敲低TRAF6对白血病K562细胞增殖和凋亡的影响及其分子机制  

The Effect of TRAF6 Knockdown on Proliferation and Apoptosis of K562 Leukemia Cells and Its Molecular Mechanism

作　　者：张帅帅[1] 鲜敬荣[1] 邹琴[1] 全静[1] 金红君[1] 高芃[1] 叶晟[1] 张伶[1] 

机构地区：[1]重庆医科大学检验医学院临床检验诊断学教育部重点实验室重庆市重点实验室,重庆400016

出　　处：《中国细胞生物学学报》2015年第12期1659-1665,共7页Chinese Journal of Cell Biology

基　　金：国家自然科学基金面上项目(批准号:81271913);重庆市人力资源和社会保障局留学人员科技活动择优资助项目(批准号:2013009)资助的课题~~

摘　　要：该文探讨了干扰肿瘤坏死因子受体相关因子6(tumor necrosis factor receptor-associated factor 6,TRAF6)表达对人白血病K562细胞增殖、凋亡的影响及其分子机制。将靶向TRAF6基因的sh RNA慢病毒载体感染K562细胞,利用荧光显微镜观察感染效率;Western blot方法检测TRAF6蛋白表达的改变;CCK-8法检测体外细胞增殖活性;流式细胞术分析细胞凋亡率;Western blot方法检测凋亡相关蛋白Bax、Bcl-2表达和AKT磷酸化水平的变化。结果显示,TRAF6-sh RNA慢病毒载体成功感染K562细胞,TRAF6蛋白表达水平明显下降。与空白对照组和TRAF6-NC组相比较,TRAF6-sh RNA组细胞增殖能力明显受抑(P<0.05),而细胞凋亡率增加(P<0.05);同时,促凋亡蛋白Bax表达升高、抗凋亡蛋白Bcl-2表达下降。此外,干扰TRAF6可下调K562细胞p-AKT(T308)和p-AKT(S473)活性,而总AKT水平未见明显变化。该研究表明,干扰TRAF6表达可抑制K562细胞增殖和诱导细胞凋亡,其机制可能与下调AKT活性有关,提示TRAF6可作为白血病治疗的一个潜在靶点。This work was aim to investigate the effect of TRAF6 knockdown on proliferation and apoptosis of human K562 leukemic cell line and its molecular mechanism. In this study, the shRNA targeting TRAF6 lentiviral vector was transfected into K562 leukemia cells. The efficiency of transfection was observed under fluorescence microscope. The change of TRAF6 protein level was confirmed by Western blot. The ability of cell proliferation were analyzed by CCK-8 method and the apoptosis rate were detected by flow cytometry. The expressions of apoptosis related proteins Bax, Bcl-2 and phosphorylated-AKT were detected by Westem blot. The results demonstrated that TRAF6-shRNA was successfully transfected into K562 cells and knockdown of TRAF6 at the protein levels was confirmed. Compared with the blank control group and the TRAF6-NC group, the cell growthin the TRAF6-shRNA group was significantly inhibited （P〈0.05）. In addition, knockdown of TRAF6 promoted apoptosis by increasing the protein level of Bax and decreasing the Bcl-2. Furthermore, TRAF6 downregulation resulted in the decreases in levels ofAKT phosphorylation at both residues Thr308 and Ser473, but no remarkable change in total AKT levels was observed. Taken together, our results revealed that TRAF6 knockdown might inhibit cell growth and induce apoptosis through downregulation of AKT activation, which indicates that TRAF6 may be a novel target for leukemia treatment.

关 键 词：TRAF6 白血病 RNA干扰 增殖 凋亡 AKT 

分 类 号：R733.7[医药卫生—肿瘤]