病细胞维甲酸受体β基因(RARβ)表达的调控作用及意义。方法应用 DNA 甲基化酶抑制剂地西他滨(DAC)和组蛋白去乙酰化酶抑制剂丙戊酸钠(VPA)与全反式维甲酸(ATRA)处理白血病细胞;RARβ基因甲基化测定和表达定量;染色质免疫沉淀。结果 56例急性髓系白血病(AML)患者 RARβ基因甲基化总阳性率为64.3%。DAC 与 VPA 可显著增加 ATRA 对 RARβ表达的激活作用。染色质免疫沉淀实验证明 DAC 和 VPA 通过 DNA 去甲基化和组蛋白乙酰化作用上调 RARβ表达。三药联合可诱导部分 U937细胞出现髓系分化标记 CD11b,使克隆形成能力明显下降。结论表观遗传学调控剂与 ATRA 联合应用可激活 RARβ的表达,具有协同抗白血病作用。

Objective To study the reactivation of retinoic acid receptor β （RARβ） expression in myeloid leukemia cells by a combination of all-trans retinoic acid （ATRA） with a DNA demethylating agent, decitabine （DAC）, and valproic acid （VPA, a histone deacetylase inhibitor）, and their effects on cell differentiation and proliferation. Methods Human myeloid leukemia cells of the line U937 were cultured and treated with all-trans retinoic acid （ATRA）, DAC, and VPA. 72 h later cell differentiation test, cloning formation test, and chromatin immunoprecipitation test were performed. Bone marrow specimens were collected fi＇om 56 patients with acute myeloblastic leukemia （AML） were cultured and treated with and 10 bone marrow specimens were used as controls. Methylation of RARβ promoter was detected with methylation specific polymerase chain reaction （MSP） after bisulfite treatment. Relative levels of RARβ mRNA were assessed with real time quantitative PCR assay. Flow cytometry assay was used to detect the myeloid differentiation marker CDllb in U937 cells. Chromatin immuno-precipitation assay （CHIP） was used to analyze the acetylated histone 3 bound to the retinoic acid response element （RARE） at the promoter region of RARβ. Results Methylation of RARβ was positive in 36 of the 56 （64.3%） AML patients and the U937 myeloid leukemia cells, however, was negative in the marrow mononuclear cells from the 10 healthy donors. The expression of RARβ in U937 cells was up-regulated after treatment with ATRA （ 1 μmol/L ） plus DAC （ 1 μmol/L ） or VPA （0.5 mmol/L ） for 72 hours, especially when the three drugs were used together. ChIP assay showed that the acetylated histone 3 bound to the RARE promoter region was increased after the cells were exposed to ATRA plus DAC/VPA. The data indicated that the reactivated expression of RAR[3 might be secondary to the drugnduced histone acetylation as well as DNA demethylation. Treatment of U937 cells with ATRA and DAC/VPA also resulted in increased myeloid differentiation （CDllb expression） and decreased plating efficiency. Conclusion The repressed expression of RAR[3 in myeloid leukemia cells is closely related to both DNA hypermethylation and historic deacetylation, and a combination of ATRA with epigenetic modulators can be beneficial in the treatment of myeloid malignancies.