DNA损伤特异性结合蛋白1在肝癌组织中的表达及其对SMMC-7721细胞同源重组修复、靶向杀伤作用的影响  

Expression of DNA damage-binding protein 1 in hepatocellular carcinoma tissues and its effect on homologous recombination repair and targeted killing in SMMC-7721 cells

作　　者：景刚[1] 关坤萍[1] 朱爱萍[1] 薛耀勤[2] Jing Gang;Guan Kunping;Zhu Aiping;Xue Yaoqin(Department of Medical Laboratory Science,the Second Hospital of Shanxi Medical University,Taiyuan 030001,China;Department of Invasive Technology,Shanxi Province Cancer Hospital,Shanxi Hospital Affiliated to Cancer Hospital,Chinese Academy of Medical Sciences,Cancer Hospital Affiliated to Shanxi Medical University,Taiyuan 030013,China)

机构地区：[1]山西医科大学第二医院检验科,太原030001 [2]山西省肿瘤医院中国医学科学院肿瘤医院山西医院山西医科大学附属肿瘤医院介入科,太原030013

出　　处：《肿瘤研究与临床》2022年第8期561-568,共8页Cancer Research and Clinic

摘　　要：目的探讨DNA损伤修复因子DNA损伤特异性结合蛋白1(DDB1)在肝癌组织中的表达情况及DDB1基因沉默对人肝癌SMMC-7721细胞DNA损伤修复和靶向杀伤作用的影响。方法利用UALCAN平台对癌症基因组图谱(TCGA)数据库中肝细胞肝癌组织(371例)和癌旁组织(50例)中DDB1的表达情况及DDB1表达与肝癌患者总生存的相关性进行生物信息学分析。向SMMC-7721细胞转染靶向DDB1的小干扰RNA(siRNA)和阴性对照siRNA, 分别为DDB1沉默组和阴性对照组。用X线照射诱导两组细胞外源性DNA双链断裂(DSB)损伤, 采用免疫荧光染色(γH2AX用于评估DSB损伤情况, RPA32s33和Rad51用于评估同源重组修复情况)和蛋白质印迹法(检测RPA32s33蛋白水平)分析DDB1基因沉默对细胞DSB损伤修复的影响。采用姐妹染色体交换(SCE)实验分析DDB1沉默组和阴性对照组细胞同源重组SCE频率。采用四甲基偶氮唑盐(MTT)法分析PARP抑制剂奥拉帕尼(10 μmol/L)、顺铂(1 μg/ml)及二者联合杀伤DDB1沉默组和阴性对照组SMMC-7721细胞的效果。结果生物信息学分析发现, 肝癌组织DDB1 mRNA水平较癌旁组织高(P<0.001), DDB1高表达患者总生存较DDB1低表达患者差(P=0.029)。X线照射后培养24 h, 阴性对照组细胞γH2AX灶点已大多消退, DDB1沉默组细胞仍较多[每个细胞中(5.1±2.0)个比(13.4±2.0)个, t=-5.08, P=0.007]。X线照射后培养4 h, 阴性对照组RPA32s33灶点数[每个细胞中(30.8±5.0)个比(13.2±1.6)个]和Rad51灶点数[每个细胞中(19.5±1.8)个比(8.3±3.3)个]均高于DDB1沉默组, 两组间差异均有统计学意义(均P<0.01)。阴性对照组SCE频率高于DDB1沉默组[(21.2±3.0)%比(11.2±1.6)%, t=5.07, P=0.007]。MTT法检测显示, 奥拉帕尼作用后, DDB1沉默组细胞的存活率低于阴性对照组[(40.3±3.6)%比(79.8±1.3)%, t=17.94, P<0.001], 奥拉帕尼联合顺铂时, 两组细胞存活率均进一步降低, 且DDB1沉默组细胞存活率低于阴性对照组[(10.2±2.8)%比Objective To investigate the expression of DNA damage repair factor DNA damage-binding protein 1(DDB1)in hepatocellular carcinoma tissues,and the effect of DDB1 gene silencing on DNA repair and targeted killing in human hepatocellular carcinoma SMMC-7721 cells.Methods The UALCAN platform was used to perform bioinformatics analysis on the expression of DDB1 in hepatocellular carcinoma tissues(371 cases)and paracancerous tissues(50 cases)in The Cancer Genome Atlas(TCGA)database and the correlation of DDB1 expression with the overall survival of liver cancer patients were analyzed by bioinformatics using the UALCAN platform.SMMC-7721 cells were transfected with small interfering RNA(siRNA)targeting DDB1 and negative control siRNA,which were DDB1 silencing group and negative control group,respectively.X-ray irradiation induced exogenous DNA double strand break(DSB)damage in the two groups of cells.Immunofluorescence staining(γH2AX was used for assessing cellular DSB damage,RPA32s33 and Rad51 were used for assessing homologous recombination repair)and Western blotting(were used to detect the level of RPA32s33 protein)were used to analyze the effect of DDB1 gene silencing on DSB damage repair.Sister chromosome exchange(SCE)experiment was used to analyze the frequency of SCE of homologous recombination of cells in DDB1 silencing group and negative control group.Tetramethylazozolium salt(MTT)method was used to analyze the killing effect of PARP inhibitor olapani(10μmol/L),cisplatin(1μg/ml)and olapani combined with cisplatin on SMMC-7721 cells in DDB1 silencing group and negative control group.Results Bioinformatics analysis showed that the level of DDB1 mRNA in liver cancer tissues was higher than that in paracancerous tissues(P<0.001),and the overall survival of patients with high expression of DDB1 was worse than that of patients with low expression of DDB1(P=0.029).When cultured for 4 hours after X-ray irradiation,the number ofγH2AX foci in cells of the negative control group had mostly disappeared,and there were

关 键 词：癌 肝细胞 DNA损伤 同源重组 DNA损伤特异性结合蛋白1 聚ADP核糖聚合酶抑制剂 

分 类 号：R735.7[医药卫生—肿瘤]