儿童非霍奇金淋巴瘤ZO-1基因甲基化状态分析及临床意义
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摘要
目的探讨紧密连接蛋白(ZO-1)基因启动子区甲基化状态在儿童非霍奇金淋巴瘤(NHL)Ⅳ期检测中的临床意义,以进一步寻找病因及早期诊断方法。方法将55例确诊为NHLⅣ期(T细胞型40例,B细胞型15例)患儿的骨髓标本作为病例组,20例非血液肿瘤患儿骨髓标本作为对照组。采用甲基化特异性PCR法(MS-PCR)检测病例组与对照组骨髓中ZO-1基因启动子区甲基化状态,并检测光密度积分值(IOD)。逆转录PCR法(RT-PCR)检测病例组与对照组ZO-1基因mRNA的表达情况。结果 MS-PCR检测结果显示病例组治疗前39例出现ZO-1基因甲基化条带,阳性率为70.9%(39/55),对照组20例均呈非甲基化状态;治疗过程中,行动态观察NHL患儿47例,其中T细胞型32例,B细胞型15例,治疗前ZO-1基因甲基化阳性率分别为72%和67%,两者差异无统计学意义(P>0.05)。T细胞型与B细胞型NHL患儿治疗前与化疗早期、中期比较差异均无统计学意义(P>0.05),而与化疗后期比较差异均有统计学意义(P<0.05)。RT-PCR检测结果显示病例组中ZO-1甲基化阳性病例均无mRNA表达,对照组患儿均表达ZO-1 mRNA。外周血白细胞总数与ZO-1基因IOD值无直线相关关系(r=0.093,P=0.575);骨髓中恶性肿瘤细胞数与ZO-1基因IOD值呈正相关关系(r=0.669,P<0.001)。结论 ZO-1基因在儿童NHL中呈特异性高甲基化状态,其程度与骨髓中恶性肿瘤细胞数呈正相关。ZO-1基因可能成为新的分子标志,为临床早期诊断、疗效判断、预后评价、微小残留检测提供新的指标。
Objective To investigate the methylation status of zonula occludens-1(ZO-1) gene promoter and its clinical significance in children with stage IV non-Hodgkin lymphoma(NHL) and to provide a basis for further etiological study and early diagnosis of this disease. Methods Fifty-five children with a confirmed diagnosis of stage IV NHL(40 cases of T-NHL and 15 cases of B-NHL) were selected as the case group, and 20 children with diseases other than hematologic malignancies were selected as the control group. Bone marrow samples were collected from these subjects. Methylation-specific PCR(MS-PCR) was applied to evaluate the methylation status of ZO-1 gene promoter, and the integrated optical density(IOD) was determined. RT-PCR was used to measure the mRNA expression of ZO-1. Results MS-PCR showed that the methylated bands of ZO-1 gene promoter were found in 39(70.9%) of 55 patients in the case group before treatment, while no ZO-1 gene promoter methylation was detected in the control group. With close tracking of 47 cases in the study group, consisting of 32 cases of T-NHL and 15 cases of B-NHL, the rates of ZO-1 gene promoter methylation prior to treatment were 72% and 67%, respectively,(P>0.572). The cases of T-NHL and B-NHL showed no significant changes in methylation rate in the early and middle phases of chemotherapy(P>0.05), but they showed significant changes in methylation rate in the late phase of chemotherapy(P<0.05). RT-PCR showed that theNHL cases carrying methylated ZO-1 gene had no mRNA expression of ZO-1, while all children in the control group had mRNA expression of ZO-1. There was no linear relationship between the total number of peripheral blood leukocytes and ZO-1 gene IOD(r=0.093, P=0.575); a positive correlation was found between the number of malignant cells in bone marrow and ZO-1 gene IOD(r=0.669, P<0.001). Conclusions ZO-1 gene shows a hypermethylation status in children with NHL, and the methylation level is positively correlated with the number of malignant cells in bone marrow. ZO-1 may be used as a novel molecular marker in early diagnosis, outcome assessment, prognostic evaluation, and detection of minimal residual disease.
Objective To investigate the methylation status of zonula occludens-1(ZO-1) gene promoter and its clinical significance in children with stage IV non-Hodgkin lymphoma(NHL) and to provide a basis for further etiological study and early diagnosis of this disease. Methods Fifty-five children with a confirmed diagnosis of stage IV NHL(40 cases of T-NHL and 15 cases of B-NHL) were selected as the case group, and 20 children with diseases other than hematologic malignancies were selected as the control group. Bone marrow samples were collected from these subjects. Methylation-specific PCR(MS-PCR) was applied to evaluate the methylation status of ZO-1 gene promoter, and the integrated optical density(IOD) was determined. RT-PCR was used to measure the mRNA expression of ZO-1. Results MS-PCR showed that the methylated bands of ZO-1 gene promoter were found in 39(70.9%) of 55 patients in the case group before treatment, while no ZO-1 gene promoter methylation was detected in the control group. With close tracking of 47 cases in the study group, consisting of 32 cases of T-NHL and 15 cases of B-NHL, the rates of ZO-1 gene promoter methylation prior to treatment were 72% and 67%, respectively,(P>0.572). The cases of T-NHL and B-NHL showed no significant changes in methylation rate in the early and middle phases of chemotherapy(P>0.05), but they showed significant changes in methylation rate in the late phase of chemotherapy(P<0.05). RT-PCR showed that theNHL cases carrying methylated ZO-1 gene had no mRNA expression of ZO-1, while all children in the control group had mRNA expression of ZO-1. There was no linear relationship between the total number of peripheral blood leukocytes and ZO-1 gene IOD(r=0.093, P=0.575); a positive correlation was found between the number of malignant cells in bone marrow and ZO-1 gene IOD(r=0.669, P<0.001). Conclusions ZO-1 gene shows a hypermethylation status in children with NHL, and the methylation level is positively correlated with the number of malignant cells in bone marrow. ZO-1 may be used as a novel molecular marker in early diagnosis, outcome assessment, prognostic evaluation, and detection of minimal residual disease.
