Xishan Z, Xianjun L, Ziying L, Guangxin C, Gang L
Clinical Research Center, Affiliated Hospital of Guangdong Medical College, Guangdong province, China.
Department of Nuclear Medicine, Weifang People's Hospital, Weifang, Shandong, P.R. China.
Cancer Gene Ther. 2014 Sep;21(9):397-404. doi: 10.1038/cgt.2014.44. Epub 2014 Sep 12.
The aim of this study was to investigate the role and mechanism of miR-23a in the regulation of BCR/ABL and to provide a new prognostic biomarker for chronic myeloid leukemia (CML). The expression levels of miR-23a and BCR/ABL were assessed in 42 newly diagnosed CML patients, 37 CML patients in first complete remission and 25 healthy controls. Quantitative real-time PCR, western blot analysis and colony formation assay were used to evaluate changes induced by overexpression or inhibition of miR-23a or BCR/ABL. MiR-23a mimic or negative control mimic was transfected into a CML cell line (K562) and two lung cancer cell lines (H157 and SKMES1) using Lipofectamine 2000, and the cells were used for real-time reverse transcription-PCR (RT-PCR) and western blot analysis. We found that the downregulation of miR-23a expression was a frequent event in both leukemia cell lines and primary leukemic cells from patients with de novo CML. The microarray results showed that most of the CML patients expressed high levels of BCR/ABL and low levels of miR-23a. Real-time RT-PCR and western blot analysis showed that the BCR/ABL levels in miR-23a-transfected cells were lower than those in the control groups. Ectopic expression of miR-23a in K562 cells led to cellular senescence. Moreover, when K562 cells were treated with 5-aza-2'-deoxycytidine, a DNA methylation inhibitor, BCR/ABL expression was upregulated, which indicates epigenetic silencing of miR-23a in leukemic cells. BCR/ABL and miR-23a expressions were inversely related to CML, and BCR/ABL expression was regulated by miR-23a in leukemic cells. The epigenetic silencing of miR-23a led to derepression of BCR/ABL expression, and consequently contributes to CML development and progression.
本研究旨在探讨miR-23a在BCR/ABL调控中的作用及机制,为慢性髓性白血病(CML)提供一种新的预后生物标志物。检测了42例新诊断的CML患者、37例首次完全缓解的CML患者和25例健康对照者中miR-23a和BCR/ABL的表达水平。采用定量实时PCR、蛋白质印迹分析和集落形成试验评估miR-23a或BCR/ABL过表达或抑制所诱导的变化。使用Lipofectamine 2000将miR-23a模拟物或阴性对照模拟物转染至CML细胞系(K562)以及两种肺癌细胞系(H157和SKMES1),然后将这些细胞用于实时逆转录PCR(RT-PCR)和蛋白质印迹分析。我们发现,miR-23a表达下调在白血病细胞系以及初发CML患者的原发性白血病细胞中均较为常见。微阵列结果显示,大多数CML患者BCR/ABL表达水平高而miR-23a表达水平低。实时RT-PCR和蛋白质印迹分析表明,转染miR-23a的细胞中BCR/ABL水平低于对照组。K562细胞中miR-23a的异位表达导致细胞衰老。此外,当K562细胞用DNA甲基化抑制剂5-氮杂-2'-脱氧胞苷处理时,BCR/ABL表达上调,这表明白血病细胞中miR-23a存在表观遗传沉默。BCR/ABL和miR-23a的表达与CML呈负相关,并且白血病细胞中BCR/ABL的表达受miR-23a调控。miR-23a的表观遗传沉默导致BCR/ABL表达去抑制,从而促进CML的发生和发展。