RNA 干扰沉默同源异型基因A5 逆转K562/ADM 细胞耐药性的研究

Effect of RNA silencing targeting HOXA5 gene on drug resistance of leukemia cell line K562 / ADM

摘要:

目的 研究RNA 干扰（RNAi）技术沉默同源异型基因A5（HOXA 5）对白血病耐阿霉素细胞株K562/ADM 细胞耐药性的影响并探讨其机制。方法 Western blot 检测各组细胞中HOXA5、p38 和p-p38 的蛋白表达，qRT-PCR 检测HOXA5、p38 的mRNA 表达；CCK8 检测各组细胞对阿霉素的敏感性变化；流式细胞术（FCM）检测各组细胞的凋亡及周期变化。结果 K562/ADM 细胞中HOXA 5 基因的 表达高于K562 细胞，且其耐药性是K562 细胞的4.94 倍。转染后实验组K562/ADM 细胞中HOXA 5 基因的表达受抑制，而实验组细胞的IC50 较对照组降低2.55 倍。同时，实验组细胞中p38 的mRNA 及p-p38的蛋白表达高于对照组。与对照组比较，实验组的细胞周期G0/G1 期增高，S 期降低。经ADM（2.5μg/ml）诱导后，实验组细胞凋亡率高于对照组。结论 RNAi 沉默HOXA5 能在一定程度上逆转白血病耐药，其机制可能与p38MAPK 信号转导通路的激活有关。

Abstract:

Objective To investigate the effect of RNA silencing targeting homeotic genes A5 (HOXA5) on drug resistance of leukemia adriamycin-resistant cell strain K562/ADM. Methods Cells were divided into siRNA group from which cells received silencing RNA of HOXA5 and control group from which cells did receive any treatments. Western blot and qRT-PCR were performed to identify expression levels of HOXA5, p38 and p-p38. Drug resistance to Adriamycin was measured through CCK8 assay. Apoptosis and cellular cycle status were determined by FCM technology. Results Expression of HOXA5 in K562/ADM cells was up-regulated significantly compared with K562 cells. CCK8 assay, which suggested that dramatic increase of drug resistance to Adriamycin (4.94 fold) was witnessed in K562/ADM in comparison with that in K562 cells. Transfection of siRNA obviously deceased expression of HOXA5 in K562/ADM cells, thus IC50 of Adriamycin was deducted (2.55 fold) compared with that in control group. Both protein and mRNA levels of p-p38 in siRNA group were increased dramatically when compared with control group. Compared with control group, number of cells in G0/ G1 stage increased and decreased in S stage in siRNA group. Cellular apoptosis rate induced by ADM (2.5 μg/ml) in siRNA group increased significantly compared with control group. Conclusions siRNA silencing of HOXA5 reverses leukemia drug resistance to Adriamycin through activation of p38-MAPK signal pathway.