Effects of inositol polyphosphate 4-phosphatase type Ⅱ overexpression on proliferation and apoptosis of human acute myeloid leukemia THP-1 cells and potential mechanism

Abstract:

Objective：To investigate the role of inositol polyphosphate 4-phosphatase typeⅡ（INPP4B） in the in vitro proliferation and apoptosis of human acute myeloid leukemia THP-1 cells and the potential mechanism. Methods：Human THP-1 cells were transfect－ed with recombinant plasmid vector pEAK-Flag/INPP4B which carried INPP4B（Flag-INPP4B group），and untreated cells were estab－lished as control group（Mock group）. Western blot and qRT-PCR were used to measure the protein and mRNA expression of INPP4B in the experimental group and the control group；CCK-8 assay was used to assess in vitro proliferative capacity；flow cytometry was used to measure cell apoptosis；Western blot was used to measure the levels of apoptosis-related proteins Bcl-2-associ－ated X/B-cell lymphoma-2（Bax/Bcl-2） and the phosphorylation levels of downstream signal molecules of phosphoinositide-3 kinase（PI3K） protein kinase B（PKB/Akt） and serum and glucocorticoid regulated kinase-3（SGK3）；ELISA was used to measure the levels of PI（3，4）P2 and PI（3）P in cells. Results：After THP-1 cells were transfected with INPP4B recombinant plasmid vector，there were significant increases in the mRNA and protein expression of INPP4B（mRNA：t=9.724，P=0.000；protein：t=19.520，P=0.000），suggesting that INPP4B was successfully overexpressed in THP-1 cells. Compared with the Mock group，the Flag-INPP4B group had a significant increase in vitro proliferative capacity（F=31.870，P=0.000）. Furthermore，INPP4B overexpression significantly reduced the apoptosis rate of THP-1 cells（t=6.999，P=0.002） and the expression of the pro-apoptotic protein Bax（t=24.710，P=0.000） and significantly increased the level of the anti-apoptotic protein Bcl-2（t=6.398，P=0.003）. In addition，INPP4B overexpression significantly increased the phosphorylation level of SGK3 in THP-1 cells（t=10.470，P=0.000），while it did not affect the phosphorylation level of AKT（t=0.285，P=0.790）. INPP4B overexpression also significantly reduced the level of PI（3，4）P2 in THP-1 cells（t=4.515，P=0.011） and significantly increased the level of PI（3）P in THP-1 cells（t=5.681，P=0.005）. Conclusion：INPP4B can promote in vitro proliferation of human acute myeloid leukemia THP-1 cells and inhibits cell apoptosis，possibly by mediating activation of the PI3K/SGK3 signaling pathway，which suggests that INPP4B might be a potential target for the treatment of acute myeloid leukemia.