沉默 UVRAG 对白血病K562/ADM 细胞自噬及耐药性的影响

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 LipofectamineTM2000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 siR-NA 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。

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

Objective To investigate the role of UV radiation resistance-associated gene(UVRAG)on autoph-agy 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 LipofectamineTM2000 mediated siRNA. In different ce1ls groups,the chemo-therapy 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 meas-ured 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 ex-pression 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 si-UVRAG-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 re-duced 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 de-creased and the degradation of P62 was reduced after inhibition expression of UVRAG gene(P all ﹤ 0. 05). Con-clusion 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.