地西他滨治疗亲嗜性病毒整合位点1高表达的慢性髓细胞性白血病分子机制研究

Molecular mechanism of decitabine in the treatment of chronic myelogenous leukemia with overexpression of ecotropic viral integration site 1

目的探索亲嗜性病毒整合位点1(ecotropic viral integration site 1,EVI1)高表达对K562细胞的影响,及DNA甲基转移酶抑制剂地西他滨治疗EVI1高表达的慢性髓细胞性白血病(chronic myelogenous leukemia,CML)的分子机制。方法使用逆转录病毒载体转染K562细胞,检测空载体组和EVI1组微小RNA(micro RNA,miR)-9表达水平、miR-9启动子序列甲基化程度及细胞克隆数。使用地西他滨和溶媒处理EVI1高表达的K562细胞,检测miR-9表达水平、miR-9启动子序列甲基化程度、细胞克隆数及细胞周期。结果与空载体组相比,EVI1组miR-9相对表达量显著降低[(0.0021±0.0003)比(0.0034±0.0003),P<0.01],miR-9启动子序列甲基化程度显著增加,细胞增殖能力增强[(232.67±6.81)比(124.67±5.03),P<0.01]。地西他滨组较溶媒组miR-9表达水平显著升高[(0.0023±0.0003)比(0.0007±0.0001),P<0.01],miR-9启动子甲基化程度显著降低[(44.67±6.57)%比(90.22±4.29)%,P<0.01],细胞增殖能力减弱[(173.67±12.06)比(219.33±10.26),P<0.01],G_(0)/G_(1)细胞比例增高[(59.25±1.20)%比(50.45±1.06)%,P<0.05]。结论EVI1通过增加miR-9启动子甲基化程度,下调miR-9表达,可能是EVI1参与CML的机制之一。地西他滨可逆转EVI1高表达所致的miR-9甲基化程度,肿瘤细胞克隆增殖能力得以遏制,为其用于EVI1高表达的CML治疗提供理论依据。

Objective To explore the effects of ecotropic viral integration site 1(EVI1)overexpression on K562 cells,and the molecular mechanism of DNA methyltransferase inhibitor decitabine in the treatment of chronic myelogenous leukemia(CML)with high expression of EVI1.Methods Retrovirus vectors were used to transfect K562 cells,and the expression level of micro RNA(miR)-9,the methylation degree of miR-9 promoter sequence and the number of cell clones in empty vector group and EVI1 group were detected.K562 cells with high EVI1 expression were treated with decitabine and solvent to detect the expression level of miR-9,methylation degree of miR-9 promoter sequence,number of cell clones and cell cycle.Results Compared with the empty vector group and EVI1 group,the relative expression level of miR-9 was significantly decreased[(0.0021±0.0003)vs(0.0034±0.0003),P<0.01],and the methylation degree of miR-9 promoter sequence was significantly increased,cell proliferation was enhanced[(232.67±6.81)vs(124.67±5.03),P<0.01].Compared with the solvent group,the expression level of miR-9 in decitabine group was significantly increased[(0.0023±0.0003)vs(0.0007±0.0001),P<0.01],and the methylation degree of miR-9 promoter was significantly decreased[(44.67±6.57)%vs(90.22±4.29)%,P<0.01],the cell proliferation ability was decreased[(173.67±12.06)vs(219.33±10.26),P<0.01],and the proportion of G_(0)/G_(1)cells was increased[(59.25±1.20)%vs(50.45±1.06)%,P<0.05].Conclusion EVI1 down-regulates the expression of miR-9 by increasing the methylation degree of miR-9 promoter,which may be one of the mechanisms of EVI1 participates in CML.Decitabine can reverse the methylation degree of miR-9 caused by EVI1 overexpression,and inhibit the cloning and proliferation ability of tumor cells,providing a theoretical basis for its use in the treatment of CML with EVI1 overexpression.