The Role Of Nanog In Acute Myeloid Leukemia And Acute Lymphoblastic Leukemia Cells

Objective:To explore gene expression of NANOG in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) cells and the effect on stemness or biological behaviors of leukemia cells.Methods:(1) RT-PCR and Western blot were respectively used to detect NANOG gene expression level and protein expression level in a variety of acute leukemia cell strains and primary leukemia cells from AML/ALL patients. To test the efficiency of RNA interference, leukemia cells were firstly infected by lentiviral vectors, which were successfully constructed with NANOG specific shRNA. Afterwards, GFP positive cells were sorted by using flow cytometry. NANOG expression levels were subsequently re-evaluated by using RT-PCR and Western blot.(2) The cell proliferation, apoptosis and colony formation of U937and THP-1cells were analyzed by CCK-8assay, flow cytometry and methylcellulose colony-forming assay. The tumorigenicity of NANOG was analyzed using a nude mice model. The expression of BMI1was detected by real-time quantitative PCR and Western blot in NANOG down-regulated U937cells. Then the U937cells stably down-regulating BMI1or overexpressing NANOGP8were constructed. The changes of proliferation, apoptosis and colony-forming in these U937cells were detected. Molecular mechanisms by which NANOGP8activated BMI1expression were confirmed using a dual luciferase reporter system.(3) The cell proliferation, apoptosis and cell cycle of MOLT-4cells were analyzed by CCK-8assay and flow cytometry. The molecular mechanisms underlying these changes were investigated by microarray analysis, and the gene expression changes were further verified by real-time quantitative PCR. Results:(1) Both NANOGP8gene and protein expression was positively detected in acute leukemia cells. The experimental data on infection of leukemia cells with lentiviral vectors revealed that both shRNA interfering sequences (shNANOG-1and shNANOG-2) could stably down-regulate NANOG gene and protein expressions. Analysis of cell proliferation indicated that the U937and THP-1cells expressing NANOGP8shRNA grew significantly slower than control cells, and this difference was time-dependent. The percentages of apoptotic cells in groups of shNANOG-1and shNANOG-2were significantly increased compared to that of sh-control group (P<0.05). The number of colonies of NANOGP8shRNAs-infected cells was lower than that of control cells (P<0.05). The tumor weight and volume in shNANOG-1group were significantly reduced than that of sh-control group (P<0.05). Further testing found that inhibition of NANOGP8expression in U937cells down-regulated BMI1expression simultaneously, but inhibition of BMI1expression did not effect NANOGP8expression. Down-regulation of NANOGP8or BMI1expression in U937cells inhibited cell proliferation, reduced colony formation and promoted apoptosis, and these effects could not be rescued in U937cells stably down-regulating BMI1and overexpressing NANOGP8. Our results also confirmed that NANOGP8occupied BMI1promoter region (-220to-210) to activate gene transcription.(3) Analysis of cell proliferation indicated that the MOLT-4cells expressing NANOGP8shRNA grew significantly slower than control cells, and this difference was time-dependent. The percentages of apoptotic cells in groups of shNANOG-1and shNANOG-2were significantly increased compared to that of sh-control group (P<0.05). The percentages of MOLT-4cells expressing NANOG shRNAs in the G0/G1-phase were higher than that of control cells (P<0.05). Microarray analysis demonstrated that the genes up-regulated or down-regulated by down-regulation of NANOGP8were involved in various signaling pathways. The highest enrichment score was associated with the P53signaling pathway. The gene expression of TP53, CDKN1A, CDKN2A, and CYCS of shNANOG-1group was augmented compared to that of sh-control group (P<0.05). Reversely, a significant down-regulation of MDM2and CCND1gene expression was observed (P<0.05).Conclusion:(1) NANOGP8can be expressed in acute leukemia cells.(2) Down-regulation of NANOGP8expression in AML cells inhibits cell proliferation, promotes apoptosis, and reduces colony formation and tumorigenicity. NANOGP8regulates BMI1through promoter occupancy and transactivation, and is involved in the stemness regulation of AML cells.(3) Down-regulation of NANOGP8expression in T-ALL cells inhibits cell proliferation, promotes apoptosis and arrests the cell cycle. NANOGP8is involved in the biological behavior regulation of T-ALL cells through P53-dependent signaling pathways.