Effects Of NLS-RARα On Proliferation And Differentiation Of Acute Promyelocytic Leukemia Cell Line NB4 And Its Mechanism

PART ⅠConstruction of NLS-RARα and RARα recombinant lentivirus vectors and the stable transfection cell lines screeningObjective: In this study, we constructed NLS-RARα and RARα recombinant lentivirus vectors, then infected NB4 cells and screening of stable transfection cell lines.Methods: NLS-RARα and RARα gene were amplified by PCR using plasmid p CMV-HA-NLS-RARα and RARα as a template. The resulting gene of NLS-RARα and RARα were subcloned into the shuttle plasmid LV5-EF1a-GFP/PURO respectively by using DNA recombinant technique. The four plasmids system were transfected into 293 T cells by liposome. The virus supernatant was harvested and concentrated, then determined the titer. The recombinant lentivirus vectors of NLS-RARα and RARα were used to infect Leukemia cell line NB4, respectively. The expression of the genes in NB4 cells was detected by Western blot. The stably transfected cell lines were selected by puromycin. The efficiency of infection was measured by flow cytometry.Results: Recombinant lentivirus plasmids were constructed correctly as proved by DNA sequencing and restriction analysis. The 293 T cells which were transfected with four plasmids emitted green fluorescence under fluorescence microscope. The titer of the lentivirus reached 2×108 Tu/m L. The flow cytometry implied that the infection efficiency of lentivirus in NB4 cells reached 86.54%. The expression level of the gene was significantly increased.Conclusion: Recombinant lentivirus of LV-NLS-RARα and LV-RARα has been constructed successfully and screen out the stable transfection cell lines.PART Ⅱ The effects of NLS-RARα on prolifertion and differentiation in leukemia cell NB4 and its mechanismObjective: To detect the effects of NLS-RARα on profleration, differentiateion and its mechanism in leukemia cell line cell NB4.Methods: The viability of cells was detected by CCK-8 assay. Cell cycle and the expression of cell differentiation antigen CD11 b were measured by flow cytometry. Wright’s staining was used to observe the nuclear morphological change. The expression level of NLS-RARα, t-AKT, p-AKT, t-GSK3β, p-GSK3β and c-myc were detected by Western blot.Results: CCK-8 assay revealed that the proliferation of NB4 cells which over-expressed NLS-RARα was enhanced. Flow cytometry showed a significant increase in the number of cells in S phase, a decrease in G2 phase of the cell cycle. The expression of CD11 b was significant decrease and the nuclear morphological change is not obvious in LV-NLS-RARα group. Over-expression of NLS-RARα could increase the AKT serine/threonine phosphorylation and cause the up-regulation of downstream proteins p-GSK3β and c-myc. The level of p-AKT, p-GSK3β and c-myc were decreased after pretreatment with PI3 K inhibitor LY294002.Conclusion: NLS-RARα can promote the proliferation of APL cells and inhibit their differentiation via the PI3K/AKT signaling pathway.