5-氮杂胞苷对骨髓瘤细胞增殖抑制作用及对XAF1基因表达的影响

5-azacytidine treatment induces tumor suppressor gene XAF1 expression and inhibits proliferation in myeloma cells

目的探讨5-氮杂胞苷处理对骨髓瘤细胞系RPMI 8226和XG-7细胞中XAF1基因表达的影响及体外抗骨髓瘤作用的机制。方法采用逆转录PCR和Western blot方法检测骨髓瘤细胞系RPMI 8226和XG-7细胞中XAF1基因和蛋白的表达。采用甲基化特异性PCR（MSP）方法检测XAF1基因启动子CpG岛甲基化状态。采用0～5μmol／L 5-氮杂胞苷处理骨髓瘤细胞株。采用CCK-8比色法检测5-氮杂胞苷处理对骨髓瘤细胞增殖抑制作用。采用Annexin V／7-AAD染色流式细胞术检测细胞凋亡。结果XG-7细胞不表达XAF1 mRNA及蛋白，RPMI 8226细胞表达XAF1 mRNA转录本1和2。XG-7和RPMI 8226细胞XAF1基因启动子CpG岛均存在过甲基化。XG-7和RPMI 8226细胞经2．5μmol／L 5-氮杂胞苷处理72h后仅表达XAF1 mRNA转录本1并表达XAF1蛋白，并且XAF1基因启动子CpG岛甲基化程度降低。5-氮杂胞苷抗骨髓瘤作用呈时间和浓度依赖性。5-氮杂胞苷处理RPMI 8226和XG-7细胞48h的，氏值分别为2．4μmol／L和2．6μmol／L。结论骨髓瘤细胞中抑癌基因XAF1表达缺失或表达异常与XAF1基因启动子CpG岛过甲基化有关。5-氮杂胞苷处理可以诱导XAF1 mRNA及蛋白表达。5-氮杂胞苷在临床上能达到的药物浓度下具有抗骨髓瘤作用，其作用机制是诱导骨髓瘤细胞凋亡。

Objective To investigate the effect of S-azacytidine on XAF1 expression in myeloma cell lines RPMI8226 and XG-7 and the in vitro anti-myeloma activity of 5-azacytidine. Methods XAF1 mRNA and protein expression was detected by semi-quantitative reverse transcriptase PCR and Western blot, respectinely. Methylation specific PCR （MSP） was used to detect methylation status of XAF1 promoter CpG islands. RPMI8226 and XG-7 cells were treated with 0 - 5 μmol/L of 5-azacytidine and Cell Counting Kit-8 colorimetric assay was used to evaluate the growth inhibitory effect. Cell apoptosis was determined with Annexin V-PE/7-AAD staining by flow cytometry. Results Untreated RPMI8226 cells expressed XAF1 mRNA isoforms 1 and 2, and untreated XG-7 cells had no XAF1 expression. Hypermethylation of XAF1 promoter CpG islands was detected in both the cell lines. After treated with 2.5 μmol/L 5-azacytidine for 72 h, both the cell lines expressed full-length XAF1 transcript and protein. 5-azacytidine treatment led to XAF1 promoter CpG islands bypomethylation and showed anti-myeloma activity in a time- and concentration-dependent manner with IC50 of 2.4 μmol/L and 2.6 μmol/L at 48 h for RPMI8226 and XG-7 cell lines, respectively. Conclusions Lack of XAF1 expression and abnormal expression of XAF1 in myeloma cell lines are associated with XAF1 gene promoter CpG islands hypermethylation. 5-azacytidine treatment can induce XAF1 mRNA and protein expression and exerts anti-myeloma activity via apoptosis at clinically achievable concentrations.