Down-regulation Of Nucleostemin On Expression Of Signal Genes Of PI3K/AKT/mTOR Pathway In HL-60Cells

Objective：The expression of NS was down-regulated in p53-deletion HL-60leukemia cellsby lentiviral transfection technique. And the expression of signal molecules ofPI3K/AKT/mTOR pathway was detected by using Real-time PCR, which willresearch the relationship between NS and PI3K/AKT/mTOR pathway. We found thatRas/Raf/Mek/Erk pathway and some grow factors were closely related toPI3K/AKT/mTOR pathway’s activity. And then the expression of genes ofRas/Raf/Mek/Erk pathway and some grow factors were detected to furtherpreliminary explore the probable causes of PI3K/AKT/mTOR pathway’s changesafter NS down-regulation, Which will provide basis to complete the functionalmechanisms of NS p53-independent pathway.Methods：（1） To control for p53wild-type K562cells and p53mutant NB4cells and theexpression of p53in HL-60were detected by using general-PCR.（2） To construct NS-siRNA lentiviral expression vector, and combined with twokinds of auxiliary packaging original vector plasmid to transfect293T cells and then proceed the package of virus and titre determination.（3） According to the MOI value of HL-60cell and the virus titre, we transfectvirus into HL-60cells to down-regulate the expression of NS. Then the transfectionefficiency was observed by inverted fluorescence microscope and the expression ofNS at the level of gene and protein was evaluated by using Real-time PCR andWestern blot.（4） The expression of signal molecules of PI3K/AKT/mTOR andRas/Raf/Mek/Erk pathways, GrB2, STAT5, NFκB,TGFβ were detected by usingReal-time PCR.（5） SPSS15.0statistical software was applied to analyze data statistics. And（x±s）, t-test and one-way ANOVA were used to compare the quantitative data, tofurther paried-comparisions using Bonferroni. α＜0.05was considered as thesignificance level.Results:（1） The general-PCR results showed that p53gene was expressed in K562andNB4cell,but not expressed in HL-60cells.（2） The lentiviral vector for NS-RNAi-GV248of NS has been transfected into293T cells to proceed packaging and detecting titre. The titre of virus was2×108TU/ml.（3） The results of inverted fluorescence microscope showed that the HL-60cellswere transfected by lentivirus vector NS-RNAi-GV248successfully and withtransfection rate up to80%. According to results of Real-time PCR detection, theinhibition rate of NS was56.5%in HL-60. The results of Western blot detectionshowed that the expression levels of NS protein were apparently decreased whateverlow MOI or high MOI.（4） Real-time PCR results demonstrated that the expression levels of signalmolecules of PI3K/AKT/mTOR and Ras/Raf/Mek/Erk pathways, GrB2, STAT5,NFκB and TGFβ mRNA were all obviously down-regulated by silencing NS, andshowed statistical difference in comparison with control.Conclusion:（1） The changes of signal molecules of PI3K/AKT/mTOR and Ras/Raf/Mek/Erk pathways,and the genes of GrB2、STAT5、NFκB and TGF were positively correlatedwith NS down-regulation.（2） The procedure of NS p53-independent may be related with the genes ofPI3K/AKT/mTOR and Ras/Raf/MekErk pathways and GrB2、STAT5、TGFβ及NFκBgenes。...