干扰RNA 沉默 ATP1A1基因对 U251胶质瘤干细胞增殖的影响

摘要

目的：探讨沉默ATP1A1基因对人U251胶质瘤干细胞增殖能力的影响。方法采用ATP1A1-shRNA慢病毒感染人U251胶质瘤干细胞，逆转录实时定量PCR（RT-qPCR）及蛋白质印迹法（Western blot）分别检测ATP1A1的mRNA和蛋白表达，流式细胞仪检测细胞周期和凋亡，M T T法检测细胞体外的增殖情况。结果通过RT-qPCR及Western blot 分析表明，感染后的细胞组ATP1A1的mRNA和蛋白表达均受到明显抑制，ATP1A1 mRNA抑制率为84．15％，蛋白表达抑制率为55．33％，细胞生长变慢，细胞凋亡比例显著增加，G1期细胞增多，而S期细胞减少，二者比较差异有统计学意义（ P＜0．05）。结论靶向ATP1A1基因干扰能够抑制人U251胶质瘤干细胞ATP1A1的表达，诱导细胞凋亡，调控细胞周期再分布，抑制细胞增殖。%Objective To investigate the effects of ATP1A1 knockdown by RNA interference(RNAi) on proliferation of human U251 glioma stem cells .Methods The human U251 glioma stem cells were infected with lentivirus expressing ATP1A1-shRNA . The mRNA and protein expressions of ATP1A1 in U251 glioma stem cells were detected by RT-qPCR and Western blotting ,re-spectively .The cell cycle and apoptosis were evaluated by flow cytometry .The proliferation of U251 glioma stem cells was deter-mined by MTT assay .Results The expressions of ATP1A1 in U251 glioma stem cells transfected with ATP1A1-shRNA were in-hibited significantly at both mRNA and protein levels ,with an inhibitory rate of 84 .15% for ATP1A1 mRNA and of 55 .33% for ATP1A1 protein respectively .The proliferation of cells was inhibited ,the cell apoptotic rate was significantly increased and the cell cycle was arrested in G1 phase and S phase decreased significantly in ATP1A1-shRNA cells(P<0 .05) .Conclusion RNAi targe-ting ATP1A1 gene could down-regulates the ATP1A1 expression ,induces cell apoptosis ,regulates cell phase redistribution and in-hibits cell proliferation in U 251 glioma stem cells .