LncRNA XIST调控miR-200b/ZEB1轴参与儿童急性髓系白血病阿柔比星耐药的机制研究

Study on mechanism of LncRNA XIST participating in arubicin resistance in children with acute myeloid leukemia by regulating miR-200b/ZEB1 axis

目的:研究LncRNA XIST调控miR-200b/ZEB1轴参与儿童急性髓系白血病(AML)阿柔比星耐药的机制。方法:以阿柔比星浓度递增法诱导建立人AML阿柔比星耐药细胞株K562/ADM,检测人AML细胞K562及K562/ADM细胞中LncRNA XIST表达。体外培养K562/ADM细胞,随机分为对照组、LncRNA XIST inhibitor组、阿柔比星+LncRNA XIST inhibitor阴性对照组、阿柔比星+LncRNA XIST inhibitor组,分组转染并以药物处理后,检测各组细胞增殖凋亡情况;检测各组细胞LncRNA XIST、miR-200b、ZEB1、MDR1 mRNA表达水平和ZEB1、耐药蛋白P-gp蛋白表达水平。结果:相比K562细胞,LncRNA XIST在K562/ADM细胞中表达明显升高(P<0.05)。与对照组相比,阿柔比星+LncRNA XIST inhibitor组细胞存活率明显降低(P<0.05),凋亡率明显升高(P<0.05);LncRNA XIST inhibitor组、阿柔比星+LncRNA XIST inhibitor阴性对照组上述指标无明显改变(P>0.05)。与LncRNA XIST inhibitor组相比,阿柔比星+LncRNA XIST inhibitor组细胞存活率明显降低(P<0.05),凋亡率明显升高(P<0.05)。与对照组、阿柔比星+LncRNA XIST inhibitor阴性对照组分别相比,阿柔比星+LncRNA XIST inhibitor组、LncRNA XIST inhibitor组LncRNA XIST、ZEB1及MDR1 mRNA表达水平、ZEB1及P-gp蛋白表达水平均明显降低(P<0.05),miR-200b表达水平均明显升高(P<0.05);阿柔比星+LncRNA XIST inhibitor阴性对照组细胞上述各指标无明显改变(P>0.05)。结论:LncRNA XIST可使儿童AML细胞产生耐药性,下调其表达可促进miR-200b表达,降低ZEB1表达,抑制耐药基因MDR1、P-gp表达,提高阿柔比星对AML细胞的杀伤。

Objective:To study the mechanism of LncRNA XIST participating in arubicin resistance in children with acute myeloid leukemia(AML)by regulating miR-200b/ZEB1 axis.Methods:Human AML arubicin-resistant cell line K562/ADM was induced by the method of increasing the concentration of arubicin.Expressions of LncRNA XIST in human AML cells K562 and K562/ADM cells were detected.K562/ADM cells were cultured in vitro and randomly divided into control group,LncRNA XIST inhibitor group,arubicin+LncRNA XIST inhibitor negative control group,and arubicin+LncRNA XIST inhibitor group.After transfection in groups and treatment with drugs,cell proliferation and apoptosis were detected in each group;expression levels of LncRNA XIST,miR-200b,ZEB1,MDR1 mRNA and expression levels of ZEB1 and drug resistance protein P-gp in each group of cells were detected.Results:Compared with K562 cells,the expression of LncRNA XIST was significantly higher in K562/ADM cells(P<0.05).Compared with control group,the cell survival rate of the arubicin+LncRNA XIST inhibitor group was significantly reduced(P<0.05),the apoptosis rate was significantly increased(P<0.05);the above indexes of cells in the LncRNA XIST inhibitor group and the arubicin+LncRNA XIST inhibitor negative control group did not change significantly(P>0.05).Compared with LncRNA XIST inhibitor group,the cell survival rate of the arubicin+LncRNA XIST inhibitor group was significantly reduced(P<0.05),the apoptosis rate was significantly increased(P<0.05).Compared with control group and the arubicin+LncRNA XIST negative control group,mRNA expression levels of LncRNA XIST,ZEB1 and MDR1,and tprotein expression levels of ZEB1 and P-gp in the arubicin+LncRNA XIST inhibitor group and LncRNA XIST inhibitor group were significantly reduced(P<0.05),expression level of miR-200b was significantly increased(P<0.05);there were no significant changes in the above indexes in the arubicin+LncRNA XIST inhibitor negative control group(P>0.05).Conclusion:LncRNA XIST can promote drug resistance in children’s AML cells,down-regulate its expression can promote the expression of miR-200b,reduce the expression of ZEB1,inhibit the expression of drug resistance genes MDR1 and P-gp,and increase the lethality of arubicin on AML cells.