Bim的异常调控在黑色素瘤细胞耐受内质网应激状态中的作用  

Role of dysregulation of Bim in resistance of melanoma cdls to endoplasmic reticulum stress-induced apoptosis

作　　者：侯丽丽[1] 金雷[2] 韩传春[2] 程冰[2] 王丽[1] 张旭东 张林杰[1] 

机构地区：[1]安徽医科大学免疫学教研室,合肥230032 [2]中国科学技术大学生命科学学院肿瘤分子生物学实验室 [3]Immunology and Oncology Unit, Calvary Mater Newcastle Hospital, Austrilia

出　　处：《中华肿瘤杂志》2011年第7期494-498,共5页Chinese Journal of Oncology

基　　金：安徽高校省级科学研究项目（KJ2011A167）

摘　　要：目的探讨Bim在黑色素瘤细胞内质网应激状态下可能存在的异常调控，及其在黑色素瘤细胞对内质网应激耐受中的作用。方法用衣霉素诱导黑色素瘤细胞Mel—RM和MM200，产生内质网应激模型。采用流式细胞仪AnnexinV／PI双染法及Hoechst染色法检测细胞凋亡；Westem blot检测caspase-3和caspase-9的活化，以及Bim、GRP78、CHOP及Foxol等的蛋白表达水平；实时荧光定量聚合酶链反应检测Bim、CHOP及Foxol的mRNA水平。以小干扰RNA（siRNA）特异性沉默人胚肾上皮细胞系HEK293的Bim基因，观察Bim的蛋白表达和细胞凋亡情况。结果衣霉素作用后，黑色素瘤细胞对内质网应激诱导的凋亡表现为耐受，HEK293细胞中caspase-3和caspase一9明显活化，但在黑色素瘤细胞中却不明显。3μmol／L衣霉素作用黑色素瘤Mel—RM和MM200细胞12、24、36h后，Bim蛋白的水平均未上调，mRNA表达的变化倍率分别为0．37±0．05、0．13±0．02、0．02±0．01和0．41±0．06、0．16±0．04、0．21±0．03，与HEK293细胞相比，均发生下调（P〈0．01）。在HEK293细胞中，特异性沉默Bim基因后，caspase-3的活化程度下降，BimsiRNA干扰组细胞凋亡率为（5．69±0．38）％，明显低于siRNA阴性对照组[（40．32±1．64）％，P〈0．01]和空白对照组[（35．46±2．01）％，P〈0．01）]。3μmol／L衣霉素作用黑色素瘤细胞6、12、24、36h后，转录因子CHOPmRNA和蛋白表达水平均发生上调，Foxol mRNA和蛋白表达水平均发生下调。结论Bim的异常调控在黑色素瘤细胞对内质网应激诱导的凋亡耐受中发挥重要作用，这可能是导致黑色素瘤对治疗不敏感的重要原因之一。转录因子CHOP和Foxol可能与Bim在内质网应激诱导的黑色素瘤细胞中的异常表达有关。Objective To establish a model of ER stress-induced apoptosis with tunicamycin and to examine whether Bim is dysregulated and its potential role in resistance of melanoma cells to apoptosis under endoplasmic reticulum（ER） stress. Methods A model of ER stress-induced apoptosis was established with tunicamycin. Apoptotic cells were quantitated using the annexin V/propidium iodide method by flow cytometry. Hoechst staining was also used to confirm the apoptotic cell death. Western blotting was used to measure the activation of caspase-3 and -9, and the expression of Bim, GRP78, CHOP, and Foxol at the protein level. The expression of Bim, CHOP and Foxol at the mRNA level was quantitated by qPCR. The siRNA technique was used to inhibit the expression of Bim. Results Treatment of the melanoma ceils with tunicamycin did not induce significant apoptosis and activation of caspase cascade, whereas it caused marked activation of caspase-3 and -9, and apoptosis in HEK293 cells which were used as a control. With exposure to tunicamycin （3μmol/L） for 12, 24, 36 hours the Bim protein levels were not increased in Mel-RM and MM200 cells. Its mRNA levels were 0.37±0.05, 0. 13 ±0.02 and 0. 02 ±0.01 in Mel-RM cells,while 0.41 ±0.06, 0.16±0.04 and 0.21 ±0.03 in MM200 cells, respeetively. The expression of Bim mRNA was significantly reduced eompared with that in the control groups of the two cell lines （ P 〈 0.01 ）. siRNA knockdown of Bim protected HEK293 cells against activation of caspase-3. The cell apoptosis of Bim siRNA group was （ 5.69 ± 0.38 ） %, significantly lower than that of the siRNA control group （40.32 1.64 ） % and blank control group （ 35.46 ± 2.01 ） % （ P 〈 0.01 ）. In the melanoma cells after exposure to tunicamycin （3 p^mol/L） for 6, 12, 24, and 36 hours the transcription factor CHOP at mRNA level were significantly increased and the expressions at protein level were also up-regulated. The expressions of another transcription factor Foxol at mRNA level significantly decrea

关 键 词：黑色素瘤 BIM 内质网应激 细胞凋亡 CHOP Foxol 小干扰RNA 

分 类 号：R739.5[医药卫生—肿瘤]