摘要
目的探究丝氨酸/苏氨酸蛋白激酶1(serine/threonine-protein kinase 1,AKT1)介导的肝癌细胞自噬对^(125)I粒籽辐照敏感性的影响。方法①利用iProX数据库和STRING12.0网站对^(125)I粒籽辐照前后肝癌蛋白组数据进行分析,探究差异表达蛋白及相关功能联系,同时借助癌症基因组图谱(the cancer genome attas,TCGA)数据库分析AKT1表达水平与肝癌患者生存期关系。②利用人肝癌细胞系HUH7和Hep3B构建体外辐照模型,^(125)I组细胞接受起始表观活度0.8 mCi/粒籽的^(125)I放射性粒籽持续辐照约120 h累计辐射剂量8 Gy,对照组(NC组)正常培养120 h。③使用自噬抑制剂氯喹(chloroquine,CQ)、诱导剂分别处理肝癌细胞系,构建CQ组和RAPA组。运用慢病毒转染技术构建过表达AKT1肝癌细胞系,即AKT1组。将同样处理的肝癌细胞系持续^(125)I粒籽辐照120 h,获得CQ+^(125)I组、RAPA+^(125)I组、AKT1+^(125)I组。④Western blot检测微管相关蛋白轻链3(microtubule-associated protein light chain 3,LC3)、p62、AKT1、p-AKT1的变化,利用细胞免疫荧光观察LC3表达;平板克隆实验和流式细胞术实验检测细胞克隆能力以及凋亡率。结果①^(125)I粒籽辐照后,相较于NC组,^(125)I组的中p62表达降低(P<0.05),LC3Ⅱ/LC3Ⅰ比值增高(P<0.05),免疫荧光显示LC3绿色荧光点增多。与^(125)I组比较,CQ+^(125)I组p62表达增高(P<0.01),LC3Ⅱ/LC3Ⅰ比值降低(P<0.01)。在细胞凋亡和克隆能力方面,CQ+^(125)I组凋亡率较^(125)I组降低(P<0.01),CQ+^(125)I组克隆形成数较^(125)I组增多(P<0.01),而RAPA+^(125)I组结果与之相反。②iProX数据库分析显示,AKT1在辐照组降低,TCGA数据库分析表明,AKT1高表达预示肝癌患者较差预后(P<0.01)。③^(125)I粒籽辐照后,^(125)I组AKT1在RNA和蛋白水平表达均降低(P<0.01)。过表达AKT1后,自噬水平降低(P<0.05),^(125)I粒籽辐照过表达AKT1的肝癌细胞可部分恢复自噬水平。相较于AKT1组,AKT1+^(125)I组的AKT1、p-AKT1、p62表达均降低(P<0.05),LC3Ⅱ/LC3Ⅰ比值增高(P<0.05)。④关于细胞凋亡和克隆能力,AKT1+^(125)I组较^(125)I组凋亡率降低(P<0.05),AKT1+^(125)I组较AKT1组凋亡率升高(P<0.05)。HUH7细胞中AKT1+^(125)I组较^(125)I组克隆能力提高(P<0.05),在Hep3B细胞中AKT1+^(125)I组较^(125)I组克隆能力提高(P<0.05),AKT1组较NC组克隆能力增高(P<0.05)。结论^(125)I粒籽辐照后的肝癌细胞通过降低AKT1的表达诱导致命性自噬,为^(125)I放射性粒籽植入治疗肝癌提供了新的理论依据。
Objective To investigate the impact of serine/threonine-protein kinase 1(AKT1)-mediated autophagy in hepatocellular carcinoma(HCC)cells on their sensitivity to^(125)I seed irradiation.Methods①iProX database and STRING12.0 website were utilized to analyze the proteomic data of HCC before and after^(125)I seed irradiation to explore the differentially expressed proteins and associated functional connections.Meanwhile,The Cancer Genomics Atlas(TCGA)database was employed to analyze the relationship between AKT1 expression level and the survival of HCC patients.②Human HCC cell lines HUH7 and Hep3B were exposed to continuous irradiation from^(125)I radioactive seeds with an initial apparent activity of 0.8 mCi per seed for approximately 120 h,accumulating a total dose of 8 Gy,while the control cells were cultured under normal condition for 120 h.③Autophagy inhibitor,chloroquine(CQ)and inducer,rapamycin(RAPA)were used to treat the HCC cells respectively to establish the CQ group and the RAPA group.The lentiviral transfection technique was employed to construct the HCC cells with overexpressed AKT1,namely the AKT1 group.The HCC cells treated in the same way were continuously irradiated with^(125)I seeds for 120 h to construct the CQ+^(125)I group,the RAPA+^(125)I group,and the AKT1+^(125)I group.④The changes in microtubule-associated protein light chain 3(LC3),p62,AKT1 and p-AKT1 were detected by Western blotting.Cell immunofluorescence assay was employed to observe the expression of autophagy related proteins,such as LC3.The colony forming ability and apoptotic rate were detected with plate cloning assay and flow cytometry.Results①Continuous irradiation with^(125)I seeds resulted in decreased expression of p62 and increased ratio of LC3Ⅱ/LC3Ⅰ(P<0.05)when compared with the negative control(NC)group.Immunofluorescence assay revealed more green fluorescence spots of LC3.When compared with the^(125)I group,the CQ+^(125)I group had significantly increased expression of p62(P<0.01),decreased ratio of LC3Ⅱ/LC3Ⅰ(P<0.01),lower apoptotic rate(P<0.01),and more colony formations(P<0.01).In contrast,the results in the RAPA+^(125)I group were opposite to those of the CQ+^(125)I group.②Analysis on the iProX database showed that the expression of AKT1 was decreased in the irradiated group,and analysis on the TCGA database indicated that high expression of AKT1 predicted a poor prognosis for HCC patients(P<0.01).③After irradiation with^(125)I seeds,the expression of AKT1 at both the RNA and protein levels was decreased in the^(125)I group(P<0.01).After overexpression of AKT1,the level of autophagy was decreased(P<0.05).Irradiation of HCC cells with overexpressed AKT1 using^(125)I seeds could partially restore the level of autophagy.In the AKT1+^(125)I group,the expression of AKT1,pAKT1 and p62 were all decreased,and the ratio of LC3Ⅱ/LC3Ⅰwas increased than the AKT1 group(P<0.05).④The apoptotic rate of the AKT1+^(125)I group was lower than that of the^(125)I group(P<0.05)and higher than that in the AKT1 group(P<0.05).In HUH7 cells,the clonogenic ability of the AKT1+^(125)I group was higher than that of the^(125)I group(P<0.05).In Hep3B cells,the clonogenic ability of the AKT1+^(125)I group was higher than that of the^(125)I group,and the clonogenic ability of the AKT1 group was higher than that of the NC group(P<0.05).Conclusion^(125)I seed irradiation induce lethal autophagy in HCC cells by reducing the expression of AKT1,providing a new theoretical basis for the implantation of^(125)I radioactive seeds in the treatment of HCC.
作者
王琛禹
吴治州
刘丽
肖云华
黄学全
WANG Chenyu;WU Zhizhou;LIU li;XIAO Yunhua;HUANG Xuequan(Department of Nuclear Medicine,First Affiliated Hospital,Army Medical Universily(Third Military Medical University),Chongqing,China)
出处
《陆军军医大学学报》
北大核心
2025年第6期539-550,共12页
Journal of Army Medical University
基金
重庆市自然科学基金面上项目(CSTB2022NSCQ-MSX1501,CSTB2023NSCQ-MSX0600)。
