I2PP2A蛋白调控PP2A/Akt信号通路对神经母细胞瘤细胞增殖和凋亡的影响

Effect of I2PP2A on the Proliferation and Apoptosis of Neuroblastoma Cells by Regulating PP2A/Akt Signaling Pathway

目的探讨蛋白磷酸酶2A抑制剂-2(protein phosphatase 2A inhibitor-2,I2PP2A)调控PP2A/Akt信号通路对人神经母细胞瘤细胞增殖和凋亡的影响。方法体外培养人神经母细胞瘤SH-SY5Y细胞转染I2PP2A-shRNA干扰质粒及对照质粒后分为siI2PP2A组及siC组。CCK-8、细胞克隆形成实验、细胞周期实验用于检测细胞增殖活性;流式细胞术检测细胞凋亡;Western blot法检测细胞内相关蛋白相对表达量;磷酸酶检测系统试剂盒测定PP2A活性;给予PP2A抑制剂冈田酸(okadaic acid,OA)处理细胞并检测增殖活性及PP2A/Akt信号通路变化。结果与siC组比较,siI2PP2A组48h及72h的吸光度(A)值下降(P<0.01),细胞克隆形成数目显著降低(P<0.01);siC组G_(0)/G_(1)期、S期、G_(2)/M期、细胞凋亡比例分别为43.29%±3.68%、31.76%±2.42%、24.96%±2.34%、1.89%±1.09%,siI2PP2A组为57.00%±3.90%、25.88%±2.06%、17.13%±2.07%、15.18%±5.60%,与siC组比较,siI2PP2A组G_(0)/G_(1)期细胞比例和凋亡细胞比例升高,S期和G_(2)/M期细胞比例下降(P<0.05)。同时,siI2PP2A组较siC组PP2A活性显著增加(P<0.05),CyclinD1、Bcl-2、p-Akt蛋白相对表达量降低(P<0.05),Bax、cleaved-PARP蛋白相对表达水平升高(P<0.01)。进一步给予PP2A抑制剂OA后,可部分恢复siI2PP2A组CyclinD1、Bcl-2、p-Akt、Bax、cleaved-PARP蛋白水平(P<0.05)并逆转下调I2PP2A导致的增殖抑制(P<0.05)。结论下调I2PP2A介导的神经母细胞瘤细胞增殖抑制和凋亡增加可能与PP2A/Akt信号通路密切相关。

Objective To investigate the effect of protein phosphatase 2A inhibitor-2(I2PP2A)on the proliferation and apoptosis of human neuroblastoma cells by regulating PP2A/Akt signaling pathway.Methods Human neuroblastoma SH-SY5Y cells transfected with I2PP2A-shRNA interference plasmid and control plasmid were divided into siI2PP2A group and siC group.CCK-8,cell clone formation assay and cell cycle assay were used to detect cell proliferation activity;flow cytometry was used to observe the changes of apoptosis;Western blot was used to detect the intracellular protein expression levels;Phosphatase assay kit was used to determine PP2A activity.The cells were treated with PP2A inhibitor okadaic acid(OA),then the proliferation activity and the changes of PP2A/Akt signaling pathway were detected.Results Compared with the siC group,the A values of the siI2PP2A group at 48h and 72h were decreased(P<0.01),and the number of clones in the siI2PP2A group was significantly lower than that in the siC group(P<0.05).The percentages of G_(0)/G_(1)phase,S phase,G_(2)/M phase and apoptosis in the siC group were 43.29%±3.68%,31.76%±2.42%,24.96%±2.34%and 1.89%±1.09%,respectively.The siI2PP2A group were 57.00%±3.90%,25.88%±2.06%,17.13%±2.07%and 15.18%±5.60%,respectively.Compared with the siC group,the proportion of cells in G_(0)/G_(1)phase and the proportion of apoptotic cells in the siI2PP2A group were increased,and the proportion of cells in S phase and G_(2)/M phase in the siI2PP2A group were decreased(P<0.05).At the same time,compared with the siC group,the PP2A activity and the relative expression level of Bax and cleaved-PARP was increased(P<0.05),while protein relative expression level of CyclinD1,Bcl-2 and p-Akt were decreased in the siI2PP2A group(P<0.05).Besides,administration of PP2A inhibitor OA,the protein relative expression levels of CyclinD1,Bcl-2,p-Akt,Bax,cleaved-PARP in siI2PP2A group were partially restored(P<0.05),and the proliferation inhibition caused by down-regulation of I2PP2A was reversed(P<0.05).Conclusion The inhibition of proliferation and increased apoptosis of neuroblastoma cells mediated by down-regulation of I2PP2A expression may be closely related to the PP2A/Akt signaling pathway.