CAJ | 학술논문

目的研究PU.1基因在慢性粒细胞性白血病(CML)患者骨髓细胞中表达下降的可能机制和意义.方法采用亚硫酸氢盐修饰后测序法检测PU.1基因富含20个CpG位点的启动子区序列,了解正常人、CML慢性期和急变期患者、经伊马替尼治疗后获得细胞遗传学完全缓解患者骨髓细胞以及CML急变细胞株K562中PU.1基因启动子区甲基化状态.采用半定量逆转录聚合酶链反应(RT-PCR)测定正常对照、CML慢性期和急变期患者骨髓细胞以及CML急变细胞株K562中PU.1基因mRNA的表达水平.采用间接免疫荧光法和Western blot法检测CML慢性期和急变期患者骨髓细胞以及CML急变细胞株K562中PU.1蛋白的表达水平.结果在CML慢性期和急变期患者骨髓细胞以及CML急变细胞株K562中,PU.1基因富含20个CpG位点的启动子区域存在异常高度甲基化状态(最多的克隆20个位点中有17个位点发生甲基化),并导致PU.1 mRNA和蛋白的表达水平下降.在正常对照者骨髓细胞中,PU.1基因启动子区域无甲基化改变,PU.1 mRNA和蛋白的表达水平也并未下降.经伊马替尼治疗后获得细胞遗传学完全缓解的CML患者骨髓细胞中仍存在PU.1基因启动子区域的高度甲基化状态.结论 CML患者骨髓细胞和K562细胞系中,PU.1基因的表观遗传学异常调控可能是导致PU.1基因表达下降的潜在机制.PU.1基因的异常高度甲基化参与了CML的发病过程,可能为CML的治疗提供新的靶点或预后标记.
목적 연구PU.1기인재만성립세포성백혈병(CML)환자골수세포중표체하강적가능궤제화의의.방법 채용아류산경염수식후측서법검측PU.1기인부함20개CpG위점적계동자구서렬,료해정상인、CML만성기화급변기환자、경이마체니치료후획득세포유전학완전완해환자골수세포이급CML급변세포주K562중PU.1기인계동자구갑기화상태.채용반정량역전록취합매련반응(RT-PCR)측정정상대조、CML만성기화급변기환자골수세포이급CML급변세포주K562중PU.1기인mRNA적표체수평.채용간접면역형광법화Western blot법검측CML만성기화급변기환자골수세포이급CML급변세포주K562중PU.1단백적표체수평.결과 재CML만성기화급변기환자골수세포이급CML급변세포주K562중,PU.1기인부함20개CpG위점적계동자구역존재이상고도갑기화상태(최다적극륭20개위점중유17개위점발생갑기화),병도치PU.1 mRNA화단백적표체수평하강.재정상대조자골수세포중,PU.1기인계동자구역무갑기화개변,PU.1 mRNA화단백적표체수평야병미하강.경이마체니치료후획득세포유전학완전완해적CML환자골수세포중잉존재PU.1기인계동자구역적고도갑기화상태.결론 CML환자골수세포화K562세포계중,PU.1기인적표관유전학이상조공가능시도치PU.1기인표체하강적잠재궤제.PU.1기인적이상고도갑기화삼여료CML적발병과정,가능위CML적치료제공신적파점혹예후표기.
Objective To investigate the underlying mechanism and clinical significance of PU.1 down-expression in chronic myeloid leukemia (CML) patients.Methods Different methylation status of PU.1 promoter region containing 20 CpG islands in normal individuals,CML chronic phase and blast crisis patients,complete cytogenetic remission patients after imatinib treatment,and blast crisis bone marrow K562 CML cells was detected by bisulfite sequencing.Semi-quantitative PCR was used to detect the PU.1 mRNA expression in normal controls,CML chronic phase and blast crisis patients,and blast crisis bone marrow K562 CML cells.Indirect immune fluorescence and Western blot were used to analyze the exprteasion of PU.1 protein in normal individuals,CML chronic phase and blast crisis patients,and blast crisis bone marrow K562 CML cells.Results Aberrant methylation in the promoter region of transcription factor PU.1 was found in both CML chronic phase and blast crisis phase bone marrow cells,as well as in CML blast K562 cells.Down-expression of PU.1 mRNA and protein levels was found in above cells.No methylation in the promoter region of PU.1 was observed in normal individuals,and the PU.1 mRNA and protein expressions were not reduced at all.Furthermore,high methylation status of bone marrow cells was even observed in the CML patients who acquired complete cytogenetic remission.Conclusions The results of our study indicate that the epigenetic modification of PU.1 in CML patients and K562 cell line might be responsible for the down-expression of PU.1.The data suggest that aberrant methylation of PU.1 plays a role in CML pathogenesis,therefore,it might serve as a useful biomarker and potential target in therapy for chronic myeloid leukemia.