Bmi-1调控非小细胞肺癌细胞对顺铂的敏感性研究  

Regulatory effects of Bmi-1 and MDR1 on the chemosensitivity of human non-small cell lung cancer cells to cisplatin

作　　者：陈明军[1] 陈奇[2] 周月鹏[3] CHEN Ming-jun;CHEN Qi;ZHOU Yue-peng(Comprehensive Office of First Clinical Medicine,Affiliated Hospital of Jiangsu University,Zhenjiang 212001,Jiangsu,China;Clinical Skills Center,Affiliated Hospital of Jiangsu University,Zhenjiang 212001,Jiangsu,China;Oncological Laboratory,Affiliated Hospital of Jiangsu University,Zhenjiang 212001,Jiangsu,China)

机构地区：[1]江苏大学附属医院(第一临床医学院)综合办公室,镇江212001 [2]江苏大学附属医院(第一临床医学院)临床技能中心,镇江212001 [3]江苏大学附属医院(第一临床医学院)肿瘤实验室,镇江212001

出　　处：《医学研究生学报》2019年第7期710-714,共5页Journal of Medical Postgraduates

摘　　要：目的为了揭示Bmi-1参与非小细胞肺癌顺铂敏感性调控相关的具体机制,文中通过顺铂处理后结合靶向干扰Bmi-1表达,观察对非小细胞肺癌细胞中多药耐药相关蛋白1(MDR1)及凋亡蛋白表达的影响。方法构建靶向Bmi-1的siRNA,利用脂质体转染到非小细胞肺癌A549细胞及顺铂耐药株A549/DDP中,取对数生长期细胞随机分为3组:Bmi-1干扰组(siRNA-Bmi-1转染)、阴性对照组(siRNA阴性对照)及空白对照组(不做任何处理)。通过RT-PCR及Westernblot实验验证干扰效果。根据A549细胞不同处理分为4组:对照组(未处理A549细胞)、Bmi-1组(siRNA-Bmi-1处理A549细胞)、DDP组(顺铂处理A549细胞)、Bmi-1+DDP组(Bmi-1干扰结合顺铂处理A549细胞)。采用CCK8实验检测A549与A549/DDP细胞中siRNA-Bmi-1处理前后,顺铂按浓度梯度(1、2、4、8、16、32、64、128、256μg/mL)加入处理24h后的细胞存活率;流式细胞检测各组凋亡水平变化,及蛋白印迹实验分析Bmi-1、MDR1和Cleaved-Caspase-3之间联系。结果RT-PCR法检测显示,Bmi-1干扰组mRNA表达水平较空白对照组降低(P<0.01)。A549细胞和A549/DDP细胞中随着顺铂浓度的升高,细胞相对存活率逐渐降低(P<0.05);Bmi-1+DDP组细胞存活率较DDP组明显降低(P<0.05)。A549细胞中,Bmi-1+DDP组细胞凋亡率[(39.65±3.41)%]较DDP组、Bmi-1组、对照组[(23.11±1.62)%、(2.05±1.56)%、(1.98±1.05)%]明显升高(P<0.05)。DDP处理24h后,流式细胞检测显示Bmi-1、MDR1高表达;靶向下调Bmi-1后A549和A549/DDP细胞中MDR1显著降低,凋亡相关Cleaved-Caspase-3蛋白表达量明显升高。结论siRNA-Bmi-1的表达可提高非小细胞肺癌顺铂敏感性,其作用机制可能与MDR1表达抑制以及凋亡相关蛋白活化有关。Objective The purpose of this study was to investigate the mechanism of Bmi-1 regulating the sensitivity of non-small cell lung cancer (NSCLC) to chemotherapy by observing its effects on multidrug-resistance protein 1 (MDR1) and apoptosis-related proteins. Methods Small interfering RNAs (siRNA) targeting Bmi-1 were transfected into A549 and A549/DDP cells of NSCLC and the logarithmic-phase cells were randomly divided into a siRNA-Bmi-1, a siRNA-negative control and a blank control group. The A549 cells were treated with siRNA-Bmi-1, DDP or Bmi-1+DDP, or left untreated (the control). CCK8 assay was employed to measure the 50% inhibitory concentration (IC50) of cisplatin in the A549 and A549/DDP cells before and after treatment. The apoptosis of the cells was detected by flow cytometry, the mRNA expression of Bmi-1 determined by RT-PCR, and the relationship of Bmi-1 with MDR1 and cleaved caspase-3 proteins analyzed by Western blot. Results After transfection, the relative mRNA and protein expressions of Bmi-1 in the A549 and A549/DDP cells were significantly lower in the siRNA-Bmi-1 than in the blank control group (P < 0.05), but higher in the A549/DDP than in the A549 cells. The survival rates of the A549 and A549/DDP cells were decreased with the increased concentration of cisplatin (P < 0.05), even lower in the Bmi-1+DDP than in the DDP subgroup (P < 0.05). The apoptosis rate of the A549 cells was markedly higher in the Bmi-1+DDP than in the DDP, Bmi-1 and control groups ([39.65±3.41]% vs [23.11±1. 62]%,[2.05±1.56]% and [1.98±1.05]%, P < 0.05). After 24 hours of treatment with DDP, both the expressions of Bmi-1 and MDR1 were remarkably elevated, while the down-regulation of Bmi-1 significantly decreased the expression of MDR1 and increased that of cleaved caspase-3. Conclusion The expression of siRNA-Bmi-1 makes non-small lung cancer cells more sensitive to cisplatin, which might be associated with its inhibition of MDR1 expression and activation of apoptosis-related proteins.

关 键 词：BMI-1 多药耐药相关蛋白1 非小细胞肺癌 顺铂 化疗敏感 

分 类 号：R734.2[医药卫生—肿瘤]