NPM1Mutation Promoting Cellmalignant Phenotype Through Regulating Akt Inacute Myloid Leukemia

Objective Nucleophosmin1(NPM1) is the most common mutated genein acute myeloid leukemia(AML), which have an important impact on thePathogenesis，treatment and prognosis of leukemia. However, the importantrole of NPM1mutations in malignant ransformation of humanAML cellsplays had not been elucidated. This paper discussed the impact of NPM1mutations in human leukemia cells malignant phenotype and the underlyingmolecular mechanism.Method Interference using lentiviral gene approach to carryingwild-type NPM1HL60cells as a negative control group, carrying NPM1mutations OCI/AML3cells in the experimental group, while theestablishment of non-infected group (Mock), load the infected group(pGIPZ) and NPM interference group (shNPM), observe the effect on themalignant phenotype of leukemia cells after disturbance NPM1mA.TheNPM1A mutation (NPM1-mA) gene expression and protein expressionwere determined by qRT-PCR, Western blot and immunocytochemistry;cell proliferation and colony forming ability were assayed through CCK-8 and colony formation in interferenced cells after NPM1mA; flowcytometry and Wright-Giemsa staining were used to test the cell cycle,apoptosis and differentiation, Western blot was used to detect the cyclerelated proteins CDK2, cyclinD1and p21, apoptosis-related proteins Bax,Bcl-2expression were also tested by qRT-PCR and Western blot analysis,the cell surface differentiation antigen CD11b and CD14expression wereassayed by streaming cytometry; Western blot analysis was used to verifyNPM1mAinfluence onAKT signaling pathway which both p-AKT and thedownstream p-FoxO3a(S253) of leukemia cells;AKT the effect ofAKTinhibitors on proliferation and apoptosis of K562cells whichoverexpressing NPM1mA were tested by CCK-8and flow cytometry.Results The results showed significant down-regulation of NPM1-mAmRNA levels and protein levels after NPM1silencing (P<0.05), andreducing of cytoplasm NPM mutant protein of OCI/AML3cells. NPM1interference in OCI/AML3cells could inhibit cell proliferation and colonyformation (P<0.05), and cell cycle was arrested in G1phase, S phase wasdecreased (P<0.05), also found that the decreased CDK2and cyclinD1protein expression, and p21protein expression was increased (P<0.05).After the interference NPM1mA the OCI/AML3cells apoptosis rate wereincreased (P<0.05), CD11b and CD14expression levels were increased(P<0.05), cell differentiation morphological changes and apoptosis bodieswere observed under light microscopy, in addition, interferenced NPM1mA OCI/AML3cells may regulate the expression of pro-apoptotic moleculesBax and downregulate the expression of anti-apoptotic Bcl-2molecules.After NPM1mA was inhibited in OCI/AML3, the expression of AKTpathway both p-AKT and the downregulated p-FoxO3a(S253) weredecreased; K562overexpressed after NPM1mA, AKT pathway activity wasincreased.Conclusion NPM1mA may regulate leukemia cells malignant phenotypeby enhancing AKT/FoxO3a signaling pathway, and promote thedevelopment and progression of leukemia. As an important signal hub,AKT could become a useful signaling pathway target which can promote tooccur of the NPMc+AML.