阿霉素、顺铂提升三阴性乳腺癌细胞RNA m6A甲基化水平及其机制探讨

Doxorubicin and cisplatin increase the level of RNA m6A methylation in triple-negative breast cancer cells and the mechanism

目的:探讨DNA损伤类化疗药物阿霉素和顺铂对三阴性乳腺癌细胞MDA-MB-231的RNA m6A甲基化水平的影响及其相关机制。方法:利用MTT法检测阿霉素和顺铂对MDA-MB-231细胞存活率的影响;通过Western blot检测γ-H2AX蛋白表达;应用Dot blot检测细胞RNA m6A甲基化水平;通过RT-qPCR和Western blot分别检测RNA m6A甲基化酶METTL3、METTL14、METTL16和RBM15以及去甲基化酶ALKBH5和FTO的mRNA和蛋白水平。结果:阿霉素和顺铂均能显著降低MDA-MB-231细胞存活率,促进细胞的DNA损伤,同时提升细胞RNA m6A甲基化水平;阿霉素和顺铂处理后细胞RNA m6A甲基化酶mRNA和蛋白表达水平无明显变化,而去甲基化酶ALKBH5的表达显著减少。结论:诱发细胞DNA损伤的化疗药物阿霉素和顺铂可提升三阴性乳腺癌细胞RNA m6A甲基化水平,其机制与m6A去甲基化酶ALKBH5的降低有关。

Objective:To explore the effects of DNA-damage chemotherapy drugs doxorubicin and cisplatin on the RNA m6A levels of triple-negative breast cancer cells MDA-MB-231 and the mechanism.Methods:The cell survival rate of MDA-MB-231 cells treated with doxorubicin and cisplatin was determined by MTT method.Western blot was used to detect the expression ofγ-H2AX.Dot blot was used to detect the change of RNA m6A methylation level.RT-qPCR and Western blot were used to compare the transcriptional and translational level of RNA m6A methyltransferases METTL3,METTL14,METTL16 and RBM15,as well as demethylases ALKBH5 and FTO respectively.Results:The treatment with doxorubicin and cisplatin could significantly decrease the cell survival rate,elevate the degree of DNA-damage,and meanwhile,increase the level of RNA m6A methylation.At transcriptional and translational level,after doxorubicin and cisplatin treatment,the changes of m6A methylase expression were undetectable,while the expressions of m6A demethylase ALKBH5 was decreased significantly.Conclusion:The chemotherapeutic drugs doxorubicin and cisplatin increase the level of RNA m6A methylation in triple-negative breast cancer cells,which may be caused by the decrease of RNA m6A demethylase ALKBH5.