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沉默AEG-1基因逆转乳腺癌MCF-7/ADM细胞耐药性

袁磊, 时冉冉, 饶淑梅, 宋金玲, 崔明辰^*

漯河医学高等专科学校分子生物学实验室，漯河 462002

摘要

本研究旨在分析星形细胞上调基因1 (astrocyte elevated gene-1, AEG-1)对乳腺癌MCF-7/ADM细胞化疗药物耐药性的影响，并探讨其作用机制。将MCF-7/ADM细胞在含1.0 mg/L阿霉素(adriamycin, ADM)的培养液中培养以维持细胞的耐药性；应用shRNA技术沉默乳腺癌MCF-7/ADM细胞中AEG-1基因表达；采用MTT比色法检测ADM对MCF-7/ADM的细胞耐毒作用，据以计算ADM的半数抑制浓度(IC50)；流式细胞术检测细胞凋亡；Western blot方法检测AEG-1、p53和多药耐药基因1 (multidrug resistance gene 1, MDR1)蛋白的表达水平以及Akt、MDM2和Bad的磷酸化水平。结果显示，乳腺癌MCF-7/ADM细胞的AEG-1蛋白水平显著高于MCF-7细胞(P < 0.05)，经shRNA干扰后AEG-1蛋白水平显著降低(P < 0.05)；沉默AEG-1基因能显著降低ADM对MCF-7/ADM细胞的IC50 (P < 0.05)，促进MCF-7/ADM细胞凋亡(P < 0.05)，并增强ADM对MCF-7/ADM细胞的促凋亡作用，抑制Akt、MDM2和Bad的磷酸化(P < 0.05)，促进p53蛋白表达(P < 0.05)，降低MDR1蛋白表达水平(P < 0.05)。结果表明，沉默AEG-1基因可通过促进MCF-7/ADM细胞凋亡和下调MDR1蛋白表达，以逆转MCF-7/ADM细胞对ADM的耐药性。

关键词： 星形细胞上调基因1; MCF-7/ADM; 细胞凋亡; 多药耐药基因1; Akt

分类号：R737.9

[Reversal of resistance to adriamycin in human breast cancer cell line MCF-7/ADM by silencing AEG-1 gene and its mechanism.] [Article in Chinese]

YUAN Lei, SHI Ran-Ran, RAO Shu-Mei, SONG Jin-ling, CUI Ming-Chen^*

Laboratory of Molecular Biology, Luohe Medical College, Luohe 462002, China

Abstract

The aim of this study was to investigate the effects of AEG-1 gene silencing on the chemoresistance of human breast cancer cell line MCF-7/ADM and its possible mechanism. MCF-7/ADM cells were incubated in the medium containing adriamycin (ADM). The recombinant pLKO.1-shAEG-1 plasmid was constructed to silence AEG-1 expression in human breast cancer MCF-7/ADM cells. MTT assay was employed to detect the anti-tumor effect of ADM on MCF-7/ADM cells, and IC50 value of ADM was calculated according to MTT. Flow cytometry was used to determine the apoptosis. Western blot was used to analyze the expression levels of AEG-1, p-Akt, p-MDM2, p-Bad, p53 and MDR1. The result showed MCF-7/ADM had a significantly higher expression level of AEG-1 compared with that of MCF-7 (P < 0.05), however, the expression of AEG-1 was decreased after AEG-1 gene silencing. The IC50 value of ADM in shAEG-1 group was significantly lower than that in shcontrol group. AEG-1 gene silencing induced cell apoptosis and enhanced the pro-apoptotic effect of ADM on MCF-7/ADM cells. After AEG-1 gene silencing, the phosphorylation of Akt, MDM2 and Bad was inhibited (P < 0.05), the protein levels of p53 and MDR1 were up-regulated (P < 0.05) and down-regulated (P < 0.05) respectively, compared with control. In conclusion, the results suggest that AEG-1 gene silencing can reverse the ADM resistance in human breast cancer cell line MCF-7/ADM by means of inducing apoptosis and down-regulating the protein level of MDR1.

Key words: astrocyte elevated gene-1; MCF-7/ADM; apoptosis; multidrug resistance gene 1; Akt

收稿日期：2014-03-07　　录用日期：2014-04-17

通讯作者：崔明辰　　E-mail: cuimingchen123@126.com

引用本文：

袁磊, 时冉冉, 饶淑梅, 宋金玲, 崔明辰. 沉默AEG-1基因逆转乳腺癌MCF-7/ADM细胞耐药性[J]. 生理学报 2014; 66 (5): 625-630.

YUAN Lei, SHI Ran-Ran, RAO Shu-Mei, SONG Jin-ling, CUI Ming-Chen. [Reversal of resistance to adriamycin in human breast cancer cell line MCF-7/ADM by silencing AEG-1 gene and its mechanism.] [Article in Chinese]. Acta Physiol Sin 2014; 66 (5): 625-630 (in Chinese with English abstract).