沉默UVRAG对白血病K562/ADM细胞自噬及耐药性的影响

Role of UVRAG on Autophagy and Drug Resistance in the K562/ADM Cells

目的探讨沉默紫外线抵抗相关基因(UVRAG)对人白血病K562/ADM细胞自噬及耐药性的影响。方法 Western Blotting检测UVRAG蛋白在K562及K562/ADM细胞中表达差异。特异性干扰UVRAG基因的UVRAG siRNA及Scramble siRNA在Lipofectamine TM2000介导下转染K562/ADM细胞,CCK-8法、MDC荧光染色及Western Blotting分别检测UVRAG siRNA转染前后K562/ADM细胞耐药性、自噬水平以及P-糖蛋白(P-gp)表达的变化。结果 Western Blotting检测显示K562/ADM细胞中UVRAG蛋白表达明显高于K562细胞(P<0.05);与K562/ADM组及Scramble siRNA转染组相比,UVRAG siRNA转染组UVRAG蛋白表达显著下降(P<0.05),以48 h效果最佳,提示UVRAG siRNA能高效沉默K562/ADM细胞UVRAG;CCK-8法显示与K562/ADM组及Scramble siRNA转染组相比,UVRAGsiRNA组对阿霉素敏感性显著增高,IC50值明显下降(P<0.05);MDC染色荧光显微镜观察到UVRAG siRNA转染后K562/ADM细胞胞浆中自噬泡明显减少;Western Blotting显示K562/ADM细胞中Beclin-1、P-gp表达及P62降解明显高于K562细胞,与K562/ADM细胞及Scramble siRNA转染组相比,UVRAG siRNA转染组Beclin-1、P-gp表达及P62降解显著降低(P均<0.05)。结论 UVRAG蛋白在K562/ADM细胞中高表达,与白血病MDR密切相关;UVRAG siRNA下调UVRAG表达可降低K562/ADM细胞耐药性,其机制可能与降低自噬水平及下调P-gp表达有关。

Objective To investigate the role of UV radiation resistance-associated gene( UVRAG) on autophagy and drugs sensitivity by down-regulation of the expression UVRAG in K562/ADM cells. Methods The UVRAG protein levels were measured by Western Blotting in the K562 cells and the K562/ADM cells. Specificity siRNA and Scramble siRNA without any gene homology were designed and synthesised aiming at UVRAG gene,K562/ADM cells were transfected by Lipofectamine TM2000 mediated siRNA. In different ce1 ls groups,the chemotherapy sensitivity of adriamycin( ADM) were determined by CCK-8 kit,the autophagic vacuoles were observed by MDC staining and fluorescence microscope,the autophagy-related protein and P-gp protein expression were measured by Western Blotting. Results The expression of UVRAG in K562/ADM cells was higher than K562 cells( P < 0.05). In comparison with K562/ADM cells group and NC-K562/ADM cells group,the UVRAG protein expression level of K562/ADM cells group was obviously decreased after transfection with UVRAG siRNA( P < 0. 05),with 48 h working best; In comparison with K562/ADM cells group and NC-K562/ADM cells group,the sensibility of siUVRAG-K562/ADM cells group to ADM was increased while IC50 obviously decreased( P < 0. 05). Autophagic vacuoles in endochylema determined by MDC staining and fluorescence microscope were significantly reduced after UVRAG siRNA transfection. Further findings showed that the expression levels of Beclin-1 and P-gp and the degradation of P62 were higher than K562 cells,the expression of Beclin-1 and P-gp were significantly decreased and the degradation of P62 was reduced after inhibition expression of UVRAG gene( P all < 0. 05). Conclusion The expression of UVRAG gene in K562/ADM cells was higher than K562 cells,suggesting that UVRAG may be closely involved in the multiple drug resistance in K562/ADM cells. Down-regulation of the expression UVRAG in K562/ADM cells can reduce drug resistance to chemotherapeutic adriamycin. It may be related to the down-regulation of autophagy and the expression of P-gp.