IL-12通过AKT／mTOR／STAT3信号通路诱导肝癌细胞自噬  

IL-12 induces autophagy via AKT/mTOR/STAT3 signaling pathway in human hepatoma cells

作　　者：刘翠颖[1] 谢昌利 林艳[1] 吴碧涛 王秦[1] 李紫薇[1] 涂植光[1] 

机构地区：[1]重庆医科大学检验医学院,临床检验诊断学教育部重点实验室,重庆400016

出　　处：《细胞与分子免疫学杂志》2016年第7期870-875,共6页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81172016)

摘　　要：目的探讨白细胞介素12(IL-12)对Hep G2和SMMC-7721肝癌细胞自噬的影响及其可能的作用机制。方法 10 ng/m L的IL-12处理肝癌细胞6 h,通过Western blot法检测自噬相关蛋白微管相关蛋白1轻链3(LC-3)和Beclin 1以及相关信号通路蛋白:蛋白激酶B(AKT)、哺乳动物雷帕霉素靶蛋白(m TOR)、信号转导子与转录激活子3(STAT3)磷酸化水平的变化,免疫荧光技术及透射电镜观察细胞自噬的发生情况;使用STATTIC或si STAT3抑制STAT3的活性以及胰岛素样生长因子1(IGF-1)活化AKT之后,再次观察IL-12诱导细胞自噬的变化情况。10 ng/m L的IL-12处理肝癌细胞1、2、3、4、5 d,CCK-8法检测细胞的增殖;10 ng/m L的IL-12处理肝癌细胞48 h,台盼蓝染色检测细胞死亡率;利用小干扰RNA(siRNA)沉默Beclin 1基因抑制自噬后,再次用CCK-8法和台盼蓝染色法检测IL-12对肝癌细胞增殖和死亡的影响。结果 IL-12能够诱导肝癌细胞发生自噬,抑制其增殖,降低其存活率;siRNA沉默Beclin 1基因后,细胞存活率降低;IL-12处理后p-AKT、p-m TOR、p-STAT3水平都显著降低;STATTIC预处理可增强IL-12诱导的细胞自噬,而IGF-1预处理后,IL-12诱导的细胞自噬减弱。结论 IL-12通过抑制AKT/m TOR/STAT3信号通路来诱导Hep G2和SMMC-7721肝癌细胞发生自噬,该自噬可减弱IL-12抑制肝癌增殖的能力。Objective To investigate the effect of IL-12 on autophagy and the relative possible mechanism in HepG2 and SMMC-7721 human hepatoma cells. Methods The hepatoma cells were treated with IL-12 （ 10 ng/mL） for 6 hours. Western blotting was applied to detect the expressions of microtubule-associated protein 1 light chain 3 （ LC-3 ）, Beclin 1 and the phosphorylated levels of protein kinase B （AKT）, mammalian target of rapamycin （mTOR）, signal transducer and activator of transcription 3 （STAT3） ; immunofluorescence assay （IFA） and transmission electron microscopy （TEM） were used to observe the formation of autophagosome. After STAT3 was inhibited by STAI-rlC or siSTAT3 and AKT was activated by insulin-like growth factor （ IGF-1 ）, Western blotting and IFA were performed again to analyze the change of IL-12-induced autophagy. After the cells were treated with IL-12 （10 ng/mL） for l, 2, 3, 4, 5 days, CCK-8 assay was used to determine the growth ability. After the hepatoma cells were treated with IL-12 （ 10 ng/mL） for48 hours, trypan blue staining was used to detect the death rate of the cells. After cell autophagy was inhibit by siBeclin 1, CCK-8 assay and trypan blue staining were performed again to study the effect of IL-12 on the proliferation and death of human hepatoma cells. Results IL-12 induced autophagy and inhibited cell growth in the hepatoma cells. Silencing Beclin 1 gene enhanced IL-12-mediated growth inhibition and cell death. Furthermore, IL-12 treatment also decreased the expressions of p-AKT, p-mTOR and p-STAT3. The pretreatment of siSTAT3 or STATTIC inhibited STAT3-enhanced IL-12-induced autophagy. Accordingly, activation of AKT with IGF-1 decreased IL-12-induced autophagy. Conclusion IL-12 could induce autophagy through AKT/mTOR/STAT3 sig- naling pathways and the induction of autophagy attenuates the growth-inhibitory effect of IL-12 on hepatoma cells.

关 键 词：白细胞介素12(IL-12) 自噬 细胞增殖 STAT3 HepG2细胞 SMMC-7721细胞 

分 类 号：R392[医药卫生—免疫学] R735.7[医药卫生—基础医学]