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目的探讨mi R-125b对儿童典型急性早幼粒细胞白血病(APL)发生发展的影响及其机制,期求新的策略治疗耐药性APL。方法用在线软件预测mi R-125b的靶基因,并通过双荧光报告及蛋白印迹技术进行验证;用q RT-PCR及western blot技术检测来自中山大学附属第一医院及华南地区儿童APL协作组其他医院2007年3月至2012年9月收治的33例患者在初诊、缓解或复发时期骨髓标本中mi R-125b及其靶基因的表达,进行体内证实;在细胞系水平过表达或抑制表达mi R-125b,用MTT、流式细胞技术等研究mi R-125b对白血病细胞增殖、凋亡及耐药的影响,进行体外验证。结果 (1)用生物学信息方法预测出BAK1是mi R-125b的可能靶基因;(2)mi R-125b在APL初诊患者中表达增高,缓解后降低,复发时再次升高,其高表达抑制BAK1蛋白的表达,同样,在NB4及HL60细胞系中,通过转染使mi R-125b过表达可抑制BAK1蛋白的表达;(3)在NB4及HL60白血病细胞株,过表达mi R-125b促进其增殖、抑制凋亡及增加耐药性,用si RNA沉默BAK1的表达后,NB4的凋亡受抑制,与过表达mi R-125b的效应相似;(4)白血病耐药细胞株NB4-R1、NB4-R2及HL-60/DOX与敏感细胞株NB4和HL-60比较,mi R-125b呈高表达(P<0.05),通过转染使mi R-125b进一步过表达,耐药细胞株对全反式维甲酸和阿霉素的IC50进一步增加。结论 mi R-125b通过对靶基因BAK1的调控影响儿童典型APL细胞的增殖、凋亡及耐药。
Objective To investigate the effect of mi R-125b on the development and progression of children with acute promyelocytic leukemia (APL) and to find new strategies for the treatment of drug-resistant APL. Methods The target gene mi R-125b was predicted by on-line software and verified by double fluorescence reporter and Western blotting. The qRT-PCR and western blot were used to detect the expression of mi R-125b from the First Affiliated Hospital of Sun Yat- Thirty-three patients admitted to our hospital from March 2007 to September 2012 were confirmed in vivo by in vivo expression of mi R-125b and its target gene in bone marrow samples from newly diagnosed, remissioned or recurrent stage. R-125b, the effects of mi R-125b on the proliferation, apoptosis and drug resistance of leukemia cells were studied by MTT and flow cytometry. Results (1) BAK1 was predicted as a potential target gene of mi R-125b by biological information method. (2) mi R-125b expression was increased in newly diagnosed APL patients, decreased after remission and then increased again in relapse Inhibited the expression of BAK1 protein. Similarly, overexpression of mi R-125b could inhibit the expression of BAK1 protein by transfection in NB4 and HL60 cell lines; (3) In NB4 and HL60 leukemia cell lines, overexpression of mi R-125b Promote the proliferation, inhibit the apoptosis and increase the drug resistance. The silencing of BAK1 by si RNA inhibited the apoptosis of NB4, which was similar to the effect of overexpression of mi R-125b. (4) The leukemic cell line NB4- Compared with NB4 and HL-60 cells, the expression of mi R-125b was highly expressed in R1, NB4-R2 and HL-60 / DOX cells (P <0.05) The IC50 was further increased for all-trans-retinoic acid and doxorubicin. Conclusion mi R-125b can affect the proliferation, apoptosis and drug resistance of typical APL cells in children by regulating the target gene BAK1.