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
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摘要
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.JAK2V617 F and MPLW515L/K gene mutations were detected by direct DNA sequencing assay.The expression of SOCS1 and SOCS3 mRNA and protein was evaluated by real-time quantitative PCR and Western blot,after MPN cells were co-cultured with GC-SF or demethylating agent5-aza-2'-deoxyazacytidin at different time points.Results The rates of SOCS1 and SOCS3 gene hypermethylation were20.0%(44/220) and 40.9%(90/220),respectively.JAK2V617 F positive mutation was detected in 156 patients(70.9%);in 3JAK2V617F-negative patients with essential thrombocytosis(ET),MPLW515 mutation was detected(2 MPLW515 L and 1 of MPLW515K) and in 2 JAK2V617F-negative patients with idiopathic myelofibrosis(IMF),MPLW515 L 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 JAK2V617 F mutation group compared to wild type group(P<0.05).After co-cultured with demethylating agent the expression of SOCS1,SOCS3 mRNA was increased significantly in hypermethylation group(P<0.05).Conclusion High-frequency JAK2V617 F gene mutation,low-frequency MPL gene mutation and SOCS gene CpG island hypermethylation are associated with myeloproliferative neoplasms,and SOCS hypermethylation might be a potential diagnostic biomarker and therapeutic target for the disease.
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.JAK2V617 F and MPLW515L/K gene mutations were detected by direct DNA sequencing assay.The expression of SOCS1 and SOCS3 mRNA and protein was evaluated by real-time quantitative PCR and Western blot,after MPN cells were co-cultured with GC-SF or demethylating agent5-aza-2'-deoxyazacytidin at different time points.Results The rates of SOCS1 and SOCS3 gene hypermethylation were20.0%(44/220) and 40.9%(90/220),respectively.JAK2V617 F positive mutation was detected in 156 patients(70.9%);in 3JAK2V617F-negative patients with essential thrombocytosis(ET),MPLW515 mutation was detected(2 MPLW515 L and 1 of MPLW515K) and in 2 JAK2V617F-negative patients with idiopathic myelofibrosis(IMF),MPLW515 L 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 JAK2V617 F mutation group compared to wild type group(P<0.05).After co-cultured with demethylating agent the expression of SOCS1,SOCS3 mRNA was increased significantly in hypermethylation group(P<0.05).Conclusion High-frequency JAK2V617 F gene mutation,low-frequency MPL gene mutation and SOCS gene CpG island hypermethylation are associated with myeloproliferative neoplasms,and SOCS hypermethylation might be a potential diagnostic biomarker and therapeutic target for the disease.
引文
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[10]Xiong H,Du W,Zhang Y J.Tricho station A,a histone deacetylase inhibitor.suppresses JAK2/STAT3 signaling via inducing the promoter-associated histone acetylation of SOCS1 and SOCS3in human colorectal cancer cells[J].Mol Carcinog,2012,51(2):174-184.
[11]Hua D,Hu Y,Wu Y Y,et al.Quantitative methylation analysis of multiple genes using methylation-sensitive restriction enzyme-based quantitative PCR for the detection of hepatocellular carcinoma[J].Exp Mol Pathol,2011,91(1):455-460.
[12]Sasi W,Jiang W G,Sharma A,et al.Higher expression levels of SOCS 1,3,4,7 are associated with earlier tumor stage and better clinical outcome in human breast cancer[J].BMC Cancer,2010,10:178.
[13]Watanabe D,Ezoe S,Fujinoto M,et al.Suppressor of cytokine signaling-1 gene silencing in acute myeloid leukaemia and human haematopoeitic cell lines[J].Br J Haenatol,2004,126(5):726-735.
[14]Wu S J,Yao M.Chou W C,et al.Clinical implications of SOCS1methylation in myelodysplastic syndrome[J].Br J Haematol,2006,135(3):317-323.
[15]Teofili L,Martini M,Cenci T,et al.Epigenetic alteration of SOCS family members is a possible patheogenetic mechanism in JAK2wild type myeloproliferative diseases[J].Int J Cancer,2008,123(7):1586-1592.
[16]Hookham M B,Elliott J,Suessmuth Y,et al.The myeloproliferative disorder-associated JAK2V617F mutant escape negative regulation by suppressor of cytokine signalingfj].Blood 2007,109(11):4924-4929.
[2]James C,Ugo v,Le Couedic J P,et al.A unique clonal JAK2 mutation leading to constitutive signaling causes polycythaemia vera[J].Nature,2005,434(7037):1144-1148.
[3]Levine R L,Wadleigh M,Cools J,et al.Activating mutation in the tyrosine kinase JAK2 in polycythemia vera,essential thrombocythemia,and myeloid metaplasia with myelofibrosis[J].Cancer Cell,2005,7(4):387-397.
[4]Scott L M,Tong W,Levine R L,et al.JAK2 exon 12 mutations in polythemia vera and idiopathic erythrocytosis[J].N Engl J Med,2007,356(5):459-468.(上接第1369页)
[5]Pardanani A D,Levine R L,Lasho T,et al.MPL515 mutations in myeloproliferative and other myeloid disorders:a study of 1182patients[J].Blood,2006,108(10):3472-3476.
[6]Valentino L.Pierre J.JAK/STAT signal transductiomregulators and implication in hematological malignancies[J].Biochem Pharmacol,2006,71(6):713-721.
[7]Krebs D L.Hilton D J.SOCS.Physiological suppressors of cytokine signaling[J].J Cell Sci.2000,113(Pt16),2813-2819.
[8]Alexander W S,Starr R,Fenner J E,et al.SOCS1 is a critical inhibitor of interferon y signaling and prevents the potentially fatal neonatal actions of this cytokine[J].Cell,1999,98(5):597-608.
[9]Boyle K,Robb L.The role of SOCS3 in modulating leukaemia inbi—hitory factor signaling during murine placental development[J].J Reprod lmmunol,2008,77(1):1-6.
[10]Xiong H,Du W,Zhang Y J.Tricho station A,a histone deacetylase inhibitor.suppresses JAK2/STAT3 signaling via inducing the promoter-associated histone acetylation of SOCS1 and SOCS3in human colorectal cancer cells[J].Mol Carcinog,2012,51(2):174-184.
[11]Hua D,Hu Y,Wu Y Y,et al.Quantitative methylation analysis of multiple genes using methylation-sensitive restriction enzyme-based quantitative PCR for the detection of hepatocellular carcinoma[J].Exp Mol Pathol,2011,91(1):455-460.
[12]Sasi W,Jiang W G,Sharma A,et al.Higher expression levels of SOCS 1,3,4,7 are associated with earlier tumor stage and better clinical outcome in human breast cancer[J].BMC Cancer,2010,10:178.
[13]Watanabe D,Ezoe S,Fujinoto M,et al.Suppressor of cytokine signaling-1 gene silencing in acute myeloid leukaemia and human haematopoeitic cell lines[J].Br J Haenatol,2004,126(5):726-735.
[14]Wu S J,Yao M.Chou W C,et al.Clinical implications of SOCS1methylation in myelodysplastic syndrome[J].Br J Haematol,2006,135(3):317-323.
[15]Teofili L,Martini M,Cenci T,et al.Epigenetic alteration of SOCS family members is a possible patheogenetic mechanism in JAK2wild type myeloproliferative diseases[J].Int J Cancer,2008,123(7):1586-1592.
[16]Hookham M B,Elliott J,Suessmuth Y,et al.The myeloproliferative disorder-associated JAK2V617F mutant escape negative regulation by suppressor of cytokine signalingfj].Blood 2007,109(11):4924-4929.