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siRNA抑制Mcl-1基因表达对TRAIL诱导的人胃腺癌SGC7901/ADR细胞凋亡的影响
         
Effect of suppression of Mcl-1 gene expression via siRNA on TRAILinduced apoptosis of human gastric cancer SGC7901/ADR cells

摘    要
目的:研究沉默髓样细胞白血病1(myeloid cell leukemia-1,Mcl-1) 基因表达对肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factor-relatedapoptosis-inducing ligand,TRAIL)诱导人胃腺癌耐多柔比星(adriamycin,ADR)SGC7901/ADR 细胞凋亡的影响, 并探讨胃癌耐药细胞株耐TRAIL 凋亡效应的可能机制.
方法: 不同质量浓度的TRAIL(10、50 和100 ng/mL) 处理SGC7901/ADR 细胞后,采用MTT 法和FCM 法分别检测细胞活力和细胞凋亡率,实时荧光定量PCR 法和蛋白质印迹法分别检测SGC7901/ADR 细胞中Mcl-1 mRNA 和蛋白的表达水平.采用脂质体转染法将Mcl-1-siRNA 转染至SGC7901/ADR 细胞,实时荧光定量PCR 和蛋白质印迹法检测Mcl-1-siRNA 对Mcl-1 mRNA 及蛋白表达水平的影响.采用TRAIL(50 ng/mL)处理Mcl-1 基因沉默后的SGC7901/ADR 细胞,MTT 法和FCM 法分别检测Mcl-1 基因沉默后TRAIL 对细胞增殖及凋亡率的影响,并进一步用蛋白质印迹法检测细胞色素C(cytochrome C,Cyt C)的表达水平以及凋亡相关蛋白caspase 3 和caspase 9 的激活情况.
结果:不同质量浓度的TRAIL(10、50 和100 ng/mL)均能抑制人胃腺癌耐药株SGC7901/ADR 细胞的增殖,并诱导其凋亡(P 值均< 0.05),但敏感性较低;TRAIL 能明显提高SGC7901/ADR 细胞中Mcl-1mRNA(P 值均< 0.001)和蛋白(P 值均< 0.05)的表达水平.Mcl-1-siRNA 转染后,SGC7901/ADR 细胞中Mcl-1 mRNA 和蛋白表达均明显下调(P 值均< 0.001).与阴性对照组(转染阴性对照-siRNA + TRAIL)相比,Mcl-1-siRNA 转染后联合应用TRAIL 组SGC7901/ADR 细胞的增殖能力明显下降(P < 0.001),细胞凋亡率明显增加(P < 0.001),Cyt C、active-caspase 3和active-caspase 9蛋白的表达水平均明显增加(P 值均<0.001).
结论:TRAIL 能上调胃癌耐药细胞株中抗凋亡蛋白Mcl-1 表达,siRNA 抑制Mcl-1 表达可增强TRAIL 对SGC7901/ADR 细胞的致凋亡作用,提示胃癌耐药细胞株SGC7901/ADR 耐TRAIL 致凋亡效应可能与Mcl-1 高表达有关.
标    签 胃肿瘤   TNF 相关凋亡诱导配体   髓样细胞白血病-1   细胞凋亡   抗药性,肿瘤   Stomach neoplasms   TNF-related apoptosis-inducing ligand   Myeloid cell leukemia-1   Apoptosis   Drug resistance, neoplasm  
 
Abstract
Objective: To study the effect of suppression of myeloid cell leukemia-1 (Mcl-1) gene expression via siRNA on apoptosis of adriamycin (ADR)-resistant human gastric cancer SGC7901/ADR cells induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and to investigate the possible mechanism of TRAIL resistance of SGC7901/ADR cells.
Methods: After treatment with different concentrations of TRAIL (10, 50 and 100 ng/mL), the cell viability and apoptosis rate of SGC7901/ADR cells were detected by MTT method and FCM, respectively. The expression levels of Mcl-1 mRNA and protein were determined by real-time fluorescent quantitative PCR and Western blotting, respectively. SGC7901/ADR cells were transfected with Mcl-1-siRNA using liposome transfection method, then the expression levels of Mcl-1 mRNA and protein were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The effects of Mcl-1 gene on cell viability and apoptosis rate of SGC7901/ADR cells treated with TRAIL (50 ng/mL) were detected by MTT method and FCM, respectively. The expression levels of Cytochrome C (Cyt C), caspase 3 and caspase 9 proteins were determined by Western blotting.
Results: The results of MTT method and FCM showed that treatment with different concentrations of TRAIL (10, 50 and 100 ng/mL) could inhibit the cell proliferation and induce apoptosis of SGC7901/ADR cells (all P < 0.05), but the sensitivity of SGC7901/ADR cells to TRAIL was lower.The different concentrations of TRAIL could significantly up-regulate the expression levels of Mcl-1 mRNA (all P < 0.001) and protein (all P < 0.05) in SGC7901/ADR cells. After transfection with Mcl-1-siRNA, the expression levels of Mcl-1 mRNA and protein in SGC7901/ADR cells were significantly down-regulated (both P < 0.001). As compared with negative control (NC)-siRNA+TRAIL group, combination of TRAIL and Mcl-1-siRNA could significantly inhibit the proliferation of SGC7901/ADR cells (P < 0.001) and also increase the apoptosis rate of SGC7901/ADR cells (P < 0.001). The expression levels of Cyt C, active-caspase 3 and active-caspase 9 proteins were significantly up-regulated (all P < 0.001).
Conclusion: TRAIL can up-regulate the expression levels of Mcl-1 mRNA and protein in SGC7901/ADR cells. Down-regulation of Mcl-1 gene by siRNA interference enhances the ability of human gastric cancer cell line SGC7901/ADR resistant to TRAIL-induced apoptosis.

中图分类号 R735.2   DOI 10.3781/j.issn.1000-7431.2016.11.798

 
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所属栏目 基础研究

基金项目 安徽省自然科学基金资助项目(编号:1308085MH167)

收稿日期 2015/10/10

修改稿日期 2016/1/20

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引用该论文: LI Qin,ZHANG Kaiguang,ZHU Xingchao,SUN Haibing,CHEN Si,WANG Qiaomin. Effect of suppression of Mcl-1 gene expression via siRNA on TRAILinduced apoptosis of human gastric cancer SGC7901/ADR cells[J]. Tumor, 2016, 36(3): 254~263
李琴,张开光,朱邢超,孙海兵,陈思,王巧民. siRNA抑制Mcl-1基因表达对TRAIL诱导的人胃腺癌SGC7901/ADR细胞凋亡的影响[J]. 肿瘤, 2016, 36(3): 254~263


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