RNAi抑制XIAP基因诱导MG63细胞凋亡、化疗增敏及其分子机制  

RNAi silencing of XIAP gene induces apoptosis and susceptibility to chemotherapy of osteosarcoma cell line MG63

作　　者：何畔[1] 梁珊[2] 

机构地区：[1]湖南省人民医院创伤外科,长沙410005 [2]湖南省人民医院临床医学研究所,长沙410005

出　　处：《第三军医大学学报》2010年第12期1312-1315,共4页Journal of Third Military Medical University

摘　　要：目的观察RNAi技术抑制X染色体连锁凋亡抑制蛋白(X-linked inhibit apoptosis protein,XIAP)基因对MG63细胞凋亡及化疗药物敏感性的影响,并探讨其分子机制。方法构建XIAP基因的shRNA稳定表达载体,将MG63细胞分为空白组(A组,不作转染),阴性对照空白质粒转染组(B组,psiRNA-Con转染组),实验组(C组,psiRNA-XIAP转染组),在此基础上又设A+阿霉素(doxorubicin,Dox,0.3μmol)、C+Dox两化疗药物干预组;Western blot检测XIAP蛋白的表达,MTT检测细胞的增殖情况,流式细胞术检测细胞周期、凋亡及Caspase-9蛋白的表达情况。结果与A、B组比较,C组细胞XIAP蛋白水平、细胞增殖活性明显降低(P<0.05),细胞凋亡率、Caspase-9蛋白表达及G2/M期细胞明显增多(P<0.05)。A+Dox组较C+Dox组细胞增殖活性更低,且Dox对A、C组细胞的抑制呈时间依赖性;Caspase-9蛋白表达明显增高(P<0.05);细胞周期出现G2/M为0,细胞阻断于S期。结论 RNAi能有效抑制XIAP基因表达,抑制骨肉瘤细胞增殖活性,诱导其凋亡,并增加其对化疗药物的敏感性,其机制可能涉及抑制DNA合成及Caspase家族凋亡信号转导通路的活化。Objective To explore the effect RNAi-mediated inhibition of X-linked inhibit apoptosis protein （XIAP） gene on apoptosis and susceptibility to chemical therapy drugs in osteosarcoma cells and its underlying mechanism.Methods shRNA plasmid of XIAP gene were constructed. MG63 cells were conventionally cultured and divided into untreated group （group A,non-transfection）,blank plasmid group （group B,psiRNA-Con transfected）,the experimental group （group C,psiRNA-XIAP transfected）,and 2 chemical drug interfered groups,non-transfection＋0.3 μmol Dox group （A＋Dox group） and psiRNA-XIAP transfection ＋0.3 μmol Dox group （C＋Dox group）. The XIAP expression in MG63 cells was detected by Western blotting. The proliferation of above-mentioned MG63 cells was examined by MTT assay. Flow cytometry （FCM） was employed to detect the cell apoptosis,cell cycle and Caspase-9 expression. Results Group C had a significantly lower protein level of XIAP and MTT value than group A and B （P0.05）,and had higher apoptotic rate,stronger Caspase-9 expression and larger percentage of G2/M arrested cells than group A and B. In C＋Dox group,MTT value was more lower and Caspase-9 expression more higher than in A＋Dox group （P0.05）. There was no cell at G2/M stage in C＋ Dox group,and its cell cycle was blocked before S stage. Conclusion RNAi could specifically suppress XIAP gene expression,effectively inhibit the proliferation and accelerate the apoptosis of MG63 cells,and enhance its susceptibility to chemical therapy drug. The mechanism may be related to the suppression of DNA synthesis and the activation of Caspase signaling pathway.

关 键 词：RNA 基因 X连锁 骨肉瘤 细胞凋亡 细胞周期 药物疗法 

分 类 号：R73-362[医药卫生—肿瘤] R738.1[医药卫生—临床医学]