乳腺癌细胞N-乙酰转移酶10高表达诱导铂类药物耐药的作用机制  

Overexpression of NAT10 induced platinum drugs resistance in breast cancer cell

作　　者：齐攀 陈雅珂 崔瑞丽 衡瑞娟 徐升 和小颖 岳爱民 康江坤 李昊翰 朱永鑫 王聪 陈雨璐 胡姱 尹延彦[2] 宣立学 宋宇[2] Qi Pan;Chen Yake;Cui Ruili;Heng Ruijuan;Xu Sheng;He Xiaoying;Yue Aimin;Kang Jiangkun;Li Haohan;Zhu Yongxin;Wang Cong;Chen Yulu;Hu Kua;Yin Yanyan;Xuan Lixue;Song Yu(Department of Head and Neck Breast,Xinxiang Central Hospital,the Fourth Affiliated Hospital of Xinxiang Medical College,Xinxiang 453000,China;College of Pharmacology,Xinxiang Medical University,Xinxiang 453000,China;Department of Breast,National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital,Chinese Academy of Medical Sciences&Peking Union Medical College,Beijing 100021,China)

机构地区：[1]新乡市中心医院头颈乳腺科新乡医学院第四临床学院,新乡453000 [2]新乡医学院药学院,新乡453000 [3]国家癌症中心国家肿瘤临床医学研究中心中国医学科学院北京协和医学院肿瘤医院乳腺科,北京100021

出　　处：《中华肿瘤杂志》2022年第6期540-549,共10页Chinese Journal of Oncology

基　　金：河南省自然科学基金(202300410325);河南省医学科技攻关计划(LHGJ20200957、LHGJ20210911、LHGJ20191312);国家自然科学基金面上项目(8157111495);河南省高等学校重点科研项目(21A350007)。

摘　　要：目的探讨在乳腺癌细胞中高表达的N-乙酰转移酶10(NAT10)引起对铂类抗肿瘤药的耐药效应, 并揭示其潜在机制。方法实验分为野生型组(MCF-7野生型细胞未经任何处理)、NAT10过表达组(h-NAT10质粒转染至MCF-7细胞)和NAT10敲低组(sh-NAT10质粒转染至MCF-7细胞)。采用Transwell实验检测各组细胞的侵袭能力, 采用免疫共沉淀实验检测NAT10与多聚ADP核糖转移酶1(PARP1)的相互作用, 并检测过表达或敲低NAT10的表达对PARP1乙酰化水平和半衰期的影响, 通过免疫组化染色和免疫沉淀实验检测乙酰化的PARP1招募相关DNA损伤修复相关蛋白的情况, 采用流式细胞术检测细胞凋亡情况。结果 Transwell实验显示, NAT10过表达组细胞侵袭数为(483.00±46.90)个, NAT10敲低组细胞侵袭数为(469.00±40.50)个, 与MCF-7野生型细胞[(445.00±35.50)个]比较, 差异均无统计学意义(均P>0.05)。在10 μmol/L奥沙利铂作用下, NAT10过表达组细胞侵袭数为(502.00±45.60)个, NAT10敲低组细胞侵袭数为(105.00±20.50)个, 与野生型细胞[(219.00±31.50)个]比较, 差异均有统计学意义(均P<0.05)。在10 μmol/L奥沙利铂作用下, 与野生型细胞比较, NAT10过表达可以增强PARP1与NAT10的结合, 而使用NAT10抑制剂Remodelin则抑制了二者的相互结合。高表达NAT10诱导PARP1乙酰化后, PARP1与X射线修复交叉互补蛋白1(XRCC1)的结合增加, 敲低NAT10的表达后, 降低了PARP1和XRCC1的结合。高表达NAT10增强了PARP1与DNA连接酶3(LIG3)的结合, 而敲低NAT10的表达则会降低PARP1与LIG3的结合。在10 μmol/L奥沙利铂处理后的细胞中, NAT10过表达细胞中γH2AX表达水平为0.38±0.02, NAT10敲低细胞中γH2AX表达水平为1.36±0.15, 与野生型细胞(1.00±0.00)比较, 差异均有统计学意义(均P<0.05)。在10 μmol/L奥沙利铂处理后的细胞中, NAT10过表达组细胞凋亡率为(6.54±0.68)%, NAT10敲低组细胞凋亡率为(12.98±2.54)%, 与野生型细胞[(9.6Objective To observe the platinum drugs resistance effect of N-acetyltransferase 10(NAT10)overexpression in breast cancer cell line and elucidate the underlining mechanisms.Methods The experiment was divided into wild-type(MCF-7 wild-type cells without any treatment)group,NAT10 overexpression group(H-NAT10 plasmid transfected into MCF-7 cells)and NAT10 knockdown group(SH-NAT10 plasmid transfected into MCF-7 cells).The invasion was detected by Transwell array,the interaction between NAT10 and PARP1 was detected by co-immunoprecipitation.The impact of NAT10 overexpression or knockdown on the acetylation level of PARP1 and its half-life was also determined.Immunostaining and IP array were used to detect the recruitment of DNA damage repair protein by acetylated PARP1.Flow cytometry was used to detect the cell apoptosis.Results Transwell invasion assay showed that the number of cell invasion was 483.00±46.90 in the NAT10 overexpression group,469.00±40.50 in the NAT10 knockdown group,and 445.00±35.50 in the MCF-7 wild-type cells,and the differences were not statistically significant(P>0.05).In the presence of 10μmol/L oxaliplatin,the number of cell invasion was 502.00±45.60 in the NAT10 overexpression group and 105.00±20.50 in the NAT10 knockdown group,both statistically significant(P<0.05)compared with 219.00±31.50 in wild-type cells.In the presence of 10μmol/L oxaliplatin,NAT10 overexpression enhanced the binding of PARP1 to NAT10 compared with wild-type cells,whereas the use of the NAT10 inhibitor Remodelin inhibited the mutual binding of the two.Overexpression of NAT10 induced PARP1 acetylation followed by increased PARP1 binding to XRCC1,and knockdown of NAT10 expression reduced PARP1 binding to XRCC1.Overexpression of NAT10 enhanced PARP1 binding to LIG3,while knockdown of NAT10 expression decreased PARP1 binding to LIG3.In 10μmol/L oxaliplatin-treated cells,theγH2AX expression level was 0.38±0.02 in NAT10 overexpressing cells and 1.36±0.15 in NAT10 knockdown cells,both statistically significant(P<0.

关 键 词：乳腺肿瘤 N-乙酰转移酶10 多聚ADP核糖转移酶1 

分 类 号：R737.9[医药卫生—肿瘤]