RNA干扰沉默EZH2基因增强人肝癌多药耐药Bel／Fu细胞对氟尿嘧啶的敏感性  

RNA interference silencing EZH2 gene strengths the sensitivity of human hepatic multidrug-resistant cancer cells to 5-Fu chemotherapy

作　　者：张易[1] 唐博[1] 梁锐[1] 刘光普[2] 刘晨[1] 王立明[1] 

机构地区：[1]大连医科大学附属第二医院普外科,116027 [2]大连医科大学附属第一医院骨外科

出　　处：《中华普通外科杂志》2012年第8期660-663,共4页Chinese Journal of General Surgery

基　　金：国家高技术研究发展计划（863计划）基金资助项目（2006AA02A309）

摘　　要：目的研究沉默人肝癌多药耐药Bel／Fu细胞的EZH2基因表达是否影响癌细胞对氟尿嘧啶（fluorouracil，5-Fu）的敏感性。方法体外培养肝癌多药耐药Bel／Fu细胞；EZH2siRNA干扰沉默EZH2基因表达；RT—PCR和Westernblot检测干扰后EZH2mRNA和蛋白的表达水平；MTT法检测细胞生长抑制率；AnnexinV—FITC／PI双标记法检测细胞凋亡率；流式细胞仪分析细胞周期；Westernblot检测多药耐药相关蛋白MDR1的表达水平。实验设对照组、5-Fu处理组、EZH2siRNA处理组、5-Fu和EZH2siRNA联合处理组。结果EZI-12siRNA干扰24h后，EZH2mRNA和蛋白的表达水平明显降低。在联合处理组中，MTF法检测其细胞凋亡抑制率为43．17％±3．81％，明显高于其他3组；流式检测联合处理组细胞凋亡率，其细胞凋亡值为30．4％±1．77％，明显高于其他3组。流式检测联合处理组细胞周期，发现联合组G1期细胞的百分比为69．16％±2．31％，明显高于其他3组，S＋G：＋M期细胞的百分比为30．76％±1．29％，明显低于其他3组，细胞周期被阻滞于G1期。检测MDR1在联合组中的表达水平，发现MDR1蛋白的表达水平明显低于其他组别。结论RNA干扰沉默EZH2基因可以增强Bel／Fu细胞对5-Fu的敏感性，其机制可能与MDR1蛋白表达降低有关。Objective To study the impact of EZH2 silence on the sensitivity of human hepatic multidrng-resistant cancer cells Bel/Fu to 5-Fu. Methods Bel/Fu cells were cultured in vitro; EZH2 siRNA was used to interfere EZH2 expression; RT-PCR and Western blot was used to detect the efficiency of interference. MTT assay was used to detect the cellular growth inhibitory rate; Annexin V-FITC/PI double staining was used to detect the apoptosis rate of cells; Flow cytometry was to analyze cell cycle ; Western blot analysis was used to detect the expression of multidrng resistance-associated protein MDR1 after silencing EZH2. The experiment set up four groups： control group, 5-Fu treatment group, EZH2 siRNA treatment group, 5-Fu combined with EZH2 siRNA treatment group. Results The expression of EZH2 was greatly decreased after 24 h in the combined group, the apoptotic inhibitory rate by MTT was 43.17% ± 3.81%, higher than other three groups ; the apoptotic rate in the combined group by Flow cytometry was 30.4% ±1.77%, markedly higher than other three groups. The cell cycle of the combined group detected by Flow cytometry was 69. 16% ± 2. 31% of cells in the combined group at G1 phase, the percentage was higher than other three group, 30. 76% ± 1.29% at S, G2 and M phases, lower than other three groups, indicating the cell cycle was blocked at G1 phase. MDR1 protein level in the combined group was lower than other groups. Conclusions Silencing EZH2 strengths the sensitivity of Bel/Fu cells to 5-Fu, probably by a mechanism decreasing the expression of MDR1.

关 键 词：肝肿瘤 基因沉默 RNA干扰 多药耐药相关蛋白类 

分 类 号：R735.7[医药卫生—肿瘤]