K562细胞及其耐药细胞株K562/A02细胞白血病耐药相关microRNA筛选

Screening for drug resistance related microRNAs in K562 and K562/A02 cell lines

目的 通过检测慢性粒细胞白血病急变细胞系K562及其阿霉素耐药株K562/A02的微小RNA（microRNA、miR）表达差异,探讨microRNA与白血病化疗耐药的关系.方法 MTT法检测K562/A02及其亲本细胞系K562的耐药性能;流式细胞术检测K562与K562/A02细胞的P-gp表达;运用microRNA芯片技术筛查K562与K562/A02细胞之间差异表达的microRNA,随后用实时荧光定量RT-PCR方法进一步证实.结果 阿霉素耐药株K562/A02相对于其亲本细胞系K562对阿霉素的耐药倍数为180倍;K562细胞P-gp的表达率为0.2%,K562/A02细胞P-gp的表达率为86%;microRNA芯片结果显示K562/A02与K562细胞之间有22种microRNA表达存在显著的差异（P〈0.01）,表达差异在2倍以上的有9种,其中miR-221、miR-155、miR-451在K562/A02细胞表达上调,而miR-98、miR-181a、let-7f、miR-424、let-7g和miR-563则表达下调.实时荧光定量RT-PCR进一步证实了上述结果,并显示miR-451、miR-155、miR-221、let-7f、miR-424在两种细胞中表达差异显著.结论 K562/A02与K562细胞存在microRNA表达差异,其中miR-451、miR-155和miR-221在K562/A02中表达显著上调,而let-7f、miR-424则显著下调,提示microRNA可能参与白血病耐药形成,差异表达的microRNA可能为逆转白血病耐药提供新的作用靶点.

Objective To explore the relationship between micmRNA and drug resistance in leukemia treatment by screening and identifying the microRNAs which ditierentially express in K562 cell line and its adriamycin resistant cells-K562/A02 cell line.Methods The drug resistance potency of K562/A02 cells was evaluated by M,TT assay.P-gp expression of K562 and K562/A02 cells were detected by flow cytometry （FCM）.The differentially expressed microRNAs in K562 and K562/A02 cells were analyzed by microarray technique and Real Time RT-PCR.Results The resistance to adriamycin （ADM） of K562/A02 cells was 180 fold greater than that of K562 cells.P-gp expression rate of K562 and K562/A02 cells was 0.2% and 86%,respectively.Twenty-two microRNAs expressed difierentially in K562 and K562/A02 cells（P〈0.01）.As compared to K562 cells,expressions of miR-221,miR-155 and miR-451 were up-regulated by more than two fold,while expression of miR-98,miR-181a,let-7f,let-7g,miR-424 and miR-563 down-regulated by more than two fold in K562/A02 cells.The results of real time RT-PCR were consistent with that of microarray.Of note,differential expressions of miR-451,miR-155.miR-221,let-7f and miR-424 were remarkable.Conclusion K562/A02 cells show a difierent microRNA expression pmfile as compared to its patental K562 cells,suggesting microRNAs including miR-221,miR-155,miR-451,let-7f and miR-424 may be involved in the mechanism of drug resistance in leukemia.These differentially expressed microRNAs provide potential novel targets for overcoming drag-resistance.