摘要
目的探讨O6-甲基鸟嘌呤-DNA-甲基转移酶(MGMT)反义RNA对人脑胶质瘤细胞MGMT表达的调节作用。方法将分别为针对MGMTmRNA5’端和全长序列的反义表达载体pLaMT5SN和pLaMTSN,分别导入U251/BCNU耐药细胞,经G418筛选产生稳定抗性克隆aMT5U和aMTU;分别将正义表达载体pLMTSN和空载体pLXSN导入U251/BCNU耐药细胞,经G418筛选产生稳定抗性克隆MTU和XU作为对照。观察转染后的U251/BCNU细胞对MGMT表达的调节作用及U251/BCNU细胞对BCNU敏感性的影响。结果经RT-PCR检测显示,pLaMT5SN和pLaMTSN可在一定程度上降低MGMTmRNA表达水平;MTT检测显示,aMT5U和aMTU细胞对BCNU的敏感性分别较未转染细胞提高7倍和2倍,表明pLaMT5SN和pLaMTSN可在一定程度上提高U251/BCNU细胞对BCNU的敏感性。结论MGMT反义RNA能够在一定程度上抑制脑胶质瘤细胞MGMT的表达水平,提高肿瘤细胞对亚硝基脲类药物的敏感性。
Objective To study the modulation effects of O6-methylguanine-DNA-methyltransferase (MGMT) antisense RNA on MGMT expression in human glioma. Methods The pLaMT5SN and pLaMTSN, antisense expression carrier targeted to 5'terminal and whole length sequence of MGMT mRNA, were transdused into U251/BCNU resistant cells respectively and the stable resistanat clones aMT5U and aMTU were screened by G418. Then the positive expression carrier pLMTSN and unloaded carrier pLXSN were transduced respectively into U251/BCNU resistant cells, the stable resistant clones MTU and XU were screened out and set for control. The modulation effects of transinfected U251/BCNU cells on MGMT expression and U251/BCNU cell sensitivity to BCNU were studied. Results It was showed by RT-PCR analysis that plaMT5SN and plaMTSN could down-regulate the MGMT mRNA expression to some extent. MTT assay demonstrated that the sensitivity of aMT5U and aMTU cells to BCNU were 7- and 2-fold higher than that of non-transinfected U251/BCNU cells, which suggested that pLaMT5SN and pLaMTSN could enhance the sensitivity of U251/BCNU cells to BCNU. Conclusion MGMT antisense RNA could down-regulate the expression of MGMT in glioma, and enhance the sensitivity of tumor cells to nitrosoureas. Thus the application of MGMT may provide an effective way to reverse the drug resistance in glioma cells, and open up a good prospects for clinical practice in the future.
基金
国家自然科学基金资助项目(30070272)
