HL-60细胞Nucleostemin表达下调对PI3K/AKT/mTOR通路相关分子的影响

Down-Regulatory Effect of Nucleostemin Expression on Signal Molecule of PI3K/AKT/mTOR Pathway in HL-60 Cells

本研究旨在探讨白血病细胞中nucleostemin(NS)基因下调对其非依赖p53通路的候选途径PI3K/AKT/mTOR的相关分子表达的影响。通过重组慢病毒表达载体NS-RNAi-GV248转染至p53缺失型HL-60细胞以干扰NS基因的表达。用Real-time PCR技术评价转染后HL-60细胞NS基因的表达情况并检测干扰前后PI3K/AKT/mTOR信号通路中相关分子水平表达的变化。结果表明: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GFP荧光水平较高,大于80%。Real-time PCR结果显示,NS基因mRNA的抑制率在HL-60细胞中为56.5%;干扰NS的表达后PI3K、AKT和GβL基因mRNA表达量分别为0.491±0.084、0.398±0.164、0.472±0.097,下调明显,与空白对照组(分别为1.002±0.171、1.000±0.411、1.001±0.206)比较差异有统计学意义(P<0.05)。结论:在HL-60细胞中PI3K/AKT/mTOR通路相关分子的变化与NS基因的下调呈正相关,两者的关联性为证明PI3K/AKT/mTOR信号通路可能是NS的一种非依赖p53作用途径提供了依据。

This study was purposed to explore the down-regulatory effect of nucleostemin（NS） expression on signal molecules of PI3K/AKT/mTOR pathway belonged to candidate ways of p53-independent signal pathway in the leukemia cells.The expression of NS was interfered by using recombinant lentivirus expression vector NS-RNAi-GV248 to transfect HL-60 cells of p53 deficiency.The exression of NS and signal molecules of PI3K/AKT/mTOR pathway were detected by using Real-time PCR.The results of showed that the HL-60 cells were transfected by recombinant lentivirus vector NS-RNAi-GV248 successfully and with transfection rate up to 80％.According to results of Real-time PCR detection,the inhibition rate of NS gene was 56.5％ in HL-60 cells.And the expression levels of PI3K,AKT and GβL mRNA （0.491 ± 0.084、0.398 ± 0.164、0.472 ± 0.097 respectively） were obviously down-regulated by silencing NS,and showed statistical difference（P ＜ 0.05） in comparison with control （1.002 ± 0.171、1.000 ± 0.411、1.001 ± 0.206 respectively）.It is concluded that the changes of signal molecules of PI3K/AKT/mTOR pathway positively correlate with NS down-regulation,which provides evidence for confirming PI3K/AKT/mTOR signal pathway possible as a type of NS p53-independent pathway.