摘要
目的:探讨HL-60白血病细胞中核干细胞因子Nucleostemin(NS)表达下调对其非依赖P53通路的候选途径PI3K/AKT/mTOR中相关蛋白表达是否有影响。方法:采用重组慢病毒表达载体NS-RNAi-GV248转染至P53缺失型HL-60细胞中干扰NS的表达。用Western blot技术评价转染后HL-60细胞NS蛋白的表达水平并检测干扰前后PI3K/AKT/mTOR信号通路中相关蛋白水平的表达变化。结果:重组慢病毒载体NS-RNAi-GV248成功感染了HL-60细胞,在荧光显微镜下可见到GFP荧光表达较强(<80%)。Western blot结果显示,NS蛋白表达明显下调,干扰表达有效;干扰HL-60细胞中NS的表达后AKT、p-AKT、p70s6k、p-p70s6k蛋白变化不明显(t_1=2.31,P=0.074;t_2=3.62,P=0.069;t_3=1.60,P=0.251;t_4=2.72,P=0.113),而mTOR复合物中的GβL蛋白表达明显下调(t=15.01,P=0.002)。结论:HL-60细胞中NS蛋白可影响mTOR复合物中GβL蛋白的表达,PI3K/AKT/mTOR通路可能为NS非依赖P53作用途径之一。
Objective： To explore the effect of nucleostemin（ NS） RNAi on the expression of signal molecules in PI3K/AKT/mTOR pathway,a candidate of p53-independent signal pathway in the leukemia HL-60 cells. Methods： The expression of NS was interfered by transfection of P53-deficient HL-60 cells with the recombinant lentivirus expression vector NS-RNAi-GV248. The exression of NS and signal molecules of PI3K/AKT/mTOR pathway were detected by Western blot. Results： The fluorescence microscopy showed that the recombinant lentivirus vector NS-RNAi-GV248 transfected HL-60 cells successfully with a 80% transfection rate. Western blot showed that the expression of NS protein was inhibited obviously in HL-60 cells,and the expression levels of AKT,p-AKT,p70s6k and p-p70s6k were not statistically different（ t1= 2. 31,P〈0. 05; t2= 3. 62,P〈0. 05; t3= 1. 60,P〉0. 05; t4= 2. 72,P〉0. 05） in comparison with control; the expression of GβL protein was statistically down-regnlated（ t = 15. 01,P = 0. 002）. Conclusion： The changes of GβL protein correlats with NS knockdown. The PI3K/AKT/mTOR pathway may be one of nucleostemin p53-independent signal pathways.
出处
《中国实验血液学杂志》
CAS
CSCD
北大核心
2017年第6期1592-1596,共5页
Journal of Experimental Hematology
基金
国家自然科学基金(81271911)
河南省医学科技攻关重点项目(201002006)资助
