DNMT1通过miRNA-148b-3p调控脑胶质瘤U87细胞替莫唑胺耐药性的作用机制

Effect of DNMT1 on temozolomide resistance via regulating miRNA-148b-3p expression in glioma U87 cells

目的探讨DNA甲基转移酶1(DNMT1)通过miRNA-148b-3p调控人脑胶质瘤U87细胞对替莫唑胺(TMZ)耐药性的作用机制。方法采用TMZ长期浓度梯度递增法体外建立U87/TMZ耐药细胞系,并检测U87细胞和U87/TMZ耐药细胞多药耐药相关基因1(MDR1)和B细胞淋巴瘤/白血病-2(Bcl-2)基因的相对表达量,采用流式细胞仪检测细胞凋亡情况。将DNMT1抑制剂5-氮杂-2’-脱氧胞苷(5-Aza-CdR)作用于U87细胞,比较作用前后U87细胞miRNA-148b-3p的相对表达量。将pcDNA-DNMT1过表达载体转染至U87/TMZ耐药细胞系,采用甲基化特异性聚合酶链反应(MSP)法检测U87/TMZ耐药细胞miRNA-148b-3p的相对表达量,并采用噻唑蓝(MTT)法检测细胞增殖能力。结果U87/TMZ耐药细胞中MDR1、Bcl-2mRNA的相对表达量均明显高于U87细胞,细胞凋亡率也明显低于U87细胞,差异均有统计学意义(P﹤0.01)。U87细胞miRNA-148b-3p的相对表达量明显低于U87/TMZ耐药细胞,DNMT1mRNA的相对表达量明显高于U87/TMZ耐药细胞,差异均有统计学意义(P﹤0.01)。5-Aza-CdR处理后的U87细胞miRNA-148b-3p表达量明显高于未经5-Aza-CdR处理的U87细胞(P﹤0.01)。pcDNA-DNMT1组U87/TMZ细胞中miRNA-148b-3p启动子区甲基化水平高于空白对照组和阴性对照组细胞,差异均有统计学意义(P﹤0.05)。不同浓度TMZ溶液处理的pcDNA-DNMT1组U87/TMZ细胞的增殖抑制率,均明显高于空白对照组和阴性对照组细胞,差异均有统计学意义(P﹤0.01)。结论DNMT1可通过调节miRNA-148b-3p启动子区甲基化水平负性调控miRNA-148b-3p的相对表达量,从而控制人脑胶质瘤细胞对TMZ的耐药性。

Objective To discuss the effect of DNA methyltransferase 1(DNMT1)on temozolomide(TMZ)resistance via regulating miRNA-148b-3p expression in glioma cells.Method U87 cells were cultured in vitro and TMZ-resistance in U87 cells was induced by the stepwise concentration gradient with TMZ.The relative expression of multidrug resistance-related gene 1(MDR1)and B cell lymphoma/leukemia-2(Bcl-2)mRNA levels of U87/TMZ-resistance cells were detected.The apoptosis of U87/TMZ-resistance cells was detected by flow cytometry.U87 cells were treated with DNMT1 inhibitor 5-Aza-2’-deoxycytidine(5-Aza-CdR),and the relative expression of miRNA-148b-3p before and after treatment in U87 cells were compared.U87/TMZ-resistance cells were transfected with pcDNA-DNMT1 overexpression vector.The relative expression of miRNA-148b-3p in U87/TMZ-resistance cells were detected by methylation specific polymerase chain reaction(MSP).Cell proliferation was determined using methylthiazolyldiphenyl tetrazolium.Result The relative expression of MDR1 mRNA,Bcl-2 mRNA in U87/TMZ cells were significantly higher compared to U87 cells(P<0.01),and the apoptosis rates were lower than that of U87 cells(P<0.01).The relative expression of miRNA-148b-3p in U87 cells was significantly lower compared with U87/TMZ-resistance cells,while the relative expression of DNMT1 mRNA was higher instead(P<0.01).After treatment of 5-Aza-CdR,the expressions of miRNA-148b-3p in U87 cells was evidently higher than that in U87 cells not treated with 5-Aza-CdR(P<0.01).The level of miRNA-148b-3p promoter methylation in U87/TMZ-resistance cells transfected with pcDNA-DNMT1 was significantly higher compared to blank control group and negative control group(P<0.05).The proliferation inhibition rates of U87/TMZ-resistance cells transfected with pcDNA-DNMT1 treated with TMZ of different concentration were all higher than that in control group and negative control group(P<0.01).Conclusion DNMT1 down-regulates miRNA-148b-3p expression by regulating miRNA-148b-3p promoter methylation levels,thus affects the TMZ resistance of glioma.