微小RNA-20a对人胶质瘤细胞替莫唑胺耐药性的影响

Effect of microRNA-20a on the temozolomide resistance of glioma cells

目的 检测微小RNA(miRNA,miR)-20a在替莫唑胺(TMZ)耐药的胶质瘤细胞株中的表达,探讨胶质瘤产生TMZ耐药的机制。方法 通过浓度梯度递增法建立人脑胶质瘤TMZ耐药模型细胞株,并命名U251/TMZ。实时荧光定量聚合酶链反应(Real-time PCR)检测细胞株中miR-20a的表达。Western blot技术检测细胞株中雷帕霉素靶蛋白(mTOR)信号及其下游信号的磷酸化。对U251/TMZ细胞株转染miR-20a inhibitor,并检测其细胞存活率、miR-20a表达及mTOR信号通路磷酸化。结果 与U251细胞株比较,U251/TMZ细胞株中miR-20a的表达水平明显升高(2.28±0.18比1.03±0.05,P<0.05),同时mTOR通路的磷酸化水平也升高。并且抑制miR-20a表达后,U251/TMZ细胞株的细胞存活率明显下降(P<0.01),mTOR信号通路的磷酸化水平下降。结论 miR-20a的表达可能与胶质瘤细胞对TMZ的耐药性相关,其可能通过mTOR信号通路发挥作用。

Objective To examine the expression of microRNA (miRNA, miR)-20a in the glioma cells with temozolomide (TMZ) resistance, and explore the potential molecular mechanisms of TMZ resistance.Methods TMZ resistant glioma cell line, called U251/TMZ cell line, was constructed by the stepwise revulsion with TMZ. The drug resistance and cell viability were detected by cell counting kit-8 (CCK-8) assay. The expression of miR-20a was detected by real-time quantitative polymerase chain reaction (Real-time PCR). The phosphorylated level of mammalian target of rapamycin (mTOR) signal was defined by Western blotting. The level of miR-20a expression in U251/TMZ cells was downregulated by miR-20a inhibitor transfection using lipidosome.Results As cmpared with U251 cells, the expression of miR-20a and the phosphorylated level of mTOR signal were significantly increased in U251/TMZ cells (2.28±0.18 vs. 1.03±0.05, P<0.05). When the expression level of miR-20a in U251/TMZ cells was downregulated by miR-20a inhibitor, the expression of miR-20a and the phosphorylated level of mTOR signal were decreased, and cell viability was decreased in U251/TMZ cells with 50 μmol/L TMZ existing (P<0.01).Conclusion The expression of miR-20a might be contributed to TMZ resistance of glioma cells, which may be related to mTOR signaling way.