基于成簇的规律性间隔的短回文重复序列关联的失活核酸酶9-脱氧核糖核酸甲基转移酶3A基因复合体的O-6-甲基鸟嘌呤-DNA甲基转移酶基因启动子区甲基化修饰对胶质瘤细胞替莫唑胺耐药性影响  

Effect of clustered,regularly interspaced,short palindromic repeat-deactivated nuclease 9 associated DNA methyltransferase 3A-based methylation modification of O^(6)-methylguanine-DNA methytransferase promoter region on temozolomide resistance in glioma cells

作　　者：陈勤[1] 孙炜[2] 张所军[2] 李朝曦[2] Chen Qin;Sun Wei;Zhang Suojun;Li Chaoxi(Department of Geriatrics,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430034,China;Department of Neurosurgery,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430034,China)

机构地区：[1]华中科技大学同济医学院附属同济医院老年病科,武汉430034 [2]华中科技大学同济医学院附属同济医院神经外科,武汉430034

出　　处：《中华实验外科杂志》2023年第7期1343-1346,共4页Chinese Journal of Experimental Surgery

基　　金：华中科技大学同济医学院附属同济医院归国启动基金(2022hgry009、2022hgry025)

摘　　要：目的探讨成簇的规律性间隔的短回文重复序列关联的失活核酸酶9-脱氧核糖核酸甲基转移酶3A基因复合体(CRISPR-dcas9-DNMT3A)介导的O-6-甲基鸟嘌呤-DNA甲基转移酶基因(MGMT)基因启动子区域DNA甲基化修饰对胶质瘤细胞替莫唑胺(TMZ)耐药性的影响。方法 CRISPR-dcas9-DNMT3A表达载体通过慢病毒包装感染TMZ耐药细胞系U251-R[U251(中国医学科院基础医学研究所)通过替莫唑胺高浓度反复间歇诱导法诱导产生], 验证其对MGMT启动子区域DNA甲基化程度, MGMT表达水平以及TMZ药物敏感性影响。结果慢病毒感染成功的U251-R细胞, MGMT启动子区域甲基化水平增高[44.7%(17/38)、63.2%(24/38)、77.1%(27/38)比7.9%(3/38), P<0.05], MGMT蛋白表达减少, 细胞对TMZ化疗的敏感性增加(P<0.05)。结论 CRISPR-dcas9-DNMT3A慢病毒感染的U251-R细胞可通过增加其MGMT基因启动子区域甲基化水平减少MGMT蛋白表达从而增加化疗敏感性。Objective To investigate the effect of clustered,regularly interspaced,short palin-dromic repeat-deactivated nuclease 9 associated DNA methyltransferase 3A(CRISPR-dcas9-DNMT3A)-me-diated DNA methylation modification in the promoter region of O^(6)-methylguanine-DNA methytransferase(MGMT)gene on temozolomide(TMZ)drug resistance in glioma cells.Methods CRISPR-dcas9-DNMT3A expression vector was infected into TMZ-resistant cell line produced from TMZ high concentration repeated intermittent induction(U251-R)by lentiviral packaging to statistically verify its effects on the de-gree of DNA methylation in the MGMT promoter region,the level of MGMT expression and TMZ drug sen-sitivity.Results Successfully lentiviral infected U251-R cells showed increased methylation levels in the MGMT promoter region[44.7%(17/38),63.2%(24/38),71.1%(27/38)vs.7.9%(3/38),P<0.05],reduced MGMT protein expression,and increased cellular sensitivity to TMZ chemotherapy(P<0.05).Conclusion CRISPR-dCas9-DNMT3A-expressing U251-R cells can reduce MGMT protein ex-pression by increasing the methylation level of their MGMT gene promoter region,thereby increasing chemotherapy sensitivity.

关 键 词：成簇的规律性间隔的短回文重复序列关联的失活核酸酶9-脱氧核糖核酸甲基转移酶3A基因复合体 DNA甲基化 O-6-甲基鸟嘌呤-DNA甲基转移酶 胶质瘤 替莫唑胺 

分 类 号：R739.41[医药卫生—肿瘤]