不同化疗药物对乳腺癌细胞MGMT表达水平的影响及其药物敏感性分析  

Analysis of the Effects of Different Chemotherapeutic Agents on the Expression Level of MGMT in Breast Cancer Cells and Their Drug Sensitivity

作　　者：邓美仪 任冰洁 杨媛媛 史慧玲 石凤灵 黄曼 王娟 陶敏[3] 梁容瑞 DENG Meiyi;REN Bingjie;YANG Yuanyuan(The Fourth Affiliated Hospital of Soochow University,Soochow,215006)

机构地区：[1]苏州大学附属第四医院,215031 [2]河南省南阳第二人民医院,215006 [3]苏州大学附属第一医院,215031

出　　处：《实用癌症杂志》2024年第11期1768-1773,共6页The Practical Journal of Cancer

基　　金：江苏省333人才工程资助项目[编号:(2022)3-2-113号]。

摘　　要：目的探讨不同化疗药物对O 6-甲基鸟嘌呤-DNA-甲基转移酶(O 6-methyl-guanine DNA methyltransferase,MGMT)表达水平的影响及其药物敏感性。方法采用Western Blotting检测环磷酰胺作用后MCF-7和MDA-MB-231细胞中MGMT蛋白水平变化;采用免疫荧光检测MGMT表达水平及其在乳腺癌细胞中内的定位情况;构建含MGMT启动子片段的荧光素酶报告载体转染入乳腺癌细胞MCF-7和MDA-MB-231,应用荧光素酶报告基因检测试剂盒检测经不同化疗药物(环磷酰胺、紫杉醇、吉西他滨)作用24 h后MGMT的转录活性;通过RT-qPCR检测化疗药物作用后MCF-7和MDA-MB-231细胞内MGMT mRNA水平变化;采用CCK8实验检测MCF-7和MDA-MB-231细胞对上述化疗药物的敏感性。结果环磷酰胺作用后两种细胞的MGMT蛋白水平均下降;荧光素酶报告的实验结果显示与对照组相比,经环磷酰胺处理后检测到MDA-MB-231细胞的发光强度增强,MCF-7细胞的发光强度减弱,具有统计学差异;RT-qPCR结果显示环磷酰胺作用后,MDA-MB-231细胞的MGMT mRNA水平升高,而MCF-7细胞的MGMT mRNA水平下降;CCK8实验结果显示在化疗药物作用下,MDA-MB-231细胞比MCF-7细胞的IC 50值高。结论在三阴性乳腺癌MDA-MB-231细胞中环磷酰胺造成肿瘤细胞耐药与促进MGMT的转录上调密切相关。Objective To investigate the effects of different chemotherapeutic drugs on the expression level of O 6-methyl-guanine DNA methyltransferase(MGMT)and its drug sensitivity.Methods Western Blotting was used to detect the changes of MGMT protein level in MCF-7 and MDA-MB-231 cells after cyclophosphamide action;immunofluorescence was used to detect the expression level of MGMT and its localization in breast cancer cells;luciferase reporter vectors containing MGMT promoter fragments were constructed and transfected into MCF-7 and MDA-MB-231 breast cancer cells.MB-231,and the luciferase reporter gene assay kit was applied to detect the transcriptional activity of MGMT after 24 h of different chemotherapeutic drugs(cyclophosphamide,paclitaxel,gemcitabine);the changes of MGMT mRNA levels in MCF-7 and MDA-MB-231 cells after chemotherapeutic drugs were detected by RT-qPCR;the CCK8 assay was used to detect the internal localization of MGMT in MCF-7 and MDA-MB-231 cells;the MGMT promoter fragments were constructed and transfected into breast cancer cells;the MGMT promoter fragments were constructed and transfected into breast cancer cells.CCK8 assay was used to detect the sensitivity of MCF-7 and MDA-MB-231 cells to the above chemotherapeutic drugs.Results The MGMT protein levels of both cells decreased after cyclophosphamide action;the results of luciferase-reported experiments showed statistically significant differences in the enhancement of luminescence intensity of MDA-MB-231 cells and the weakening of luminescence intensity of MCF-7 cells detected after cyclophosphamide treatment when compared with the control group;the results of RT-qPCR showed that the MGMT mRNA levels of MDA-MB-231 cells were significantly higher in the MGMT mRNA levels of MCF-7 cells and MCF-7 cells after cyclophosphamide treatment,compared with the control group.MGMT mRNA level of MDA-MB-231 cells increased while that of MCF-7 cells decreased;CCK8 assay results showed that under the effect of chemotherapeutic drugs,MDA-MB-231 cells had higher IC 5

关 键 词：乳腺癌 MGMT 环磷酰胺 化疗耐药 

分 类 号：R737.9[医药卫生—肿瘤]