RNA干扰Apollon基因逆转白血病K562细胞多药耐药

doi:10.3969/j.issn.1007-3969.2013.09.004

中国癌症杂志 ›› 2013, Vol. 23 ›› Issue (9): 713-720.doi: 10.3969/j.issn.1007-3969.2013.09.004

RNA干扰Apollon基因逆转白血病K562细胞多药耐药

贾秀红,孝飞飞,李建厂

滨州医学院附属医院儿科，山东滨州 256603

出版日期:2013-09-25 发布日期:2014-02-20
通信作者: 贾秀红　E-mail:jiaxiuhong001@163.com
基金资助:
山东省科学技术发展计划项目(No：2010GSF10264)

Reversion of multidrug resistance in leukemia K562 cells by RNA interference targeting Apollon gene

JIA Xiu-hong,XIAO Fei-fei,LI Jian-chang

Department of Pediatrics, Affiliated Hospital of Binzhou Medical University, Binzhou Shandong 256603, China

Published:2013-09-25 Online:2014-02-20
Contact: JIA Xiu-hong E-mail: jiaxiuhong001@163.com

摘要/Abstract

摘要：

背景与目的：Apollon基因在白血病等多种肿瘤内高表达。本试验通过构建高效干扰Apollon基因的短发夹RNA(short hairpin RNA，shRNA)真核表达载体，探讨RNA干扰技术能否逆转人髓系白血病K562细胞多药耐药。方法：构建靶向Apollon基因真核表达载体pGPHI-GFP-Neo-Apollon，应用Lipofectamine^TM2000转染K562细胞，G418稳定筛选。采用反转录-聚合酶链反应(RT-PCR)法、细胞免疫荧光法分别检测重组载体稳定转染K562细胞后Apollon mRNA及蛋白的表达情况；四甲基偶氮唑盐(MTT)、流式细胞术检测细胞转染前后对长春新碱(leurocristine，VCR)、足叶乙苷(etoposide，VP16)的敏感性及凋亡率的变化。结果：成功构建了pGPHI-GFP-Neo-Apollon载体并在K562细胞内稳定表达的细胞克隆，经G418筛选后，重组载体能有效沉默Apollon的表达，Apollon mRNA及蛋白表达水平明显下降。MTT结果显示，基因干扰组细胞对VCR、VP16的敏感性明显增强，其半数抑制浓度(half-inhibitory，IC₅₀)值分别为(0.144±0.018)mg/L、(17.336±3.571)mg/L，明显低于细胞对照组(P<0.05)。流式细胞术检测结果表明，基因干扰组细胞联合化疗药物后细胞凋亡率显著升高(P<0.05)，而转染阴性对照组细胞凋亡率与正常对照组比较差异无统计学意义(P>0.05)。结论：pGPHI-GFP-Neo-Apollon载体能显著增强VCR和VP16对白血病K562细胞的诱导凋亡作用，提高K562细胞对化疗药物的敏感性，提示RNA干扰Apollon基因表达能一定程度逆转白血病细胞的多药耐药。

关键词: 白血病, RNA干扰, Apollon基因, 多药耐药性, 细胞凋亡, K562细胞

Abstract:

Background and purpose: Apollon gene is highly expressed in leukemia and other tumors. The study aimed to discuss whether RNAi technology can reverse multidrug resistance of chronic myeloid leukemia cell line K562 through constructing a eukaryotic vector of short hairpin RNA (shRNA) targeting at Apollon gene. Methods: The eukaryotic vector pGPHI-GFP-Neo-Apollon with shRNA targeting at Apollon gene was constructed and then transfected into K562 cells by Lipofectamine^TM2000, and G418 pressure selection. Reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence were used to detect the expression of Apollon mRNA and protein after Apollon was transfected stably in K562 cells. The changes of sensitivity of K562 cells to leurocristine (VCR) and etoposide (VP16) after transfection with shRNA-Apollon were detected by MTT method, and the apoptosis rate was detected by flow cytometry. Results: pGPHI-GFP-Neo-Apollon carrier was constructed successfully and expressed stably in K562 cells, and after G418 screening, it silenced Apollon mRNA and protein expression effectively. According to the result of MTT, the sensitivity of K562 cells to VCR and VP16 increased significantly in the group of gene interference, with half of its inhibition concentration (half-inhibitory, IC₅₀) value significantly lower than the control group (P<0.05); Flow cytometry showed that the cell apoptosis rate was increased significantly (P<0.05), but there was no statistically significant difference in the apoptosis rate between shRNA negative control group and normal control group (P>0.05). Conclusion: pGPHI-GFP-Neo-Apollon carrier can enhance the abilities of VCR and VP16 to induce the apoptosis of K562 cells, namely an increase of sensitivity to these chemotherapeutics in K562 cells, it is hinted that RNA interference targeting Apollon gene may reverse the multidrug resistance of leukemia cells in some degree.

Key words: Leukemia, RNA interference, Apollon genes, Multiple drug resistance, Apoptosis, K562 cells

贾秀红,孝飞飞,李建厂. RNA干扰Apollon基因逆转白血病K562细胞多药耐药[J]. 中国癌症杂志, 2013, 23(9): 713-720.

JIA Xiu-hong,XIAO Fei-fei,LI Jian-chang. Reversion of multidrug resistance in leukemia K562 cells by RNA interference targeting Apollon gene[J]. China Oncology, 2013, 23(9): 713-720.

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RNA干扰Apollon基因逆转白血病K562细胞多药耐药

Reversion of multidrug resistance in leukemia K562 cells by RNA interference targeting Apollon gene

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