儿童神经母细胞瘤ZO-1基因甲基化状态临床意义分析

ZO-1 gene methylation status in children neuroblastoma and its clinical significance

目的:探讨紧密连接蛋白(zonula occludens protein-1,ZO-1)基因启动子区甲基化状态在儿童神经母细胞瘤检测中的临床意义。方法:将2002-01-15-2012-12-15河北医科大学第四医院儿科35例确诊为神经母细胞瘤(Ⅳ期)患儿的骨髓标本作为研究组,20例非血液肿瘤患儿骨髓作为对照组。采用甲基化特异性聚合酶链反应(methylation specific PCR,MS-PCR)方法动态检测研究组与对照组骨髓中ZO-1基因启动子区甲基化状态。逆转录聚合酶链反应(reverse transcription PCR,RT-PCR)法检测两组ZO-1基因mRNA的表达情况,同时用ZO-1基因经MS-PCR扩增后的甲基化条带进行凝胶成像系统荧光强度半定量分析后的荧光强度(integrated option density,IOD)积分值,观察研究组化疗过程中IOD的动态变化。结果:MS-PCR检测结果显示,研究组治疗前32例出现ZO-1基因甲基化条带,阳性率为91.4%(32/35),对照组20例,均呈非甲基化状态,χ2=15.11,P<0.01;动态观察研究组31例ZO-1基因甲基化阳性率,治疗前为90.3%(28/31),化疗早期为80.6%(25/31),化疗后期为71.0%(22/31)。临床完全缓解3例,仍有2例可检出。治疗前与化疗早期、后期比较差异均无统计学意义,χ2=0.4,P=0.819。RT-PCR检测结果显示,研究组治疗前35例提取总RNA,3例见到ZO-1基因表达,阳性率为8.6%(3/35),对照组20例均有表达,阳性率为100.0%(20/20),χ2=16.616,P<0.001。动态观察研究组31例ZO-1基因表达阳性率,治疗前为9.7%(3/31),化疗早期为19.4%(6/31),化疗后期为29.0%(9/31)。临床完全缓解3例,2例未见到ZO-1基因表达。治疗前与化疗早期、化疗后期比较差异均无统计学意义,χ2=2.53,P=0.500。性别(t=0.530,P=0.25)、年龄(r=0.080,P=0.25)与IOD值线性无关。外周血白细胞总数与IOD值线性无关,r=0.093,P=0.25。骨髓中瘤细胞数与IOD值线性相关,且呈正相关,r=0.669,P=0.036 5。结论:ZO-1基因在儿童神经母细胞瘤中呈特异性高甲基化状态,导致该基因表达沉默,动态观察甲基化消失不明显,这与肿瘤恶性程度、发生、发展及转归密切相关,可能是预后不良因素之一。

OBJECTIVE . To investigate the methylation status of zonula occludens protein-l（ZO-1 ）gene promotor in children neuroblastoma（NB） and its clinical significance. METHODS.. Thirty-five cases that had been diagnosed NB（stage IV ）in Department of Pediatrics,Fourth Hospital of Hebei Medical University from Jan. 15,2002 to Dec. 15,2012 were se- lected as study group, 20 non-blood tumor cases were selected as control group. Methylation specific PCR （MS-PCR） method was used dynamically to detect the bone marrow specimen in both study group and control group. Reversed tran- scription PCR （RT-PCR） method was used to detect the expression of metbylated ZO-1 gene mRNA in both groups. The bands of ZO-1 methylation DNA were amplified by the MS-PCR, then semi-quantitatively was analyzed with gel imaging system to obtain integrated optical density （IOD values）, and observe the dynamic changes of IOD values in research group. RESULTS： MS-PCR showed that the 91.4% （32/35） NB（stage IV）cases were found in ZO-1 gene promoter in bone marrow harboring aberrant methylation, no ZO-1 gene promoter methylation was detected in control group（χ2= 15. 11,P〈0.01）. In dynamic observing study group（31 cases）, the methylation-positive rate before treatment was 90.3% （28/31） ,early stage of chemotherapy was 80.6% （25/31） ,later stage of chemotherapy was 71.0% （22/31）. There were 3 cases of clinical complete remission,and 2 cases were still checked out. There was no significant difference between the pre treatment and early stage or advanced stage of chemotherapy（χ2=0.4 ,P 0. 819）. RT-PCR showed that there were 3 examples expressed the ZO- 1 gene before treatment in study group,the positive rate was 8.6% （3/35）,the control group were all expressed 100%（20/20）（χ2=16. 616,PQ0. 001）. I）ynamieally observed the positive rate of ZO-1 gene＇s expression, it was 9.7%（3/31） before treatment, 19. 4%（6/31） in early stage of chemotherapy, 29. 0%（9/31） in ad- vanced stage of chemotherapy. There were 3 patients of Clinical complete remission,2 cases of ZO-1 gene expression was not detected. There was no significant difference between the pre-treatment and early stage of chemotherapy or advanced stage of chemotherapy（χ2=2.53, P = 0. 500）. IOD value： Both gender and age were linearly independent with IOD（t= 0. 530, P= 0.25;r=0. 080, P 0.25）, total white blood ceils in the peripheral blood leucocyte was also linearly independ ent with IOD（r= 0. 093, P= 0.25）, but the number of neoplastic cell in the bone marrow was linearly dependent with IOD, and it was positive（r= 0. 669, P= 0. 036 5）. CONCLUSIONS ： ZO- 1 gene shows specific hyper-methylation status in children neuroblastoma,which leads to silent expression,and the disappearance of methylation status is not obviou during the dynamical observation. This may closely dependent on the malignant degree of the tumor,and the occurrence,develop ment and turnover,may be one of the negative factors of prognosis.