The Effect Of Down-Regulated Nucleostemin On P73 In P53-Deficient HL-60 Cells

Objective:We have illustrated the effect of down-regulation Nucleostemin on genome expression in HL-60 cells and screened out some of very ralavant signaling pathways by bioinformatics. We also have confirmed that PI3K/AKT/m TOR pathway participated the NS P53-independent pathway at the level of m RNA and protein expression. Subsequent studies indicate m TOR-mediated autophagy also involved.However, we don’t know more details about mechanism.P73 is a member of the P53 family which is gaining increasing importance in the field of cancer and Rare mutations. Its structural homology with P53 led to the assumption that it coμld act as a new tumour suppressor gene.P73 is likely a key gene in NS P53-independent pathway.This study try to provide the basis for the theory by down-regulation Nucleostemin in HL-60 cells.Methods:( 1) Recombinat lentivirus plasmid which encodes lentivirus DNA and two kinds of auxiliary packing original vector plasmid were made and then transfect into293 T cells. The supernatant contain lentivirus was collected and its titer was dected.(2)HL-60 cells were transfected with different levels of lentiviral vectors in96-well plates to confirm appropriate levels of vectors.(3)Transfection efficiency and expression of NS were dected with furescence inversion microscope, real-time PCR and western blot respecively after lentiviral vectors were transfected in HL-60 cells in different times.(4)Transfection efficiency and expression of NS, △Np73,TAp73 were dectedwith real-time PCR and western blot respectively after lentiviral vectors were transfected in HL-60 cells in different time points compared with negative control group.( 5) The datas were analyzed by SPSS19.0 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.Resμlt:(1)NS-RNAi-GV248 lentiviral vectors were packed successfμlly and its titer is 4×108TU/ml.(2)The appropriate MOI value of HL-60 cells is 100. Under this condition, the transfection efficiency of lentiviral vectors is beyond 80% and the expression level of NS in experimental groups is samewhen the MOI value is 150.(3)Real-time PCR results showed that Down-regulating NS expression induced a significant time-dependent increase of the transactivation of the TAp73 and a significant time-dependent decrease of the transactivation of the△Np73 at the gene level.(4)Weatern blot results showed that Down-regulating NS expression induced a significant time-dependent increase of the expression of the TAp73 and a significant time-dependent decrease of the expression of the △Np73 on protion level.Conclusion:( 1) Down-regulating NS expression induced a significant time-dependent increase of the expression of the TAp73 and a significant time-dependent decrease of the expression of the △Np73.(2)P73 is likely a key gene in NS P53-independent pathways.