miRNA-146a在全反式维甲酸诱导NB4细胞分化中的变化及作用  

Change of miRNA-146a in all-trans retinoic acid-induced differentiation of NB4 cell lines and its effects

作　　者：杨爽[1] 钟华[1] 陈芳源[1] 王海嵘[1] 王利民[2] 钟济华[1] 黄洪晖[1] 

机构地区：[1]上海交通大学医学院附属仁济医院血液科,上海200127 [2]上海交通大学医学院附属仁济医院中心实验室,上海200127

出　　处：《肿瘤》2009年第5期423-427,共5页Tumor

基　　金：上海市教育委员会科研资助项目(编号:06BZ031);上海市科委基金资助项目(编号:04ZR14106)

摘　　要：目的:检测全反式维甲酸(all-trans retinoic acid,ATRA)诱导的急性早幼粒细胞白血病(acute promyelocytic leukemia,APL)细胞株NB4分化前后表达发生显著变化的微小RNA(miRNA),并对其靶基因进行预测及功能研究,进而探讨miRNA在APL发生中的作用机制。方法:ATRA(1μmol/L终浓度)处理NB4细胞,分别于4、24、72和96h时收集细胞,用miRNA基因芯片检测细胞分化前后各时间点的表达谱差异,找出其中关键的miRNA;实时荧光定量-PCR(real-time fluorogentic quanti-tative PCR,RFQ-PCR)(TaqMan探针)方法验证其表达,并对其靶基因进行预测;应用RFQ-PCR及Western印迹法检测靶基因及靶蛋白的表达情况。结果:miRNAs芯片检测结果显示:表达显著上调的miRNAs有8个,显著下调的有15个;其中miRNA-146a在ATRA处理NB4细胞4、24、72和96h后的表达量,分别是处理前的0.82、0.83、0.44和0.37倍。利用TargetScan软件对其靶基因进行预测,发现TGF-β1信号转导途径中的公共调节型Smad4是其中一个靶基因。ATRA处理后Smad4mRNA的表达量上调,蛋白表达水平也呈逐渐上升趋势。结论:抑制miRNA-146a的表达有可能通过上调其靶基因Smad4的表达,恢复TGF-β1信号转导通路,在ATRA诱导APL细胞分化中发挥重要作用。Objective：To detect the expression pattern of microRNA （miRNA） in the differentiation of acute promyelocytic leukemia （APL） cell lines NB4 induced by all-trans retinoic acid （ATRA） and predict its target genes as well as study to functions and finally investigate the action mechanism of miRNAs in the carcinogenesis of APL. Methods：NB4 cells were treated with 1 μmol/L ATRA for 4, 24, 72 and 96 h. The cells were collected and the expression patterns of miRNAs were detected by using miRNAs array at these time points. The key miRNA was identified, and their expression was verified by using real-time fluorogentic quantitative （RFQ） PCR （TaqMan probe） and the target gene was predicted. RFQ-PCR and Western blotting were used to detect the mRNA and protein expression of targent genes, respectively. Results：MiRNA array showed that eight miRNAs were up-regulated and fifteen miRNA were down-regulated. The relative expressions of miRNA-146a were 0.82, 0.83, 0.44, and 0.37 folds at 4, 24, 72, and 96 h after ATRA treatment compared with the control group. This study predicted the target genes of miRNA-146a by using TargetScan software and found that one of its target genes was Smad 4, one of the common regulating genes in transforming growth factor-β1（TGF-β1） signaling transduction pathway. The expression level of Smad 4 mRNA in NB4 cells was up-regulated after ATRA treatment, and its protein expression level tended to increase simultaneously. Conclusion：Inhibition of expression of miRNA-146a may play an important role in the differentiation of APL cells by up-regulating its target gene Smad 4 and restoring TGF-β1 signaling transduction pathway.

关 键 词：白血病 早幼粒细胞 急性 维甲酸 细胞分化 微RNAS 

分 类 号：R733.71[医药卫生—肿瘤]