【作者】 朱明月； 夏华； 李伟； 鲁琰； 董栩； 陈栘； 符史干； 谢协驹； 李孟森；

【Author】 Ming-Yue ZHU;Jun-Li Guo;Hua XIA;Wei Li;Yan Lu;Xu Dong;Yi Chen;Shi-Gan Fu;Xieju XIE;Meng-Sen Li;Hainan Provincial Key Laboratory of Carcinogenesis and Intervention,Hainan Medical College;Key Laboratory of Molecular Biology,Hainan Medical College;School of Postgraduate,Guangxi Medical University;Department of Pathophysiology,Hainan Medical College;Institution of Tumor,Hainan Medical College;

【机构】 海南省肿瘤发生和干预重点实验室； 海南医学院分子生物学重点实验室； 广西医科大学研究生学院； 海南医学院病理生理学教研室； 海南医学院肿瘤研究所；

【摘要】 目的:研究甲胎蛋白(Alpha-fetoprotein,AFP)对肝癌细胞内PI3K/AKT信号传递的影响,及其在癌细胞耐受全反式维甲酸(All trans retinoic acid,ATRA)的作用。方法:MTT法检测ATRA对人肝癌Bel7402细胞增殖的影响:显微照相观察细胞形态学的改变;流式细胞分析细胞凋亡;激光共聚焦显微镜观察AFP与PTEN的共定位;免疫共沉淀(Co-IP)技术研究AFP与PTEN相互作用;Western Blotting法分析磷酸化的蛋白激酶B(pAKT)和Src表达,构建干扰AFP表达的载体(AFP-siRNA)并转染Bel7402细胞;用PI3K特异性阻断剂Ly294002处理细胞,分析Ly294002的作用效果。结果:MTT分析显示,人肝癌Bel7402耐受ATRA的细胞毒性;共聚焦显微镜观察显示AFP与PTEN共定位于细胞浆;Co-IP技术研究发现AFP能与PTEN结合;干扰AFP表达后能显著的增加Bel 7402细胞对ATRA敏感性,并能抑制pAKT和Src的表达;Ly294002能抑制AFP促进Bel 7402细胞表达pAKT和Src的作用。结论:肝癌细胞内表达的AFP能与PTEN结合并抑制PTEN对AKT的去磷酸化作用,肝癌细胞内高表达的AFP能激活PI3K/AKT信息通路对抗ATRA诱导的凋亡。更多还原

【Abstract】 Objective:Alpha-fetoprotein(AFP) has a property to maintain the growth of hepetocellular cancinoma cells(HCC) in vivo,but the critical functional step of AFP still obscurity,the present investigation in order to explore the effect of AFP on transduction of PI3K/AKT signal in HCC,and AFP played role in resistance cytotoxicity of all trans retinoic acid(ATRA) of HCC.Methods:The effects of ATRA on proliferation of human liver cancer cells line,Bel 7402 was detected by MTT;For showing the change of morphology in Bel 7402 cells,microscopical photograph was done;Apoptosis of Bel 7402 cells were analyzed by flow cytometry;Laser confocal microscope and co-immunoprecipitation(Co-IP) were applied to detected the co-location and interaction of AFP with PTEN;Western blotting was utilized to evidence the expression of phosphoryated protein kinase B(pAKT) and Src;Short small RNA interfering(RNAi)technique was applied to knockdown the expression of AFP in Bel 7402 cells;PI3K specific inhibitor was used to monitor the influence of AFP in transduction of PI3 K signal pathway.Results:Human hepatoma cells line Bel 7402 resisted cytotoxicity of ATRA;PTEN and AFP was observed co-localize in cytoplasm and co-IP indicated that AFP has a property to interact with PTEN in Bel 7402 cells;RNAi could knockdown the expression of AFP,the expression of pAKT(Ser473) and Src were decreased,and the sensitivity of Bel 7402 cells to ATRA were promoted when repressed the expression of AFP by RNAi;It also indicated that Ly294002 been able to reduce the role of AFP in stimulating expression of pAKT(Ser473) and Src in Bel 7402 cells.Conclusions:These data provide the evidence that AFP has a capacity to activate the transduction of PI3K/AKT signal;Expression of AFP in hepatoma cells was a pivotal event for resisting apoptosis induced by ATRA.更多还原