CAJ | 학술논문

目的探讨SHP-1基因表达与启动子甲基化状态在慢性髓性白血病(CML)疾病进展中的作用及其机制.方法 SYBR Green荧光定量PCR及Western blot方法检测94例CML患者骨髓或外周血单个核细胞中SHP-1基因的表达;甲基化特异性PCR(MSP)方法检测SHP-1基因启动子区CpG岛的甲基化状态;慢病毒表达载体感染K562细胞使SHP-1基因过表达,CCK-8法检测细胞增殖活性,流式细胞术检测细胞的凋亡率及细胞周期分布;荧光探针定量PCR检测BCR-ABL mRNA水平,Western blot方法检测蛋白及磷酸化蛋白的水平.结果疾病进展期CML(包括加速期和急变期)患者SHP-1 mRNA的相对表达水平为0.79±0.37,较慢性期CML(CP-CML)的1.18±0.64明显降低(P=0.009),疾病进展期CML患者SHP-1蛋白相对表达水平(0.57±0.02)较CP-CML患者降低(1.02±0.04)(P=0.039).CP-CML患者SHP-1基因启动子的甲基化阳性率(10/42,23.8％)明显低于疾病进展期CML患者(52/52,100.0％) (P＜0.01).过表达SHP-1基因的K562-SHP-1细胞与转染空载体的K562-EGFP细胞相比,增殖受抑,凋亡率增加,并出现G0/G1期的细胞阻滞.过表达SHP-1基因降低K562细胞中BCR-ABL蛋白的水平,但不影响BCR-ABL mRNA的转录;过表达SHP-1基因能降低K562细胞MYC蛋白及磷酸化Akt、MAPK、JAK2、STAT5蛋白的表达水平,而对K562细胞中Akt、MAPK、JAK2蛋白总表达水平无影响.结论 SHP-1基因启动子CpG岛甲基化及其基因沉默导致BCR-ABL、Akt、MAPK、JAK2、STAT5、MYC信号通路的异常激活与表达可能与CML疾病进展有关.
목적 탐토SHP-1기인표체여계동자갑기화상태재만성수성백혈병(CML)질병진전중적작용급기궤제.방법 SYBR Green형광정량PCR급Western blot방법검측94례CML환자골수혹외주혈단개핵세포중SHP-1기인적표체;갑기화특이성PCR(MSP)방법검측SHP-1기인계동자구CpG도적갑기화상태;만병독표체재체감염K562세포사SHP-1기인과표체,CCK-8법검측세포증식활성,류식세포술검측세포적조망솔급세포주기분포;형광탐침정량PCR검측BCR-ABL mRNA수평,Western blot방법검측단백급린산화단백적수평.결과 질병진전기CML(포괄가속기화급변기)환자SHP-1 mRNA적상대표체수평위0.79±0.37,교만성기CML(CP-CML)적1.18±0.64명현강저(P=0.009),질병진전기CML환자SHP-1단백상대표체수평(0.57±0.02)교CP-CML환자강저(1.02±0.04)(P=0.039).CP-CML환자SHP-1기인계동자적갑기화양성솔(10/42,23.8％)명현저우질병진전기CML환자(52/52,100.0％) (P＜0.01).과표체SHP-1기인적K562-SHP-1세포여전염공재체적K562-EGFP세포상비,증식수억,조망솔증가,병출현G0/G1기적세포조체.과표체SHP-1기인강저K562세포중BCR-ABL단백적수평,단불영향BCR-ABL mRNA적전록;과표체SHP-1기인능강저K562세포MYC단백급린산화Akt、MAPK、JAK2、STAT5단백적표체수평,이대K562세포중Akt、MAPK、JAK2단백총표체수평무영향.결론 SHP-1기인계동자CpG도갑기화급기기인침묵도치BCR-ABL、Akt、MAPK、JAK2、STAT5、MYC신호통로적이상격활여표체가능여CML질병진전유관.
Objective To investigate the profile of promoter methylation and expression of SHP-1 gene in the progression of chronic myeloid leukemia (CML).Methods The expression level of SHP-1 mRNA and protein in bone marrow or peripheral blood mononuclear cells from CML patients were detected by Western blot and SYBR Green-based qRT-PCR.The methylation status of SHP-1 were assessed by methylation-specific polymerase chain reaction (MSP) assay.K562 cells were infected with the lentiviral plasmids pEX-SHP-1-puro-Lv105 (K562-SHP-1) or pEX-EGFP-puro-Lv 105 (K562-EGFP).The levels of proteins and phosphorylated proteins were detected by Western blot.qRT-PCR assay was used to test the level of BCR-ABL mRNA.Results The relative levels of SHP-1 mRNA were sharply decreased in advanced stages CML compared to chronic phase (CP)-CML (0.79±0.37 vs 1.18±0.64,P=0.009).The level of SHP-1 protein was lower in advanced stages CML compared to CP-CML (0.57±0.02 vs 1.02±0.04,P=0.039).The frequency of SHP-1 gene promoter methylation at selected loci in CP-CML was 23.8％ (10/42),and the methylated regions were detected in all advanced CML samples (P＜0.01).SHP-1 was stably transfected into K562 cells and selected with puromycin.Overexpression of SHP-1 inhibited the proliferation and induced the apoptosis of K562 cells,meanwhile leaded to G0/G1 phase arrest.After transfection,the level of BCR-ABL mRNA was not affected in K562-SHP-1 cells (1.32 ± 0.34) compared to K562-EGFP cells (1.18 ± 0.20,P=0.644),but overexpression of SHP-1 caused a slight decrease in BCR-ABL protein in K562-SHP-1 cells compared to K562-EGFP cells (0.78±0.15 vs 1.27± 0.24,P=0.040).Overexpression of SHP-1 resulted in a remarkable decrease in MYC protein,phosphorylated forms of JAK2,STAT5,Akt and MAPK.However,the un-phosphorylated forms of these molecules were not significantly affected.Conclusion Decreased expression of SHP-1 caused by aberrant promoter hypermethylation may play a key role in the progression of CML by dysregulation of BCR-ABL,Akt,MAPK,MYC,JAK2 and STAT5 signaling.