1、XBP1调控胶质瘤细胞氧化应激的实验研究刘耀华赵世光陈晓丰梁元郑秉杰哈尔滨医科大学附属第一医院神经外科ROSThe Achilles Heel of cancer cells(Cancer Cell.2019;14(6):427-9 IF 24.962)CatalaseSODCancer Cell 14,December 9,2019Exogenous ROS:The last straw breaks the camels back.研究背景活性氧（ROS）与胶质瘤?肿瘤，包括胶质瘤可以产生高水平活性氧；?肿瘤细胞的Akt过度激活，决定了其对活性氧敏感;?大量活性氧可以杀伤肿瘤，很多化疗药物
2、为活性氧诱导剂;?调控胶质瘤细胞氧化应激水平可能对其治疗具有积极意义。研究背景：内质网应激、氧化应激、XBP1HypoxiaROSNutrients DeprivationXBP1?OxidativeStressER StressXBP1Protective moleculesantioxidant genes(Catalase,SOD1,etc)CCAATNF-Y?XBP1transcriptstranscriptstranscriptsCCAATNF-Ytarget genesERSEXBP1NF-Ytranscriptstranscriptstranscripts实验设计?胶质瘤细胞系U
3、251MG进行RNA干扰，抑制XBP1基因表达后比较细胞对外源性活性氧H2O2的敏感性；?流式细胞技术检测ROS蓄积和线粒体膜电位；?检测比较细胞内抗氧化分子表达水平；?将细胞进行XBP1基因过表达，观察能否起到相反的效果；?应用启动子突变分析探讨XBP1促抗氧化分子转录活性的机制。实验结果一对H2O2的敏感性比较100survival fraction(%of total)806040200*00.10.250.5H2O2 concentration(mM)RandomSiXBP11H2O2处理后trypan blue检测细胞生存情况*P0.05,*P0.01实验结果一对H2O2的敏感性比较
4、-CH2O2-0.5Random7.1100101102FL1-H103104100101102FL1-H29.429.4103104-CH2O2-0.5SiXBP15.4100101102FL1-H103104100101102FL1-H60.310310413.0流式细胞技术检测线粒体膜电位（MMP）实验结果二抑制XBP1特异性增强细胞对氧化应激的敏感性 100survival fraction(%of total)80604020*0051020PTL concentration(uM)RandomSiXBP140对活性氧诱导剂PTL的敏感性比较（*P0.05,*P0.01)实验结果二抑
5、制XBP1特异性增强细胞对氧化应激的敏感性50cell death(%of total)randomSiXBP1403020100CVP16FK228对其它2种化疗药物诱导的细胞死亡没有明显区别实验结果二XBP1缺失特异性增强细胞对氧化应激的敏感性Random-C.001Random-PTL-20Random-FK228Random-VP169.0100101102FL1-HSiXBP1-C103104100101102FL1-H15.2103104100101102FL1-H28.6103104100101102FL1-H26.9103104SiXBP1-PTL-20SiXBP1-FK228
6、SiXBP1-VP166.7100101102FL1-H103104100101102FL1-H41.5103104100101102FL1-H23.5103104100101102FL1-H21.3103104CPTLFK228VP16实验结果三抑制XBP1细胞内活性氧（ROS）明显增高RandomROS(fold increase)C(4.6)3h12.1654321001h2hRandomSiXBP1*100101FL1-H102SiXBP1C(3.8)3h*24.93h100101FL1-H102DCFH-DA孵育细胞，流式细胞技术检测细胞内ROS堆积：*P0.05,*P0.01实验结
7、果三抑制XBP1细胞内活性氧（ROS）明显增高H2O2RandomSiXBP1C 1h2h 3h 4h C 1h2h 3h 4hP-JNKJNK-1P-P38P38PTLC 1h 2h 4h 6hRandomC 1h 2h 4h 6hSiXBP1P-P38P38实验结果四XBP1与细胞抗氧化分子的表达Random XBP1catalase transcripts(fold)Catalase1.00.80.60.40.20.0randomSiXBP1SOD1TRX1GPXGAPDH*抑制XBP1后细胞中抗氧化分子的mRNA水平(*P0.01)实验结果四XBP1与细胞抗氧化分子的表达RandomC
8、 1h 2h3hSiXBP1C 1h 2h 3hCatalaseSOD1TRX1GAPDHRandomC 3h 6h 9hSiXBP1C 3h 6h 9hCatalase14-3-3H2O2处理细胞后未见Catalase、SOD1 和TRX1表达变化实验结果四XBP1与细胞抗氧化分子的表达cell death(%of total)100806040*P0.01CAT0.9M10.9M10200200400FL2-A6000200400FL2-A600CAT H2O2AT+H2O2H2O2AT-H2O2.01817.0%M162%M1H2O2 C1h 2h 3hAT C1h 2h 3hPP38P
9、380200400FL2-A6000200400FL2-A600Catalase特异性抑制剂AT预处理的影响实验结果五XBP1可增强Catalase转录活性U251 XBP1过表达过表达mock XBP1CatalaseSOD1b-actinXBP1ROS(fold increase)76543210H2O2P0.01*CmockXBP1实验结果六XBP1可增强Catalase转录活性mockXBP1CAT(-1394/+68)*P0.05CAT(-191/+68)*P0.01vehicle02040relative activity(fold)60荧光素酶（luciferase）活性分析实验
10、结果六XBP1可增强Catalase转录活性mockwild typeGC box2CCAAT box2CCAAT box(2/3)0246XBP1*P0.01*P0.01*P0.01relative activity(fold)Catalase启动子突变实验结果七XBP1促进NF-Y与Catalase启动子结合凝胶电泳迁移分析（EMSA）实验结果七XBP1促进NF-Y与Catalase启动子结合染色质免疫沉降小 结OxidativestressOxidativestressOxidativestressXBP1ROSROSROSXBP1XBP1CellDeathCatalaseCatalaseCatalasetranscriptsNF-YCCAATNF-YCCAATtranscriptstranscriptsCatalase gene