引文
[1]杜瑜,王畅,康慧媛,等.ZO-1基因甲基化状态在非霍奇金淋巴瘤骨髓检测中的临床意义[J].山东医药,2010,50(8):26-28.
[2]王畅,于力,谭亚辉,等.慢性粒细胞白血病ZO-1基因启动子区甲基化状态分析及临床意义探讨[J].山东医药,2011,51(32):33-35.
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[8]董丽钧,顾晓怡,姜藻.结直肠癌患者血清SFRP1基因甲基化的检测及临床意义[J].江苏医药,2010,36(18):2153-2155.
[9]Costello JF,Plass C.Methylation matters[J].J Med Genet,2001,38(5):285-303.
[10]Fanning AS,Ma TY,Anderson JM.Isolation and functional characterization of the actin binding region in the tight junction protein ZO-1[J].FASEB J,2002,16(13):1835.
[11]刘毅.46例恶性淋巴瘤临床病理特征和预后分析[J].中国医学创新,2013,10(9):104-106.
[12]潘慈,汤静燕,薛惠良,等.儿童非霍奇金淋巴瘤肾脏浸润的临床观察[J].中国当代儿科杂志,2003,5(4):345-347.
[13]康慧媛,于力,王莉莉.闭锁小带蛋白1研究进展[J].生物技术通讯,2009,20(4):576-578.
[14]王畅,王冠军,谭业辉,等.急性白血病ZO-1基因启动子区甲基化状态分析及临床意义[J].中华内科杂志,2008,47(2):111-113.
[15]张玲,盛树力,秦川.表观遗传学药物的研究进展[J].中国药理学通报,2013,29(3):297-300.
[16]Bradner JE,West N,Grachan ML,et al.Chemical phylogenetics of histone deacetylases[J].Nature Chen Biol,2010,6(3):238.
[2]王畅,于力,谭亚辉,等.慢性粒细胞白血病ZO-1基因启动子区甲基化状态分析及临床意义探讨[J].山东医药,2011,51(32):33-35.
[3]陈树宝.小儿内科疾病临床治疗与合理用药[M].北京:科学技术文献出版社,2007:402-406.
[4]Baylin SB,Herman JG.DNA hypermethylation in tumorigenesis:epigenetics joins genetics[J].Trends Genet,2000,16(4):168-174.
[5]王丽,汪竹,袁昕,等.结直肠癌中CDH13基因甲基化及其临床意义[J].江苏医药,2012,38(2):199-201.
[6]Jeltsch A.Beyond Watson and Crick:DNA methylation and molecular enzymology of DNA methyltransferases[J].Chembiochem,2002,3(5):274-293.
[7]Cheng X,Roberts RJ.AdoMet-dependent methylation,DNA methylt-ransferases and base flipping[J].Nucleic Acids Res,2001,29(18):3784-3795.
[8]董丽钧,顾晓怡,姜藻.结直肠癌患者血清SFRP1基因甲基化的检测及临床意义[J].江苏医药,2010,36(18):2153-2155.
[9]Costello JF,Plass C.Methylation matters[J].J Med Genet,2001,38(5):285-303.
[10]Fanning AS,Ma TY,Anderson JM.Isolation and functional characterization of the actin binding region in the tight junction protein ZO-1[J].FASEB J,2002,16(13):1835.
[11]刘毅.46例恶性淋巴瘤临床病理特征和预后分析[J].中国医学创新,2013,10(9):104-106.
[12]潘慈,汤静燕,薛惠良,等.儿童非霍奇金淋巴瘤肾脏浸润的临床观察[J].中国当代儿科杂志,2003,5(4):345-347.
[13]康慧媛,于力,王莉莉.闭锁小带蛋白1研究进展[J].生物技术通讯,2009,20(4):576-578.
[14]王畅,王冠军,谭业辉,等.急性白血病ZO-1基因启动子区甲基化状态分析及临床意义[J].中华内科杂志,2008,47(2):111-113.
[15]张玲,盛树力,秦川.表观遗传学药物的研究进展[J].中国药理学通报,2013,29(3):297-300.
[16]Bradner JE,West N,Grachan ML,et al.Chemical phylogenetics of histone deacetylases[J].Nature Chen Biol,2010,6(3):238.