RNAi沉默STAT3基因联合mTOR抑制剂rapamycin诱导BEL-7402肝癌细胞凋亡  

Apoptosis of hepatocarcinoma cells BEL-7402 induced with STAT3 gene silencing combined with rapamycin

作　　者：张毅[1,2] 张君薇[3] 谢淑丽[4] 王广义[5] 

机构地区：[1]辽宁医学院附属第一医院微创肝胆外科,辽宁锦州121000 [2]辽宁省盘锦市中心医院普通外四科 [3]辽宁医学院附属第三医院肿瘤科,辽宁锦州121000 [4]吉林大学第一医院普通外科实验室,吉林长春130021 [5]吉林大学第一医院肝胆胰外科,吉林长春130021

出　　处：《吉林大学学报（医学版）》2013年第5期903-908,I0003,共7页Journal of Jilin University：Medicine Edition

基　　金：辽宁省科技厅科学技术计划项目资助课题(201300384)

摘　　要：目的:探讨PI3K/AKT/mTOR和JAK/STAT3 2条信号转导途径共同作用对肝癌细胞凋亡的影响,为肝癌基因治疗提供依据。方法:选取对数生长期BEL-7402细胞,随机分为对照组、mTOR抑制剂rapamycin(Rapa)组、阴性质粒组、阴性质粒+Rapa组、STAT3-siRNA质粒组和STAT3-siRNA质粒+Rapa组,应用LipofectamineTM2000转染试剂将含有目的基因的质粒转染BEL-7402细胞,同时应用rapamycin,分别采用流式细胞术和Hoechst33258荧光染色检测细胞凋亡率和形态学的变化,JC-1荧光染色观察线粒体膜电位(ΔΨm)变化,Western blotting法检测活性caspase-3蛋白表达水平。结果:STAT3-siRNA+Rapa组细胞凋亡率为60.22%±0.87%,明显高于其他各组(P<0.05),且细胞ΔΨm明显降低(27.28%±1.82%,P<0.05);Hoechst33258荧光染色检测,见STAT3-siRNA有大量细胞出现细胞核聚集、边缘化和核碎裂等典型细胞凋亡形态;Western blotting检测,STAT3-siRNA+Rapa组活性caspase-3蛋白表达水平明显高于其他各组(P<0.05)。结论:RNAi沉默BEL-7402肝癌细胞STAT3基因联合rapamycin可促进BEL-7402肝癌细胞的凋亡,二者具有明显的协同作用。Objective To explore the influence of PI3K/AKT/mTOR and JAK/STAT3signaling pathway in apoptosis of the hepatocarcioma cells（HCC）,and to provide basis for gene therapy for liver cancer.Methods BEL-7402cells at inlogarithm growth phase were selected and randomly divided into control group,scramble-siRNA group,STAT3-siRNA group,scramble-siRNA ＋rapamycin（Rapa）group,Rapagroup and STAT3-siRNA＋Rapagroup.The plasmids pGCsi.U6/neoRFP-STAT3designed for expression of STAT3siRNA was transfected into BEL-7402cells by LipofectamineTM2000.The cells with or without transfection siRNA were treated in wells in the absence or presence of rapamycin.The apoptotic rate was detected by flow cytometry（FCM） and AnnexinV/PI apoptosis detection kit staining.The morphological changes of the cells were assessed by Hoechst33258immunofluorescence staining.Simultaneously,the mitochondrial membrane potential（ΔΨm）was visualized by the JC-1fluorescence staining and inverted fluorescence microscope.Further more,the expression level of active caspase-3protein was analyzed by Western blotting method.Results The apoptotic rate of BEL7402cells in STAT3-siRNA＋Rapagroup（60.22%±0.87%）was higher than those in other groups（P0.05）;and theΔΨm was significantly decreased（27.28%±1.82%,P0.05）.The Hoechst33258fluoresence staining results showed that the characteristic changes of chromatin condensation and nuclear fragmentation were observed in STAT3-siRNA ＋ Rapagroup.The Western blotting results showed that the protein expression level of active caspase-3in STAT3-siRNA ＋ Rapagroup was significantly higher than those in the other groups（P0.05）.Conclusion Combined treatment of rapamycin and STAT3gene silencing can significantly promote the apoptosis of BEL-7402cells and they display synergistic effects.

关 键 词：mTOR蛋白 STAT3基因 RNA干扰 RAPAMYCIN 细胞凋亡 肝细胞癌 

分 类 号：R735.7[医药卫生—肿瘤]