Objective: To investigate the effect of glycolytic enzyme pyruvate kinase type 2 (PKM2) on the proliferation and apoptosis of human leukemia HL-60 cells.
Methods: si-PKM2 plasmid was transfected into HL-60 cells (set as si-PKM2 group), and blank vector transfected cells were set as control group (si-Ctl group). The expression levels of PKM2 mRNA and protein in si-Ctl group and si-PKM2 group were detected by RT-qPCR and Western blot. CCK-8 cell detection kit was used to detect the proliferation ability of the cells in the two groups. Flow cytometry was used to detect the changes of cell cycle and apoptosis. Western blot and RT-qPCR were used to detect the changes of p-Akt and p-mTOR protein levels in PI3K/Akt/mTOR signaling pathway and the changes of glycolysis-related mRNA levels of the cells in the two groups. The changes in glucose consumption and lactic acid production of the cells were assayed. Over expressed PKM2, HL-60 cells were treated with PI3K inhibitor LY294002 or galactose, the changes in cell proliferation ability, cell cycle and apoptosis, as well as changes in glucose consumption and lactic acid production were detected.
Results: Interfered by si-PKM2, mRNA and protein levels of PKM2 in si-PKM2 group significantly decreased, and proliferation ability of the cells was also reduced (P<0.05). After PKM2 knockdown, the cells were significantly blocked at G1 phase, and cell apoptosis was obviously induced (P<0.05). p-Akt and p-mTOR levels were lower in si-PKM2 group than those in si-Ctl group. The glucose consumption and lactic acid production significantly decreased in si-PKM2 cells. Overexpressed PKM2, HL-60 cells were treated with PI3K inhibitor LY294002. The glucose consumption and lactate acid production induced by overexpressed PKM2 were reduced. Overexpressed PKM2, HL-60 cells showed no significant changes in cell proliferation, cycle and apoptosis when cultured with galactose.
Conclusion: PKM2 knockdown can inhibit the proliferation and induce apoptosis of HL-60 cells, and its molecular mechanism may be related to the PKM2-mediated PI3K/Akt/mTOR-glycolysis, which suggesting that PKM2 may serve as a molecular target for the prevention and treatment of leukemia.
题目: 干扰PKM2对急性白血病细胞增殖和凋亡的影响及分子机制.
目的: 探讨糖酵解酶丙酮酸激酶M2型( PKM2)对人白血病细胞HL-60增殖和凋亡的影响及其分子机制。.
方法: 以干扰PKM2的质粒转染HL-60细胞株( si-PKM2 组) ，同时将转染空载体的HL-60细胞设为对照组( si-Ctl组)。采用RT-qPCR和Western blot 技术分别检测si-Ctl组和si-PKM2 组PKM2 mRNA和蛋白水平的变化； CCK-8检测两组细胞的增殖能力；采用流式细胞术检测两组细胞周期和凋亡的变化；Western blot 和RT-qPCR技术检测两组细胞中PI3K/Akt/mTOR信号通路的变化及糖酵解相关基因的表达变化，并检测两组细胞中葡萄糖消耗和乳酸生成的变化情况。过表达PKM2的细胞给予PI3K抑制剂LY294002或给予半乳糖培养后检测细胞的增殖能力、周期和凋亡的变化以及葡萄糖消耗和乳酸生成的变化。.
结果: 干扰PKM2表达后，si-PKM2 组中PKM2的mRNA （t=45.21，P<0.001）和蛋白水平（t=13.56，P<0.01）显著降低，si-PKM2 组的细胞增殖能力较对照组明显下降（F= 253.59，P<0.05），敲低PKM2后细胞被显著阻滞于G1期，且明显诱导了细胞的凋亡（t=56.38，P<0.05）。与对照组相比，si-PKM2 组p-Akt和p-mTOR水平显著降低（t=50.23、13.51，P<0.01），葡萄糖消耗和乳酸生成显著下降（t=23.16、15.20，P<0.05）。补救实验结果显示，过表达PKM2给予PI3K抑制剂LY294002后减弱了其诱导的葡萄糖消耗和乳酸的产生（t=45.13、26.35，P<0.05），半乳糖培养过表达PKM2的HL-60细胞对细胞增殖能力（t=16.52，P>0.05）、周期及凋亡无显著影响（t=48.63，P>0.05）。.
结论: 敲低PKM2可以抑制白血病HL-60细胞的增殖并诱导其凋亡，其分子机制可能与PKM2 介导的PI3K/Akt/mTOR糖酵解轴抑制有关，提示PKM2可能作为白血病防治的一个分子靶点。.