miR-370调控MGMT增强替莫唑胺对胶质瘤细胞化疗敏感性的实验研究

Mechanism of microRNA-370 ontemozolomide sensitivityvia regulating MGMT expressionin glioma cells

目的探讨microRNA-370(miR-370)通过调控O^(6)-甲基鸟嘌呤-DNA甲基转移酶(MGMT)增强替莫唑胺(TMZ)对胶质瘤化疗敏感性的机制研究。方法采用TMZ浓度梯度递增法体外建立U87/TMZR耐药细胞株,分别转染阴性空质粒和miR-370 mimics作为miR-NC组和miR-370 mimics组,另将未经处理的U87/TMZR细胞作为对照组。采用实时荧光定量PCR(qPCR)和Western blotting检测miR-370和MGMT表达。TargetScan在线预测和双荧光素酶报告实验分析miR-370靶基因。采用MTT法和克隆形成实验检测各组细胞对替莫唑胺的敏感性。结果体外成功建立U87/TMZR耐药细胞株,U87/TMZR细胞miR-370表达量较U87细胞降低(0.52±0.08 vs.1.00±0.05,P<0.05);miR-370 mimics组miR-370表达量为5.65±0.77,高于对照组和miR-NC组(P<0.05);而miR-370 mimics组细胞MGMT蛋白表达量低于对照组(0.28±0.03 vs.0.63±0.05,P<0.05)。经TargetScan在线预测和双荧光素酶报告实验结果显示,MGMT是miR-370的靶基因。经不同浓度TMZ作用后,miR-370 mimics组U87/TMZR细胞IC 50为(72.73±5.19)μmol/L,克隆形成数为179±35,均低于对照组和miR-NC组(P<0.05)。结论TMZ耐药肿瘤细胞中MGMT表达量增加,同时miR-370表达量降低。上调miR-370表达可通过抑制MGMT转录提高U87/TMZR耐药细胞对TMZ的敏感性。

Objective To discuss the effect and mechanism of microRNA-370(miR-370)on temozolomide(TMZ)sensitivityvia regulating O 6-methylguanine-DNA methyltransferase(MGMT)expressionin glioma cells.Methods U87 cells were cultured invitro and TMZ-resistance in U87 cells was initiated by the stepwise revulsion with TMZ.U87/TMZR cells were transfected by negative vector or miR-370 mimics as miR-NC group and miR-370 mimics group.And then U87/TMZR cells without any treatment were as control group.qPCR and Western blotting methods were used for the detection of miR-370 and MGMT expressions.TargetScan online forecasting software and double luciferase reporter gene assay were used to analyze the target gene of miR-370.MTT assay and colony formation method were used to detect the sensitivity of U87/TMZR cells to temozolomide.Results U87/TMZR-resistance cells were successfully built by the stepwise revulsion with TMZ.Compared with the U87 cells,miR-370 expressions in U87/TMZR cells were lower(0.52±0.08 vs.1.00±0.05,P<0.05).The expression of miR-370 mimics gorup was 5.65±0.77m which was higher than taht of control group and miR-NC group(P<0.05).The expression of MGMT protein in miR-370 mimics group was lower than that in control group(0.28±0.03 vs.0.63±0.05,P<0.05).TargetScan online prediction and double luciferase reporter gene test showed that MGMT was the target gene of miR-370.After treatment by different concentrations of TMZ,IC 50 of U87/TMZR cells was(72.73±5.19)μmol/L,while the colonies formation number was 179±35,both lower than that of control group and miR-NC group(P<0.05).Conclusion The expression of miR-370 in TMZ resistant cells lower,while the expression of MGMT is higher.Up-regulation of miR-370 in U87/TMZR cells can increase the sensitivity of glioma cells to TMZ by inhibiting MGMT expression.