SOCS超甲基化在经典型慢性骨髓增殖性肿瘤发病中的作用研究

Association between SOCS gene hypermethylation and classical myeloproliferative neoplasms

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、SOCS3mRNA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、SOCS3mRNA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。

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＆amp;nbsp;biomarker and therapeutic target for the disease.