泛素特异性肽酶13对食管鳞状细胞癌放疗抵抗的影响及其机制  

Effect of ubiquitin-specific peptidase 13 on radioresistance of esophageal squamous cell carcinoma and its mechanism

作　　者：邹睿 罗鸣[1] 陈甜甜 杨曦 傅裕伦 李勇[1] 宋志旺 ZOU Rui;LUO Ming;CHEN Tiantian;YANG Xi;FU Yulun;LI Yong;SONG Zhiwang(Department of Oncology,Nanchang University,Jiangxi Medical College,First Affiliated Hospital of Nanchang University,Nanchang,Jiangxi 330006,China)

机构地区：[1]南昌大学第一附属医院肿瘤科,江西医学院,南昌大学,江西南昌330006

出　　处：《中华肿瘤防治杂志》2024年第23期1421-1428,共8页Chinese Journal of Cancer Prevention and Treatment

摘　　要：目的分析泛素特异性肽酶13(USP13)在食管鳞状细胞癌(ESCC)组织和细胞中的表达情况及其对放疗抵抗的影响,初步探讨其潜在机制。方法使用公开的TIMER 2.0数据库中的癌症基因组图谱(TCGA)模块和基因表达综合(GEO)数据库,分析USP13在ESCC组织中的表达情况。应用蛋白质印迹实验检测南昌大学第一附属医院经病理科确诊的10例ESCC患者癌组织和癌旁组织,以及ESCC细胞系ECA-109、KYSE-30、KYSE-105和正常食管上皮细胞系HAT-1A的USP13蛋白表达情况。体外培养ESCC细胞,通过慢病毒转染技术将USP13-shRNA导入KYSE-30和KYSE-105细胞,建立USP13低表达的细胞模型[sh-USP13(KYSE-30)和sh-USP13(KYSE-150)]。同时,转染空载体(NC-shRNA)构建对照组[NC(KYSE-30)和NC(KYSE-150)],使用蛋白质印迹实验检测转染后USP13蛋白水平。在不同照射剂量(0、2、4、6、8 Gy)下,通过细胞计数试剂盒8(CCK8)实验及克隆形成实验评估各组细胞的活性及增殖情况。在动物实验中,将10只裸鼠随机分为sh-USP13放疗组和NC放疗组,分别皮下注射sh-USP13和NC细胞以建立皮下荷瘤模型,并施加相同放射线照射,比较2组裸鼠的肿瘤体积和质量。最后,通过免疫荧光实验比较USP13低表达组和对照组的γH2AX及RAD51焦点数,以反映DNA损伤及修复情况。对于符合正态分布的数据,采用t检验进行2组间比较,多组间样本分析采用方差分析;对于不符合正态分布的数据,2组组间比较采用曼-惠特尼U检验,多组组间比较采用克鲁斯卡尔-沃利斯检验。结果TIMER 2.0数据库显示,ESCC组织中USP13表达水平高于癌旁组织,P<0.05。GEO数据库分析表明,USP13在ESCC组织中的表达水平同样高于正常组织,差异有统计学意义,t=3.032,P=0.004。蛋白质印迹检测分析显示,10例ESCC组织中USP13蛋白表达量高于癌旁组织。与正常食管上皮细胞系HAT-1A相比,ESCC细胞系(ECA-109、KYSE-30、KYSE-105)的USP13蛋白表达增加,t值分�Objective To analyze the expression of ubiquitin-specific peptidase 13(USP13)in esophageal squamous cell carcinoma(ESCC)tissues and cells,as well as its impact on resistance to radiotherapy,while preliminarily exploring its potential mechanisms.Methods The expression of USP13 in ESCC tissues was analyzed by using publicly available data from the TIMER 2.0 database(The Cancer Genome Atlas,TCGA module)and the Gene Expression Omnibus(GEO)database.Western blotting was employed to assess the expression levels of USP13 in cancerous and adjacent normal tissues from ten ESCC patients diagnosed at the First Affiliated Hospital of Nanchang University,along with ESCC cell lines(ECA-109,KYSE-30,KYSE-105)and the normal esophageal epithelial cell line HAT-1A.ESCC cells were cultured in vitro,and lentiviral transfection was used to introduce USP13-shRNA into KYSE-30 and KYSE-105 cells to establish models of reduced USP13 expression[sh-USP13(KYSE-30)and sh-USP13(KYSE-150)].Control groups were established by using empty vector(NC-shRNA)transfection[NC(KYSE-30)and NC(KYSE-150)].USP13 protein levels were measured by Western blotting.The viability and proliferation of cells under different radiation doses(0,2,4,6,8 Gy)were assessed by using cell counting Kit-8(CCK8)assays and colony formation assays.In animal experiments,ten nude mice were randomly divided into sh-USP13 radiotherapy and NC radiotherapy groups,receiving subcutaneous injections of sh-USP13 and NC cells,respectively,to establish subcutaneous tumor models and subjected to the same radiation treatment for comparison of tumor volumes and weights between the two groups.Finally,immunofluorescence assays compared the number ofγH2AX and RAD51 foci between the low USP13 expression group and the control group to reflect DNA damage and repair capacity.Data following a normal distribution were analyzed using t-tests for comparisons between two groups and ANOVA for multiple groups;non-normally distributed data were analyzed by using the Mann-Whitney U test for two-group compariso

关 键 词：食管鳞状细胞癌 泛素特异性肽酶13 放疗抵抗 DNA双链断裂 DNA损伤修复 

分 类 号：R735.1[医药卫生—肿瘤]