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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
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