下调PRPS2基因表达对HCT116细胞增殖和凋亡的影响

Effects of down-regulated PRPS2 gene expression on HCT116 cell proliferation and apoptosis

目的:探讨下调磷酸核糖焦磷酸合成酶亚基II(PRPS2)基因表达对人结肠癌HCT116细胞增殖和凋亡的影响。方法:构建PRPS2基因干扰载体sh-PRPS2,正常对照组不转染载体,阴性对照组转染阴性对照载体sh-PRPS2-0,3个实验组分别转染干扰载体sh-PRPS2-1、sh-PRPS2-2和sh-PRPS2-3,经DNA测序鉴定后转染人结肠癌HCT116细胞,分别采用RT-PCR和Western blot法检测各载体转染后细胞PRPS2 m RNA和蛋白水平的表达变化,CCK8试剂盒检测细胞增殖能力,流式细胞术检测细胞周期及凋亡的变化。结果:经DNA测序证实成功构建sh-PRPS2干扰载体3个,分别为sh-PRPS2-1、sh-PRPS2-2和sh-PRPS2-3。将上述3个干扰载体分别转染HCT116细胞72 h后,RT-PCR结果显示,PRPS2 m RNA相对表达量依次为0.61±0.03、0.89±0.02、0.27±0.05,与对照组比较,均显著下降(P<0.05)。Western blot结果显示,PRPS2蛋白相对表达量依次为0.37±0.06、0.84±0.05、0.30±0.04,与对照组比较均显著下降(P<0.05)。转染sh-PRPS2-3载体72 h后细胞增殖抑制率达19.8%±2.4%,凋亡率上升至68.4%±4.6%,G0/G1期细胞比例上升为12.9%±3.8%。结论:PRPS2的低表达可以有效抑制细胞增殖,促进细胞凋亡,细胞阻滞于G0/G1期,本研究为肿瘤的靶向治疗提供了研究基础。

OBJECTIVE： To investigate the effects on HCT116 cell proliferation and apoptosis by down-regulating PRPS2 gene expression. METHODS： PRPS2 gene interference vector sh-PRPS2 was constructed. The vector was not the transfected in normal control; the negative vector was transfected in negative control and the RNAi vector was transfected in experimental groups. The vectors were transfected to HCTll6 cells after identified by DNA sequencing, and the level of PRPS2 mRNA and protein were detected by RT-PCR and Western blot, respectively. The ability of cell proliferation was evaluated by CCK8 kit. The cell cycle and apoptosis were assessed by flow cytometry. RESULTS： The RNAi expression vector of PRPS2 was successfully constructed as identified by DNA sequencing： sh-PRPS2-1, sh-PRPS2-2 and sh- PRPS2-3. The relative expression levels of PRPS2 mRNA were 0.61 ± 0.03, 0.89 ± 0.02 and 0.27 ± 0.05 in HCT116 cells 72 h after transfected with sh-PRPS2-1, sh-PRPS2-2 and sh-PRPS2-3, respectively, all decreased significantly compared with control （P〈0.05）. The relative expression levels of PRPS2 protein were 0.374±0.06, 0.844±0.05 and 0.30 ± 0.04, respectively, and also decreased significantly compared with control （P〈0.05）. 72 h after transfection, the inhibition rate of sh-PRPS2-3 proliferation was 19.8% ± 2.4%. The apoptosis rate of HCT116 cell was increased to 68.4% ±4.6% 72 h post transfection. Sub-G1 population percentage was increased to 12.9% ± 3.8%. CONCLUSION： Down-regulation of PRPS2 expression level could effectively inhibit cell proliferation as well as promote apoptosis. HCT116 cell was arrested in G0/G1 phase. This study provided a research basis for tumor targeted therapy.