摘要
目的:探讨二型磷脂酰肌醇4磷酸酶(inositol polyphosphate 4-phosphatase typeⅡ,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p EAK-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。
Objective:To investigate the role of inositol polyphosphate 4-phosphatase typeⅡ(INPP4 B)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 transfected with recombinant plasmid vector pEAK-Flag/INPP4 B which carried INPP4 B(Flag-INPP4 B group),and untreated cells were established as control group(Mock group). Western blot and qRT-PCR were used to measure the protein and mRNA expression of INPP4 B 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-associated X/B-cell lymphoma-2(Bax/Bcl-2)and the phosphorylation levels of downstream signal molecules of phosphoinositide-3 kinase(PI3 K) 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 INPP4 B recombinant plasmid vector,there were significant increases in the mRNA and protein expression of INPP4 B(mRNA:t=9.724,P=0.000;protein:t=19.520,P=0.000),suggesting that INPP4 B was successfully overexpressed in THP-1 cells. Compared with the Mock group,the Flag-INPP4 B group had a significant increase in vitro proliferative capacity(F=31.870,P=0.000). Furthermore,INPP4 B 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,INPP4 B 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). INPP4 B 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:INPP4 B can promote in vitro proliferation of human acute myeloid leukemia THP-1 cells and inhibits cell apoptosis,possibly by mediating activation of the PI3 K/SGK3 signaling pathway,which suggests that INPP4 B might be a potential target for the treatment of acute myeloid leukemia.
作者
金红君
杨丽媛
汪路
唐雨婷
陶瑶
黄圣博
敬一佩
杨再林
张伶
Jin Hongjun;Yang Liyuan;Wang Lu;Tang Yuting;Tao Yao;Haang Shengbo;Jing Yipei;Yang Zailin;Zhang Lin(College of Laboratory Medicine ,Key Laboratory of Laboratory Medical Diagnostics Designated by the Ministry of Education,Chongqing Medical University;Department of Clinical Laboratory, The Third Affiliated Hospital of Chongqing Medical University)
出处
《重庆医科大学学报》
CAS
CSCD
北大核心
2019年第2期133-138,共6页
Journal of Chongqing Medical University
基金
国家自然科学基金面上资助项目(编号:81271913)
重庆市研究生科研创新资助项目(编号:CYS17155)
关键词
二型磷脂酰肌醇4磷酸酶
白血病
增殖
凋亡
PI3K信号通路
血清糖皮质激素调节激酶-3
Iinositol polyphosphate 4-phosphatase type Ⅱ
leukemia
proliferation apoptosis
phosphoinositide-3 kinase signaling pathway
serum and glucocorticoid regulated kinase-3