过表达INPP4B对人急性髓系白血病THP-1细胞增殖和凋亡的影响及其潜在机制

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

摘要:

目的：探讨二型磷脂酰肌醇4磷酸酶（inositol polyphosphate 4-phosphatase type Ⅱ，INPP4B）对人急性髓系白血病THP-1细胞体外增殖和凋亡的影响及潜在机制。方法：将携带INPP4B的重组质粒载体pEAK-Flag/INPP4B转染人THP-1细胞系（Flag-INPP4B组），同时设立未处理组为对照（Mock组）。采用qRT-PCR和Western blot分别检测实验组和对照组细胞INPP4B mRNA和蛋白的表达水平；CCK-8实验检测细胞体外增殖能力；流式细胞术检测细胞凋亡情况；Western blot检测凋亡相关蛋白Bax/Bcl-2（Bcl-2-associated X/B-cell lymphoma-2，Bax/Bcl-2）以及磷脂酰肌醇-3激酶（phosphoinositide-3 kinase，PI3K）下游信号分子蛋白激酶B（protein kinase B，PKB/AKT）和血清糖皮质激素调节激酶-3（serum and glucocorticoid regulated kinase-3，SGK3）蛋白的磷酸化水平；酶联免疫吸附试验（ELISA）检测细胞内PI（3，4）P2和PI（3）P的水平。结果：INPP4B重组质粒载体转染THP-1细胞后，INPP4B的mRNA和蛋白水平均明显上升（mRNA：t=9.724，P=0.000；蛋白：t=19.520，P=0.000），提示在THP-1细胞中成功过表达INPP4B。与Mock组相比，Flag-INPP4B组细胞体外增殖能力明显增强（F=31.870，P=0.000）。同时，过表达INPP4B可明显降低细胞凋亡率（t=6.999，P=0.002）；使促凋亡蛋白Bax的表达水平下降（t=24.710，P=0.000）、抗凋亡蛋白Bcl-2的蛋白表达水平增加（t=6.398，P=0.003）。此外，过表达INPP4B可增强THP-1细胞SGK3蛋白的磷酸化水平（t=10.470，P=0.000），而对AKT蛋白的磷酸化水平无明显影响（t=0.285，P=0.790）。最后，过表达INPP4B还可明显降低THP-1细胞中PI（3，4）P2的水平（t=4.515，P=0.011），同时明显升高PI（3）P的水平（t=5.681，P=0.005）。结论：本研究结果表明过表达INPP4B可促进人急性髓系白血病THP-1细胞的体外增殖并抵抗凋亡，其机制可能与INPP4B介导激活PI3K/SGK3信号通路有关，这提示INPP4B可作为急性髓系白血病治疗的一个潜在靶点。

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.