柠檬酸转运体SLC25A1对食管鳞癌细胞体外放射敏感性的影响及其分子机制  

Role of mitochondrial citrate transporter SLC25A1 in radiosensitivity of human esophageal squamous cell carcinoma in vitro

作　　者：师盼 林重华 舒易方 瞿家权 宋淑亮[1] SHI Pan;LIN Chonghua;SHU Yifang;QU Jiaquan;SONG Shuliang(Marine College,Shandong University,Weihai 264209,Shandong;Cholestatic Liver Diseases Center and Department of Gastroenterology,Southwest Hospital,Third Military Medical University,Chongqing 400038;Department of Imaging Technology,Jishou University,Jishou 416000,Hunan;Department of Oncology,Southwest Hospital,Third Military Medical University,Chongqing 400038,China)

机构地区：[1]山东大学海洋学院,山东威海264209 [2]陆军军医大学第一附属医院消化内科胆汁淤积肝病中心,重庆400038 [3]吉首大学医学院医学影像技术系,湖南吉首416000 [4]陆军军医大学第一附属医院肿瘤科放疗中心,重庆400038

出　　处：《癌变．畸变．突变》2020年第2期132-138,共7页Carcinogenesis,Teratogenesis & Mutagenesis

基　　金：国家自然科学基金(81703043,81770583);山东省自然科学基金(ZR2017MH040);湖南省卫生厅科研课题(B2017070)。

摘　　要：目的:探讨线粒体柠檬酸转运体SLC25A1对食管癌细胞体外放射敏感性的影响及其分子机制。方法:采用癌症基因组图谱(TCGA)数据库及UALCAN交互式网络工具比较分析184例食管癌组织与11例正常组织的SLC25A1 mRNA表达水平的差异;采用亚致死剂量X射线多次暴露法建立放射抵抗及对照组食管癌细胞系,放射克隆存活法线性二次靶模型分析两株食管癌细胞株放疗反应性的差异;多次X射线照射(总剂量6.0 Gy)上述两株食管癌细胞后,Hoechst33258染色荧光显微镜观察细胞凋亡形态学变化,流式细胞术检测细胞活性氧水平;免疫印迹法分析慢病毒小发夹RNA稳定转染敲低SLC25A1表达对DNA损伤修复关键信号分子H2AX和DNA-PKcs磷酸化水平的影响。结果:TCGA肿瘤组织数据库比较分析证实,与正常食管组织比较,食管癌组织SLC25A1 mRNA表达水平升高1.65倍(P=1.64×10^-4);多轮亚致死剂量暴露法构建的放射抵抗食管鳞癌细胞株SLC25A1蛋白表达水平较对照组升高2.36倍(P<0.05);与对照组比较,稳定敲低SLC25A1表达的放射抵抗食管癌细胞株凋亡率增加1.58倍(P<0.05);活性氧水平下调(65.3±14.3)%(P=0.007);并下调磷酸化H2AX(Ser139)和磷酸化DNA-PKcs(Ser2056)水平(P均<0.05)。结论:SLC25A1可能是一种参与食管鳞癌发病和放射抵抗的癌基因,通过下调活性氧水平,抑制DNA损伤修复,诱导细胞凋亡并可作为食管鳞癌放射增敏的潜在分子靶标。OBJECTIVE: To investigate the effect and molecular mechanism of mitochondrial citrate transporter SLC25 A1 on sensitivity of esophageal squamous cell carcinoma to radiotherapy in vitro.METHODS:The Cancer Genome Atlas(TCGA) tumor tissue database and UALCAN interactive network tool were used to compare differences in SLC25 A1 expression between 184 cases of esophageal cancer and 11 cases of normal tissues. Radioresistant esophageal carcinoma cell line and the control were established by multiple round of X-ray sublethal dose exposures. Radioresistance and radiosensitivities were characterized by the radiation clonal survival assay and the linear quadratic target model. Morphologic changes of cell apoptosis were observed by fluorescence microscopy using the Hoechst 33258 staining after multiple X-ray exposures at a total dose of 6.0 Gy. Flow cytometry was used to detect levels of reactive oxygen species after the same Xray exposures. Western blotting was used to analyze the expression alteration of levels of phosphor-H2 AX and phosphor-DNA-PKcs,and the key signal kinases in DNA repair by SLC25 A1 knockdown with lentivirus vector carrying small hairpin interfering RNA. RESULTS: A comparative analysis of TCGA tumor tissue databaseconfirmed that the expression level of SLC25 A1 m RNA in esophageal cancer tissues was 1.65-folds higher thanthat in normal esophageal tissues(P=1.64×10^-4). The expression level of SLC25 A1 protein was increased by2.36-folds when compared with the control(P<0.05). The apoptosis rate of esophageal cancer cell lines withstable SLC25 A1 expression compared to the control was increased by 1.58-folds(P<0.05). ROS level in theSLC25 A1 knockdown radioresistant esophageal carcinoma cell line was decreased by(65.3±14.3)%(P=0.007).Levels of phosphorylation of H2 AX(Ser139) and DNA-PKCs(Ser2056) in SLC25 A1 knockdown radioresistantesophageal carcinoma cell lines were significantly reduced when compared with the control(all P<0.05).CONCLUSION: SLC25 A1 may be an oncogene involved in tumor pathogenesis

关 键 词：线粒体柠檬酸转运体 食管鳞癌 放射增敏 活性氧产物 DNA损伤修复 

分 类 号：R735.1[医药卫生—肿瘤]