miR-708在儿童普通急性淋巴细胞白血病中的表达及调控机制  

The expression and regulatory mechanism of miR-708 in pediatric common B-cell acute lvmDhoblastic leukemia

作　　者：李雪[1] 李栋[1] 庄泳[1] 时庆[1] 魏伟[1] 张红[1] 鞠秀丽[1] 

机构地区：[1]山东大学齐鲁医院儿科、低温医学研究室,济南250012

出　　处：《中华血液学杂志》2013年第2期138-143,共6页Chinese Journal of Hematology

基　　金：山东省自然科学基金面上项目（ZR2011HM007）

摘　　要：目的检测普通急性淋巴细胞白血病（common-ALL）患儿中微小RNA（miRNA，miR）的差异性表达，并探讨miR-708的调控机制。方法研究对象为34例common-AlJIJ患儿及5例行骨关节病手术并排除肿瘤和血液系统疾病患儿的骨髓标本。应用微阵列基因芯片技术筛选common-ALL患儿样本中差异性表达的miRNA，应用茎环状逆转录引物的stem-loop实时荧光定量PCR技术进行验证。应用生物信息学预测、双报告基因检测、RT-PCR和Westernblot等方法验证miR-708调控的靶基因及其表达。结果在common-ALL患儿样本中检测的2006个miRNA中，miR-708、miR-181b和miR-210表达量分别为16．886±16．854、5．7104±4．652和9．7894-1．178，与正常对照组（1．8724±0．339、1．2764±0．531和1．0054±0．080）相比表达均上调，差异均有统计学意义（P〈0．05）。miR-27a和miR-345表达量为0．5244±0．085和0．6754±0．086，与正常对照组（1．1234±0．066和1．2044±0．140）相比均下调，差异均有统计学意义（P〈0．05）。miR-708和miR-181b在高危组中表达水平为44．9904±6．379和12．9804-1．889，高于中危组和标危组（P〈0．05）。转染miR-708模拟物的人胚胎细胞株中CNTFR、NNAT和GNG12的表达水平均下降，而转染miR-708抑制剂组其表达升高。miR-708与CNTFR的结合区域位于3＇-UTR端394-400bp。结论miRNA在调控儿童common-ALL发生发展的过程中发挥重要的作用，其中miR-708是高危型common-ALL重要的调控因子。miR-708通过结合靶基因的3＇-UTR端，降低其靶基因表达水平。Objective To evaluate the expression of microRNAs and reveal the regulatory mechanism of miRNA-708 in pediatric common acute lymphoblastic leukemia（ALL） （common-ALL）. Methods The expressions of mieroRNAs in common-ALL patients were detected by microarrays in 3 pediatric common-ALL samples, and then verified by stem-loop quantitative RT-PCR in 34 common-ALL samples. The target genes of miR-708 were found by bioinformatics software, and verified by dual-luciferases reporter assay, RT-PCR and Western blot. Results Compared to normal bone marrow samples, of all the 2006 detected miRNAs, the expression of miR-708, miR-181b and miR-210 were 16. 886 ± 16. 854, 5. 710 ± 4. 652, and 9. 789±1. 178 ,retrospectively, being significantly up-regulated expressed than those in normal control （1. 872 ±0. 339,1. 276 ± 0.531 and 1. 005± 0.080, retrospectively） （P 〈 0.05 ）, while miR-27b and miR-345 were the two most down-regulated ones （ 0. 524 ±0. 085 and 0. 675 ± 0. 086, retrospectively） （ normal control： 1. 123 ± 0.066 and 1. 204 ± 0. 140, retrospectively） （ P 〈 0.05 ）. And the expression of miR-708 and miR- 181b were significantly correlated with the clinical types in common-ALL. In high risk common-ALL, miR- 708 and miR-181 b were much higher than in standard and middle risk common-ALL （P 〈 0.05 ）. The further verification research in 293 cell line showed that miR-708 decreased the expression level of its target genes CNTFR, NNAT and GNG12 by combining with 3＇-UTR of the 3 genes, moreover, miR-708 combined with CNTFR 3＇-UTR in 394 - 400 bp sequence region. Conclusion MicroRNAs plays an important regulatory role during the occurrence and development of the pediatric common-ALL and miR-708 is an important factor for high risk common-ALL.

关 键 词：白血病 淋巴细胞 急性 微RNA 微阵列分析 靶基因 

分 类 号：R733.71[医药卫生—肿瘤]