靶向抑制miRNA-21提高白血病K562细胞对阿糖胞苷的敏感性

Enhanced Chemosensitivty of Leukemic Cells to Cytarabine by Targeted Suppression of miRNA-21

为研究以miRNA-21为靶标的反义核酸(AMO-miR-21)提高白血病K562细胞对阿糖胞苷(Ara-C)的敏感性及可能的作用机制,在LipofectamineTM2000介导下,将化学合成的AMO-miR-21转染K562细胞,四甲基偶氮唑蓝(MTT)法检测单独使用AMO-miR-21、Ara-C以及两者联合使用对细胞增殖抑制作用,并计算抑制率和IC50;流式细胞仪检测细胞凋亡;实时定量PCR(Real-time PCR)检测miRNA-21及其候选靶基因Pdcd4 mRNA的表达水平;免疫印迹(Western blot)检测Pdcd4蛋白的表达水平.结果显示Ara-C与AMO-miR-21联合使用后,IC50从1.95μmol/L降低到0.84μmol/L,敏感性提高到单用Ara-C的2.3倍.AMO-miR-21联合Ara-C组细胞凋亡明显增加(P<0.05).AMO-miR-21显著抑制K562细胞中miRNA-21的表达水平(P<0.05),同时Pdcd4的表达明显增加(P<0.05).提示AMO-miR-21可以提高K562细胞对Ara-C的敏感性,其作用机制与靶向抑制miRNA-21,进而诱导细胞凋亡有关.

Our goal is to investigate the influences C chemosensitivity in human leukemic K562 cells, of anti-miRNA-21 oligonucleotide （AMO-miR-21） on Arawhich were incubated with different concentration of Ara- C alone or in combination with AMO-miR-21. The growth-inhibitory potencies were measured by MTT assay. The inhibition rate and IC5o was calculated. The cell apoptosis was assessed by flow cytometry. The expression of miRNA-21 and its potential target gene Pdcd4 was detected by real-time PCR and Western blot in K562 cells. Transfection of K562 cells with AMO-miR-21 enhanced the chemosensitivity of Ara-C by up to 2.3-fold and promoted Ara-C-induced cell apoptosis. Suppressing the cellular levels of miRNA-21 signifi- cantly upregulated the expression of Pdcd4. The results support a substantial role for AMO-miR-21 that enhance Ara-C sensitivity in K562 cells. The mechanism appears to be related to anti-miRNA-21-induced apoptosis, suggesting a novel potential approach to treatment of leukemia.