CAJ | 학술논문

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-ALL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Western blot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、5.710±4.652和9.789±1.178,与正常对照组(1.872±0.339、1.276±0.531和1.005±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0.524±0.085和0.675±0.086,与正常对照组(1.123±0.066和1.204±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44.990±6.379和12.980±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394~400 bp.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.
목적 검측보통급성림파세포백혈병(common-ALL)환인중미소RNA(miRNA,miR)적차이성표체,병탐토miR-708적조공궤제.방법 연구대상위34례common-ALL환인급5례행골관절병수술병배제종류화혈액계통질병환인적골수표본.응용미진렬기인심편기술사선common-ALL환인양본중차이성표체적miRNA,응용경배상역전록인물적stem-loop실시형광정량PCR기술진행험증.응용생물신식학예측、쌍보고기인검측、RT-PCR화Western blot등방법험증miR-708조공적파기인급기표체.결과 재common-ALL환인양본중검측적2006개miRNA중,miR-708、miR-181b화miR-210표체량분별위16.886±16.854、5.710±4.652화9.789±1.178,여정상대조조(1.872±0.339、1.276±0.531화1.005±0.080)상비표체균상조,차이균유통계학의의(P<0.05).miR-27a화miR-345표체량위0.524±0.085화0.675±0.086,여정상대조조(1.123±0.066화1.204±0.140)상비균하조,차이균유통계학의의(P<0.05).miR-708화miR-181b재고위조중표체수평위44.990±6.379화12.980±1.889,고우중위조화표위조(P<0.05).전염miR-708모의물적인배태세포주중CNTFR、NNAT화GNG12적표체수평균하강,이전염miR-708억제제조기표체승고.miR-708여CNTFR적결합구역위우3′-UTR단394~400 bp.결론 miRNA재조공인동common-ALL발생발전적과정중발휘중요적작용,기중miR-708시고위형common-ALL중요적조공인자.miR-708통과결합파기인적3′-UTR단,강저기파기인표체수평.
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 microRNAs 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-181b 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.