CAJ | 학술논문

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XAFI基因启动子CpG岛甲基化状态.采用0～5 μmoL/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XAFl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72 h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48 h的IC50值分别为2.4 μmol/L和2.6 μmol/L.结论骨髓瘤细胞中抑癌基因XAFI表达缺失或表达异常与XAF1基凶启动子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.
목적 탐토5-담잡포감처리대골수류세포계RPMI 8226화XG-7세포중XAF1기인표체적영향급체외항골수류작용적궤제.방법 채용역전록PCR화Western blot방법검측골수류세포계RPMI 8226화XG-7세포중XAF1기인화단백적표체.채용갑기화특이성PCR(MSP)방법검측XAFI기인계동자CpG도갑기화상태.채용0～5 μmoL/L 5-담잡포감처리골수류세포주.채용CCK-8비색법검측5-담잡포감처리대골수류세포증식억제작용.채용Annexin V/7-AAD염색류식세포술검측세포조망.결과 XG-7세포불표체XAF1 mRNA급단백,RPMI 8226세포표체XAF1 mRNA전록본1화2.XG-7화RPMI 8226세포XAFl기인계동자CpG도균존재과갑기화.XG-7화RPMI 8226세포경2.5μmol/L 5-담잡포감처리72 h후부표체XAF1 mRNA전록본1병표체XAF1단백,병차XAF1기인계동자CpG도갑기화정도강저.5-담잡포감항골수류작용정시간화농도의뢰성.5-담잡포감처리RPMI 8226화XG-7세포48 h적IC50치분별위2.4 μmol/L화2.6 μmol/L.결론 골수류세포중억암기인XAFI표체결실혹표체이상여XAF1기흉계동자CpG도과갑기화유관.5-담잡포감처리가이유도XAF1 mRNA급단백표체.5-담잡포감재림상상능체도적약물농도하구유항골수류작용,기작용궤제시유도골수류세포조망.
Objective To investigate the effect of 5-azacytidine on XAFI expression in myeloma cell lines RPMI8226 and XG-7 and the in vitro anti-myeloma activity of 5-azacytidine.Methods XAFI mRNA and protein expression was detected by semi-quantitative reverse transeriptage PCR and Western blot.respectinely.Methylation specific PCR(MSP)was used to detect methylation status of XAFl promoter CpG islands.RPMI8226 and XG-7 cells were treated with 0-5μmol/L of 5-azaeytidine and Cell Counting Kit-8colorimetric 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 wag 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-azaeytidine treatment led to XAF1 promoter CpG islands hypomethylation and showed anti-myeloma activity in a time-and concentration-dependent manner with IC50 of2.4 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 title agseciated with XAF1 gene promoter CpG islands hypermethylation.5-azacytidine treatment can induce XAF1 mRNA and protein expression and execs anti-myeloma activity via apoptosis at clinically achievable concentrations.