Down-regulatory Effect Of Nucleostemin On Autophagy Activities Mediated By MTOR Signal Pathway In P53 Null HL-60 Leukemic Cells

Objective:Based on previous reaserch results about PI3K/AKT/m TOR signal pathway, to investigate further down-regulatory effect of nucleostemin(NS) on autophagy activities mediated by m TOR signal pathway in p53 null HL-60 leukemic cells,which lays the foundation on studying mechanisms of p53-independent signal pathway of NS in details. Methods:(1) Recombinat lentivirus plasmid which encodes lentivirus DNA and two kinds of auxiliary packing original vector plasmid were made and then transfect into 293 T cells. The supernatant contain lentivirus was collected and its titer was dected with 293 T cells after concentrating.(2) HL-60 cells were transfected with different levels of lentiviral vectors to comfirm the appropriate levels of vectors.(3) Transfection efficiency and the expression of NS were dected with flurescence inversion microscope and western blot respecively after lentiviral vectors were transfected into HL-60 cells.(4) Acid vesicular organella(AVO) were counted and compared among blank groups、negative control groups and experimental groups with acidine orange staining.(5) Proteins which were extracted from HL-60 cells treated with Rapamycin as positive control, the changes of LC3Ⅱ/LC3Ⅰwere dected and compared among blank groups、negative control groups and experimental groups after transfected lentiviral vectors into HL-60 cells.(6) The number of autophagosomes were counted with transmission electron mcrioscope and compared among blank groups、negative control groups and experimental groups.(7) The gray value of protein bands were analyzed with Image J image processing sofeware. The datas were analyzed by SPSS 17.0 statistical software and were showed in the form of x ± s. One-way ANOA was used to compare qucntitative datas, to further paried-comparisions using Bonferroni, α,=0.0167 was considered as calibration inspection level. Results:(1) NS-RNAi-GV248 lentiviral vectors were packed successfully and its titer is 4×108TU/ml.(2) The appropriate MOI value of HL-60 cells is 80. Under this condition, the transfection efficiency of lentiviral vectors is beyond 80% and the expression level of NS in experimental groups is lower than them in the other groups.(3) Tht result of acidine orange staining shows the number of AVO in experimental groups [(22.4±0.76)%] is higher than blank groups[(3.1±0.28)%] and negative control groups[(6.2±0.64)%](p<0.05).Western blot shows the ration of LC3Ⅱ/LC3Ⅰin experimental groups(1.537±0.072) is higher than them in the other groups [(1.010±0.039) and(0.608±0.008)].The result of transmission electron mcrioscope technology also shows the number of autophagosomes in experimental groups(8.7±3.1)is higher than them in the other groups[(4.2±1.2)and(2.3±0.5)]. Conclusions:(1)Autophagy activties can be enhanced after the level of NS was down regulated.(2)The change of autophagy activties after NS was down regulated indicates PI3K/AKT/m TOR pathway may be one of the pathways of p53-independent pathway of NS, which lay a foundation on continuing studying details in mechanisms of p53-independent signal pathway of NS.