肾母细胞瘤患儿SIX2基因的转录表达及其启动子甲基化状态的意义分析

The significance of transcriptional expression of SIX2 gene and its promote methylation status in pediatric patients with nephroblastoma

目的探究小儿肾母细胞瘤（WT）组织和血液中SIX2基因在mRNA水平的相对表达量及启动子是否发生甲基化,并观察其与患儿的基本临床资料之间的关系,以期为临床靶向基因治疗提供科学的依据。方法选择2011年3月至2015年7月就诊并确诊的64例WT患儿[男30例,女34例;年龄3~67（26.9±8.6）月]为研究对象,运用实时荧光定量PCR（qRT-PCR）和甲基化特异性聚合酶链反应（MSP）分别检测患儿WT组织、瘤旁组织和外周血以及18例健康儿童外周血中SIX2基因在mRNA水平的相对表达量及启动子甲基化状态。结果 WT患儿瘤组织SIX2 mRNA的相对表达量明显高于瘤旁正常组织（P〈0.01）,SIX2 mRNA启动子的甲基化阳性率明显低于瘤旁正常组织（P〈0.01）;WT患儿外周血中SIX2 mRNA的表达量明显高于对照组外周血（P〈0.01）,SIX2基因启动子的甲基化阳性率明显低于对照组外周血（P〈0.01）。WT患儿组织SIX2基因相对表达量与患儿肿瘤大小、临床分期、病理类型、淋巴转移、血管浸润有关（P〈0.05,P〈0.01）,而与性别、年龄和复发无明显关系（P均〉0.05）。SIX2基因启动子的甲基化与上述临床资料均无明显关系（P均〉0.05）。随访36个月,死亡42例,存活22例,3年存活率34.4%;SIX2 mRNA相对表达量〉2.8和≤2.8的患儿存活率（23.8%vs 54.5%,P〈0.05）和存活时间[（13.3±5.2）月vs（22.2±8.7）月,P〈0.01]比较,差异均有统计学意义。SIX2启动子甲基化表达阴性和阳性的患儿3年存活率（14.3%vs 44.2%,P〈0.05）和存活时间[（14.5±4.5）月vs（22.1±4.9）月,P〈0.01]比较,差异均有统计学意义。结论 SIX2基因的高表达和其启动子的未甲基化可能是WT的发病基础,SIX2基因或可作为WT基因治疗的靶目标。

Objective To investigate the relative expression quantity of SIX2 mRNA in tissues and blood in pediatric patients with nephroblastoma（Wilm tumor,WT）and promoter methylalion status,and observe their association with clinicopathological features of WT patients in order to provide a scientific basis for the clinical target gene therapy.Methods A total of 64 confirmed WT pediatric patients [30 cases in male,34 cases in female,age：3-67（26.9 ± 8.6）months]visited from March 2011 to July 2015 were selected as research objects,and 18 healthy children were selected as control（control group）.Real time fluorescence quantitative polymerase chain reaction（qRT-PCR）and methylation specific polymerase chain reaction（MSP）were used to respectively detect the relative expression quantity and promoter methylalion status of SIX2 mRNA in WT tissues,tumor adjacent tissues and peripheral blood of WT patients and in peripheral blood of healthy children.Results For WT patients,the relative expression quantity of SIX2 gene in tumor tissues was significantly higher than that in tumor adjacent normal tissues（P 0.01）,and the promoter methylalion positive rate of SIX2 gene in tumor tissues was significantly lower than that in tumor adjacent normal tissues（P 0.01）.The relative expression quantity of SIX2 gene in peripheral blood of WT patients was significantly higher than that in peripheral blood of control group（P 0.01）,and the promoter methylalion positive rate of SIX2 gene in peripheral blood of WT patients was significantly lower than that in peripheral blood of control group（P 0.01）.The relative expression quantity of SIX2 gene in tumor tissues was associated with tumor size,clinical stage,pathological type,lymph node metastasis and vascular invasion（P 0.05,P 0.01）,but was not significantly associated with sex,age and relapse（all P 0.05）.The promoter methylalion status of SIX2 gene was not associated with all the aforementioned clinical data（all P 0.05）.In follow-up period of 36 months,42 cases died,and 22 cases survived,thus the 3-year survival rate was 34.4%.There were significant differences in 3-year survival rates（23.8% vs 54.5%,P 0.05）and survival time [（13.3 ± 5.2）months vs（22.2 ± 8.7）months,P 0.01]between patients with mean SIX2 gene expression quantity of more than 2.8 and less than or equal to 2.8.There were significant differences in 3-year survival rates（14.3% vs 44.2%,P 0.05）and survival time [（14.5 ± 4.5）months vs（22.1 ± 4.9）months,P 0.01]between patients with negative and positive SIX2 gene promoter methylalion.Conclusion The high-expression of SIX2 gene and promoter no-methylation may be the pathogenic foundation of WT,and perhaps SIX2 gene can be served as a target of gene therapy.