Knockdown of nucleophosmin by RNA interference reverses multidrug resistance in resistant leukemic HL-60 cells
Knockdown of nucleophosmin by RNA interference reverses multidrug resistance in resistant leukemic HL-60 cells
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RNA干扰敲低核磷蛋白可逆转耐药性白血病HL-60细胞的多药耐药性
DOI：
10.1016/j.imbio.2013.04.001
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发表时间：
2013-09-01
期刊：
影响因子：
2.8
通讯作者：
Chen, Xinji
中科院分区：
文献类型：
--
作者：
Lin, Minhui;Hu, Jianda;Chen, Xinji
核磷蛋白是一种多功能的核仁磷蛋白，参与多种细胞活动。然而，NPM在白血病耐药中的作用尚未得到探讨。我们设计并筛选了一条靶向NPM基因的shRNA，通过慢病毒载体转染HL-60和HL-60/ADR细胞（阿霉素耐药细胞株）。检测细胞增殖、凋亡和分化情况。采用实时荧光定量RT-PCR和Western blotting检测相关基因和蛋白的表达。结果显示，NPM RNAi后NPM mRNA和蛋白水平明显下调。NPM靶向的RNAi也导致许多细胞变化，如抑制细胞增殖和诱导细胞分化。下调NPM基因可使细胞周期阻滞，G 0/G1期比例增加。NPM基因沉默还可以诱导促凋亡基因和蛋白的表达，抑制抗凋亡基因/蛋白的表达。阿霉素和阿霉素、柔红霉素和柔红霉素对HL-60和HL-60/ADR细胞的IC_（50）均降低，对HL-60/ADR细胞的IC_（50）降低更为明显。ADR对HL-60/ADR细胞的IC 50从12.544 ± 0.851 μ mol/L（NPM RNAi前）降至6.331 ± 0.522 μ mol/L（NPM RNAi后），DNR的IC 50从2.152 ± 0.143 μ mol/L（NPM RNAi前）降至1.116 ± 0.093 μ mol/L（NPM RNAi后）。HL-60/ADR细胞对ADR的相对逆转率为50.2%，对柔红霉素的相对逆转率为48.9%。总之，我们的研究结果表明，shRNA表达载体可以有效地减少NPM的表达，并恢复耐药白血病细胞对常规化疗药物的药物敏感性。(C)2013 Elsevier GmbH. All rights reserved.
Nucleophosmin, a multifunctional nucleolar phosphoprotein, is involved in many cellular activities. However, the role of NPM in drug-resistance of leukemia has not yet been explored. We designed and selected one shRNA targeting on NPM gene transduction into HL-60 and HL-60/ADR cell lines (an adriamycin resistant cell line) by lentivirus. Cell proliferation, apoptosis and differentiation were assessed. The expressions of the related genes and proteins were detected by real-time quantitative RT-PCR and Western blotting. The results showed obvious down-regulation of NPM mRNA and protein levels after NPM RNAi. NPM-targeted RNAi also resulted in many cellular changes, such as, suppressing cell proliferation and inducing cell differentiation. Down-regulation of NPM gene could arrest the cell cycle progression, an increase in the proportion of G0/G1 phase in knockdown groups. NPM gene silencing could also induce pro-apoptotic genes and proteins expression, and inhibit anti-apoptotic genes/proteins expression. Furthermore, IC50 of two chemotherapeutic agents (adriamycin and ADR; daunorubicin and DNR) to HL-60 and HL-60/ADR cells decreased, especially more remarkable on HL-60/ADR cells. IC50 of ADR on HL-60/ADR cells was reduced from 12.544 +/- 0.851 mu mol/L (before NPM RNAi) to 6.331 +/- 0.522 mu mol/L (after NPM RNAi), IC50 of DNR was reduced from 2.152 +/- 0.143 mu mol/L (before NPM RNAi) to 1.116 +/- 0.093 mu mol/L (after NPM RNAi). The relative reversal rate of HL-60/ADR cells on ADR was 50.2%, and on DNR was 48.9%. In conclusion, our results demonstrated that shRNA expression vectors could effectively reduce NPM expression and restore the drug sensitivity of resistant leukemic cells to conventional chemotherapeutic agents. (C) 2013 Elsevier GmbH. All rights reserved.