CAJ | 학술논문

目的探讨细胞因子信号传导抑制蛋白（SOCS）基因超甲基化在经典型慢性骨髓增殖性肿瘤（MPN）发病机制中的作用。方法采用甲基化特异性PCR法检测220例MPN患者骨髓标本中SOCS1、SOCS3基因启动子区CpG岛甲基化发生情况，直接测序法检测MPN患者JAK2V617F、MPLW515L/K基因突变情况。应用粒细胞集落刺激因子化学诱导MPN细胞生长不同时间后，采用实时定量PCR和蛋白印迹法检测SOCS1、SOCS3 mRNA及其蛋白表达情况。同时，加入去甲基化试剂培养MPN细胞不同时间后，实时定量PCR法检测SOCS1、SOCS3 mRNA表达情况。结果 MPN患者中44例（20.0%）存在SOCS1基因超甲基化，90例（40.9%）存在SOCS3基因超甲基化，156例（70.9%）JAK2V617F突变阳性，3例JAK2V617F未突变的原发性血小板增多症患者检测到MPLW515突变（其中2例为MPLW515L，1例为MPLW515K），2例JAK2V617F未突变原发性骨髓纤维化患者检测到MPLW515L突变；SOCS1、SOCS3基因超甲基化组与未甲基化组相比，其SOCS1、SOCS3 mRNA和蛋白表达量明显减少（P＜0.05）；JAK2V617突变组与无突变组相比，其SOCS1、SOCS3 mRNA和蛋白表达量明显减少（P＜0.05）；SOCS1、SOCS3基因超甲基化组，加入去甲基化试剂后SOCS1、SOCS3 mRNA表达量明显升高（P＜0.05）。结论 MPN患者中存在高频率的JAK2V617F基因突变和低频率的MPL基因突变以及SOCS基因启动子区CpG岛超甲基化；SOCS超甲基化和JAK2V617F突变导致SOCSmRNA和蛋白表达水平降低，异常激活JAK/STAT信号传导通路，引起细胞代谢失常而最终影响MPN的发生、发展；SOCS超甲基化是一种潜在的MPN诊断生物分子标志物及治疗靶标。
목적탐토세포인자신호전도억제단백（SOCS）기인초갑기화재경전형만성골수증식성종류（MPN）발병궤제중적작용。방법채용갑기화특이성PCR법검측220례MPN환자골수표본중SOCS1、SOCS3기인계동자구CpG도갑기화발생정황，직접측서법검측MPN환자JAK2V617F、MPLW515L/K기인돌변정황。응용립세포집락자격인자화학유도MPN세포생장불동시간후，채용실시정량PCR화단백인적법검측SOCS1、SOCS3 mRNA급기단백표체정황。동시，가입거갑기화시제배양MPN세포불동시간후，실시정량PCR법검측SOCS1、SOCS3 mRNA표체정황。결과 MPN환자중44례（20.0%）존재SOCS1기인초갑기화，90례（40.9%）존재SOCS3기인초갑기화，156례（70.9%）JAK2V617F돌변양성，3례JAK2V617F미돌변적원발성혈소판증다증환자검측도MPLW515돌변（기중2례위MPLW515L，1례위MPLW515K），2례JAK2V617F미돌변원발성골수섬유화환자검측도MPLW515L돌변；SOCS1、SOCS3기인초갑기화조여미갑기화조상비，기SOCS1、SOCS3 mRNA화단백표체량명현감소（P＜0.05）；JAK2V617돌변조여무돌변조상비，기SOCS1、SOCS3 mRNA화단백표체량명현감소（P＜0.05）；SOCS1、SOCS3기인초갑기화조，가입거갑기화시제후SOCS1、SOCS3 mRNA표체량명현승고（P＜0.05）。결론 MPN환자중존재고빈솔적JAK2V617F기인돌변화저빈솔적MPL기인돌변이급SOCS기인계동자구CpG도초갑기화；SOCS초갑기화화JAK2V617F돌변도치SOCSmRNA화단백표체수평강저，이상격활JAK/STAT신호전도통로，인기세포대사실상이최종영향MPN적발생、발전；SOCS초갑기화시일충잠재적MPN진단생물분자표지물급치료파표。
Objective To investigate the association between hypermethylation of suppressor of cytokine signaling(SOCS) gene and typical myeloproliferative neoplasms (MPN). Methods Methylation- specific PCR was used to detect CpG island methylation status of SOCS1 and SOCS3 genes in bone marrow samples from 220 MPN patients. JAK2V617F and MPLW515L/K gene mutations were detected by direct DNA sequencing assay. The expression of SOCS1 and SOCS3mRNA and protein was e-valuated by real- time quantitative PCR and Western blot, after MPN cells were co- cultured with GC- SF or demethylating agent 5- aza- 2′- deoxyazacytidin at different time points. Results The rates of SOCS1 and SOCS3 gene hypermethylation were 20.0%(44/220) and 40.9%(90/220), respectively. JAK2V617F positive mutation was detected in 156 patients (70.9%); in 3 JAK2V617F- negative patients with essential thrombocytosis (ET), MPLW515 mutation was detected (2 MPLW515L and 1 of MPLW515K) and in 2 JAK2V617F- negative patients with idiopathic myelofibrosis (IMF), MPLW515L mutation was detected. The expression of SOCS1,SOCS3 mRNA and protein was significantly decreased in hypermethylation group compared to non- methylation group(P<0.05);and in JAK2V617F mutation group compared to wild type group(P<0.05). After co- cultured with demethylating agent the expression of SOCS1,SOCS3mRNA was increased significantly in hypermethylation group (P<0.05). Conclusion High- frequency JAK2V617F gene mutation, low- frequency MPL gene mutation and SOCS gene CpG island hyper-methylation are associated with myeloproliferative neoplasms, and SOCS hypermethylation might be a potential diagnostic biomarker and therapeutic target for the disease.