WT1基因表达下调对K562/A02细胞阿霉素敏感性的影响

目的 探讨WT1基因表达下调对人红白血病耐药细胞系K562/A02阿霉素敏感性的影响.方法 将WT1mRNA的短发夹RNA(short hair RNA,shRNA)构建至真核表达载体后转染K562/A02细胞,流式细胞术检测转染效率;荧光定量RT-PCR和Western blot法分析WT1基因在转染前后表达的差异;pWT1shRNA转染K562/A02细胞48 h并经阿霉素作用后,MTT法检测各组细胞阿霉素IC50值;流式细胞术检测细胞阿霉素累积量及细胞凋亡率.结果 与未转染对照组及空载体组相比,转染WT1shRNA质粒的K562/A02细胞WT1mRNA和蛋白水平均明显降低;经阿霉素诱导24 h后,其对阿霉素的敏感性明显增高,相对逆转率为71.5%,细胞内阿霉素的累积量较各对照组明显增高(P值均<0.05),细胞凋亡率明显升高(P<0.05).结论 靶向WT1shRNA干扰质粒可有效抑制K562/A02细胞WT1基因表达,增强K562/A02细胞对阿霉素的敏感性.

The effects of WT1 gene down regulation on the sensitivity of K562/A02 cells to adrimycin

Objective To investigate the effects of WT1 gene down regulation on the sensitivity of K562/A02 cells to adrimycin (ADM). Methods Short hair RNA (shRNA) plasmid vector targeting WT1 gene with enhanced green fluorescent protein (EGFP) was transfected into K562/A02 cells. Transfection effi-ciency was detected and transfected cells were sorted by flow cytometery. Expression of WT1 gene was detec-ted by real time fluorescence RT-PCR and Western blot. The IC50 of ADM on K562/A02 cells was measured by MTT assay, Intracellular ADM accumulation and ADM-induced apoptosis of K562/A02 cells were detected by flow cytometry. Results 48 h after transfection, the transfection efficiency was (68.2±4.5 ) %. Both the mRNA and protein of WT1 were significantly decreased in K562/A02 cells transfected with WT1 shRNA plas-mid than in control. After 24 h ADM induction, the relative reverse rate of sensitivity of K562/A02 cells to ADM was 71.5%. The intracellular accumulation of ADM was greatly increased (P<0.05) and ADM-in-duced apoptosis rate elevated from 10.4% to 67.3% (P<0.05). Conclusion shRNA targeting WT1 gene can efficiently inhibit the WT1 expression in K562/A02 cells and enhance the drug sensitivity of K562 cells to ADM.