MiR-34b在儿童急性白血病中的表达及临床意义

Expression and clinical significance of miR-34b in children with acute leukemia

目的 检测miR-34b在儿童急性白血病（AL）中的甲基化状态及表达情况,并探讨其临床意义.方法 应用甲基化特异性聚合酶链反应（MSP）检测miR-34b启动子区CpG岛在儿童AL中的甲基化状态,用实时荧光定量聚合酶链反应（qRT-PCR）检测miR-34b的相对表达量并与正常对照组比较,分析其与临床指标间的关系.结果 8种白血病细胞株（U937、HL-60、MV4-11、M2R、K562、Raji、CCRF、DAMI）均出现甲基化现象,其甲基化阳性率为100％.儿童初诊急性淋巴细胞白血病（ALL）患儿31例,其中24例出现甲基化现象,甲基化阳性率为77.42％（24/31例）,儿童初诊急性髓细胞白血病（AML）患儿19例,其中8例出现甲基化现象,甲基化阳性率为42.11％（8/19例）,23例正常对照患儿,无一例出现甲基化现象.miR-34b在正常对照组、白血病细胞株组、ALL组、AML组及混合谱系白血病基因重排阳性（MLL＋）组的相对表达量分别为5.22±1.15、0.03 ±0.03、1.65±0.69、0.18 ±0.06、0.64 ±0.34,ALL组（P＜0.05）、白血病细胞株组、AML组及MLL＋组（P均＜0.01）与正常对照组相比差异均有统计学意义.miR-34b的相对表达量与儿童初诊AML患儿的性别、年龄、初诊时白细胞（WBC）计数、染色体、融合基因、MLL基因重排及初诊时乳酸脱氢酶（LDH）水平差异均无统计学意义（P均＞0.05）.miR-34b的相对表达量与儿童初诊ALL患儿的性别、年龄、初诊时WBC计数、免疫分型、染色体、融合基因、MLL基因重排、TEL/AML1基因、危险度分层、第33天微小残留病（MRD）及初诊时LDH水平与miR-34b的相对表达量之间差异均无统计学意义（P均＞0.05）.但在泼尼松试验反应方面,敏感组与不敏感组之间差异有统计学意义（P＜0.05）.结论 miR-34b在儿童AL表达量明显减低并受甲基化机制调控,提示miR-34b可能作为抑癌基因参与儿童AL的形成.miR-34b的表达量影响儿童初诊ALL早期治疗反应,提示其可能成为判定儿童初诊ALL危险度和不良预后的一个指标.

Objective To research the expression,methylated regulation and clinical significances of miR-34b in chidren with acute leukemia（AL）.Methods The methylation status of miR-34b promoter CpG islands were detected with methylation-specific polymerase chain reaction （MSP） in patients with AL.Then the expression of miR-34b was compared,which was detected by Taqman real-time fluorescence quantitative polymerase chain reaction （qRT-PCR）,between the AL patients and normal group,in order to analyze their relationship with the clinical indicators.Resuits In 8 leukemia cell lines （U937,HL-60,MV4-11,M2R,K562,Raji,CCRF,DAMI） showed methylation,the positive rate of the methyl was 100%.Thirty-one acute lymphocytic leukemia（ALL） pediatric patients were newly diagnosed,and 24 cases showed methylation,the methylation positive rate 77.42% （24/31）.Nineteen acute myeloid leukemia（AML） patients were newly diagnosed,and 8 cases showed methylation,the methylation positive rate was 42.11% （8/19 cases）.There was no methylation in the 23 cases of normal children.The relative expression levels of miR-34b in the normal group,the group of leukemia cell lines,the group of ALL pediatric patients with newly diagnosed,the AML group and the group with mixed lineage leukemia gene rearrangement MLL＋ were 5.22 ± 1.15,0.03 ± 0.03,1.65 ± 0.69,0.18 ± 0.06,0.64 ± 0.34,respectively.The findings indicated that there were significant differences in the relative expression levels of miR-34b between the normal group and the group of leukemia cell lines,the ALL group,the AML group,and the MLL＋ group.The relative expression and methylated level of miR-34b had no statistically difference in gender,age at diagnosis,WBC count,chromosome,fusion gene,MLL gene rearrangement,and the minimal residual disease（LDH） levels in newly diagnosed AML patients（all P 〉 0.05）.And the relative expression and methylated level of miR-34b had no statistically difference in gender,age at diagnosis,WBC count,immunophenotype,chromosome,fusion gene,MLL gene rearrangement,TEL/AML1 gene,risk stratification,the minimal residual disease （MRD） in thirty-three days and the LDH levels in newly diagnosed ALL patients（all P 〉 0.05）.But as for the response to prednisone experiment,there was a significant difference between the sensitive group and the non sensitive group（P 〈 0.05）.Conclusions The expression level of miR-34b in AL was significantly lower and it was regulated by methylation mechanism,which implies that miR-34b may play a role of a tumor suppressor gene in the pathogenesis of leukemia.MiR-34b may affect the early treatment response of ALL patients,and it may be an indicator of risk stratification and poor prognosis in pediatric ALL.