敲低叉头盒蛋白Ml抑制骨肉瘤细胞糖酵解

Knocking down forkhead box M1 reduces glycolytic level in osteosarcoma cells

目的观察叉头盒蛋白Ml ( FOXM1 )对骨肉瘤细胞糖酵解水平的影响。方法FOXM1小干扰RNA(siRNA)慢病毒、对照siRNA慢病毒感染骨肉瘤细胞,记为FOXM1 siRNA和siRNA Control组,把未经慢病毒感染的细胞作为Control。噻唑蓝(MTT)检测细胞增殖能力,平板克隆实验检测细胞克隆形成能力,试剂盒检测培养液上清中乳酸含量和细胞中三磷酸腺苷(ATP)含量.Western blot检测细胞中己糖激酶2 ( HK2 )、丙酮酸激酶M2亚型(PKM2 )蛋白水平。结果FOXM1 siRNA 细胞中 FOXM1 mRNA 和蛋白水平均低于 siRNA Control 和 Control 组(FOXM1 mRNA:0.31 ±0. 03 比 0.98 ±0. 13 比 1.00 ±0. 09;FOXM1 蛋白:0. 21 ±0. 02 比 0. 63 ±0. 08 比 0. 62 ± 0. 06,P<0.05)。与 Control,siRNA Control 组比较.FOXM1 siRNA 细胞存活率降低[(64. 17 ±3.25)%比(97. 56 ± 10. 48)%比 100. 00%, P< 0.05],细胞克隆形成率降低[(21.26 ± 8.66)%比(49. 89 ±7.36)比(45. 16±9.23)%,P<0.05],上清中乳酸含量下降[(2. 18 ± 0. 27 ) mmol/L 比(4.62 ±0. 68 ) mmol/L 比(4. 56 ±0.51) mmol/L,P <0. 05],细胞中 ATP 含量也降低[(16. 20 ±1.65) mmol/L比(33. 58 ±4. 15) mmol/L 比(31. 57 ±2. 63) mmol/L,P<0. 05],细胞中 HK2、PKM2 蛋白水平下降[HK2:0. 20 ±0. 02 比 0. 38 ±0. 05 比 0. 35 ±0. O3;PKM2:O. 32 ±0.04 比 0. 78 ±0.09 比 0. 75 ±0. 08,P<0.05]。结论敲低F0XM1能够降低骨肉瘤细胞增殖和克隆形成能力,降低骨肉瘤细胞糖酵解水平。

Objective To study the effects of fork head box M1 ( FOXM1 ) on glycolysis of osteosarcoma cells. Methods FOXM1 small interfering RNA (siRNA) lentivirus and control siRNA lentivims infect osteosarcoma cells, named as FOXM 1 siRNA and siRNA control, the cells that were not infected with lentivirus were used as Control. Methylthiazolyldiphenyl - tetrazolium bromide ( MTT) detection of cell proliferation ability, the colony forming ability of cells was detected hy plate cloning test, the contenl of lactic acid and the content of adenosine - triphosphate ( ATP) in the supernatant of the culture medium were detected by the kit, Western blotting was used to detect the level of HK2 and M2 - pyruvate kinase (PKM2) protein in the cells. Results The levels of FOXM 1 mRNA and protein in F0XM1 siRNA cells were lower than those in siRNA control ( FOXM 1 mRNA: 0. 31 ± 0. 03 vs. 0. 98 ± 0. 13 , 1.00 ± 0. 09;FOXM1 protein: 0.21 ±0. 02 vs. 0. 63 ±0. 08, 0. 62 ±0.06, P<0. 05). Compared with Control, siRNA control, the survival rate of FOXM 1 siRNA cells was reduced [( 64. 17 ± 3. 25 )% vs.( 97. 56 ± 10. 48)%,( 100. 00 ± 10. 25 )%, P < 0. 05 ], the cell clone formation rate decreased [( 21. 26 ±8. 66)% vs.(49. 89 ±7. 36)%,(45. 16 ±9. 23)%, P <0. 05]. the content of lactic acid in the supernatant decreased [(2. 18 ± 0. 27) mmol/L vs.(4. 62 ± 0. 68 ) mmol/L,(4. 56 ± 0. 51) mmol/L, P < 0. 05 ], the content of ATP in cells also decreased [( 16. 20 ± 1.65) mmol/L vs.( 33. 58 ± 4. 15 ) mmol/L,(31. 57 ±2.63) mmol/L, P < 0. 05], the level of HK2 and PKM2 protein in the cells decreased〔 HK2 : 0. 20 ± 0. 02 vs. 0. 38 ± 0. 05 , 0. 35 ± 0. 03;PKM2 : 0. 32 ± 0. 04 vs. 0. 78 ± 0. 09, 0. 75 ±0. 08 , P<0. 05]. Conclusion Knocking down FOXM1 lowers the ability of proliferate and clone in osteosarcoma cells, the glycolysis level of osteosarcoma cells was reduced.