He Jin-Hua, Li Yu-Min, Li Yu-Guang, Xie Xing-Yi, Wang Li, Chun Shun-Yi, Cheng Wu-Jia
Department of Laboratory, Central Hospital of Panyu District, Guangzhou, Guangdong 511400;
Exp Ther Med. 2013 May;5(5):1315-1321. doi: 10.3892/etm.2013.981. Epub 2013 Feb 27.
The aim of this study was to investigate the effect of a eukaryotic expression vector expressing hsa-miR-203 on the sensitivity of K562 leukemia cells to arsenic trioxide (ATO) and the possible mechanism of action. The eukaryotic expression vector expressing the hsa-miR-203 plasmid (PmiR-203) was transfected into K562 cells using Lipofectamine 2000. bcr/abl 3' untranslated region (UTR) and bcr/abl mutated 3'UTR dual luciferase report vectors (psi-CHECK-2) were used to validate the regulation of bcr/abl by miR-203. The inhibitory effects of ATO and PmiR-203, used singly or in combination, on cell proliferation were detected by MTT assay. Apoptosis of the K562 cells was detected by flow cytometry using double-staining with Annexin V and propidium iodide (PI). The activities of caspase-3 and caspase-9 were detected by a colorimetric method and the cytochrome protein levels were detected by western blotting. When used in combination with PmiR-203, the IC of ATO was reduced from 6.49 to 2.45 g/ml and the sensitivity of cells to ATO increased 2.64-fold. In addition, PmiR-203 and ATO caused growth inhibition, apoptosis and G1-phase arrest in K562 cells. Furthermore, PmiR-203 significantly promoted ATO-mediated growth inhibition and apoptosis, affecting the G1 phase. JC-1 fluorescent staining revealed that the membrane potential of the mitochondria had changed. The activities of caspase-3 and caspase-9 increased, the expression levels of cytochrome were upregulated and the expression level of bcr/abl mRNA was significantly suppressed. Furthermore, the dual-luciferase reporter vector, containing tandem miR-203 binding sites from the bcr/abl 3'UTR, demonstrated that bcr/abl was directly regulated by miR-203. PmiR-203 sensitized K562 leukemia cells to ATO by inducing apoptosis and downregulating bcr/ abl gene levels. The induction of apoptosis may occur through the mitochondrial pathway. The combination of ATO and PmiR-203 presents therapeutic potential for chronic myelogenous leukemia.
本研究旨在探讨表达hsa-miR-203的真核表达载体对K562白血病细胞对三氧化二砷（ATO）敏感性的影响及其可能的作用机制。使用Lipofectamine 2000将表达hsa-miR-203质粒的真核表达载体（PmiR-203）转染至K562细胞。使用bcr/abl 3'非翻译区（UTR）和bcr/abl突变的3'UTR双荧光素酶报告载体（psi-CHECK-2）来验证miR-203对bcr/abl的调控作用。采用MTT法检测ATO和PmiR-203单独或联合使用对细胞增殖的抑制作用。使用Annexin V和碘化丙啶（PI）双染，通过流式细胞术检测K562细胞的凋亡情况。采用比色法检测caspase-3和caspase-9的活性，通过蛋白质印迹法检测细胞色素蛋白水平。与PmiR-203联合使用时，ATO的半数抑制浓度（IC）从6.49降至2.45μg/ml，细胞对ATO的敏感性提高了2.64倍。此外，PmiR-203和ATO导致K562细胞生长抑制、凋亡和G1期阻滞。此外，PmiR-203显著促进ATO介导的生长抑制和凋亡，影响G1期。JC-1荧光染色显示线粒体膜电位发生了变化。caspase-3和caspase-9的活性增加，细胞色素的表达水平上调，bcr/abl mRNA的表达水平显著下调。此外，包含来自bcr/abl 3'UTR的串联miR-203结合位点的双荧光素酶报告载体表明bcr/abl受miR-203直接调控。PmiR-203通过诱导凋亡和下调bcr/abl基因水平使K562白血病细胞对ATO敏感。凋亡的诱导可能通过线粒体途径发生。ATO与PmiR-203的联合应用为慢性粒细胞白血病提供了治疗潜力。
