siRNA沉默Twist1增强吉西他滨诱导胰腺癌细胞凋亡的研究  

Study on the Twist1 silencing by small interfering RNA enhanced Gemcitabine-induced cell death in pancreatic cancer cells

作　　者：邢人鑫[1] 冯敏[1] 

机构地区：[1]四川省医学科学院四川省人民医院城东病区,四川成都610101

出　　处：《华西药学杂志》2015年第2期192-195,共4页West China Journal of Pharmaceutical Sciences

摘　　要：目的通过抑制Twist1来观察胰腺癌细胞对吉西他滨化疗敏感性的变化。方法通过靶向人Twist1基因小干扰RNA(siRNA)抑制Twist1的表达;用Western法检测Twist1的表达、Annexin V/PI染色和流式细胞术检测吉西他滨诱导的细胞凋亡;通过Western blot检测caspase及可能的丝裂原活化蛋白激酶(MAPK)和线粒体途径;用激光扫描共聚焦显微镜检测细胞内活性氧和线粒体膜的电位。结果 Twist1沉默显著增加了Panc-1细胞对吉西他滨的敏感性,JNK/线粒体的途径被激活,Twist1 siRNA诱导线粒体膜去极化同时显著上调线粒体分裂相关蛋白Fis1并降低融合相关蛋白Mfn1的表达。Twist1 siRNA提高了细胞内活性氧(ROS)的水平,与吉西他滨联合治疗进一步增加ROS。结论 siRNA介导的Twist1沉默在PANC-1细胞对吉西他滨化疗耐受中扮演了关键角色,Twist1沉默可能作为胰腺癌治疗的一种有效方法。OBJECTIVE To observe the increased chemosensitivity of pancreatic cancer cells to Gemcitabine and examine the effects of inhibiting Twist1 expression on Gemcitabine - induced cytotoxicity in vitro. METHODS Expression of Twist1 was suppressed using a small interfering RNA （ siRNA ） targeting human Twist1 mRNA. Twist1 expression was quantified by Western blots. Gemcitabine - induced apoptosis was characterized by Annexin V - PI staining and flow cytometry. The caspase, MAPKs and mitochondrial pathways were determined by Western blot. The intracellular ROS and mitochondrial membrane potential were detected using laser confocal scan- ning microscopy. RESULTS The data showed that Twist1 depletion significantly sensitized PANC - 1 cells to gemcitabine by inducing activation of JNK/mitochondrial pathway but not ERK or p - 38 pathways, Moreover, treatment of cells with Twistl siRNA induced mito- chondrial membrane depolarization and significantly upregulated the mitochondrial fission - related proteins Fisl and decreased the expression of fusion - related proteins Mfnl. Intracellular reactive oxygen species （ ROS ） levels were increased in cells treated with Twist1 siRNA and further increased by co - treatment with Gemcitabine. CONCLUSION The resuhs shows that siRNA - mediated Twistl knockdown plays a critical roles in PANC - 1 cell chemoresistance to gemcitabine and puts forward the possibility of Twist depletion as a promising approach to pancreatic cancer therapy.

关 键 词：胰腺癌 TWIST1 吉西他滨 线粒体 增敏 小干扰RNA 辣根过氧化酶 细胞调亡 基因表达 Twist1沉默 

分 类 号：R96[医药卫生—药理学]