阻断mTOR表达对结肠癌LoVo细胞增殖的影响及机制

Effects of Knock-down of mTOR Gene on Growth and Mechanism of Colon Cancer LoVo Cell

目的观察RNA干扰(RNAi)特异性阻断哺乳动物雷帕霉素靶蛋白(mTOR)基因表达对结肠癌LoVo细胞体内外增殖的影响及机制。方法实验分3组:正常培养组:未转染的正常培养LoVo细胞;阳性实验组:转染mTOR-RNA干扰质粒的LoVo细胞;阴性对照组:转染空载体质粒的LoVo细胞。MTT法检测LoVo细胞体外生长曲线;平板克隆形成试验分析细胞克隆形成能力;流式细胞仪检测细胞周期和凋亡;高效液相色谱法(HPLC)测定细胞内三磷酸腺苷(ATP)含量。结果阳性实验组细胞生长速度较正常对照组减慢。正常培养组、阳性实验组和阴性对照组细胞克隆形成率分别为(26.8±4.9)%、(11.8±2.1)%和(21.3±4.7)%;与正常培养组比较,阳性实验组细胞克隆形成率显著减少(P<0.01)。与正常培养组相比,阳性实验组G1期细胞数增加8.6%,S期的细胞数减少8.0%,细胞凋亡率增加7.0%(均P<0.01)。正常培养组、阳性实验组和阴性对照组细胞内ATP含量分别为(2.47±0.20)nmol/106、(1.62±0.18)nmol/106和(2.37±0.21)nmol/106;阳性实验组ATP含量明显低于正常培养组(P<0.01)。结论特异性阻断mTOR基因表达可通过遏制能量代谢和诱导细胞凋亡来抑制结肠癌LoVo细胞增殖。

Objective To investigate the effects and mechanism of knock-down the expression of mammalian target of rapamycin （mTOR） gene with RNA interference （RNAi） technique on growth and mechanism of colon cancer LoVo cell. Methods There were three groups in the study. Group 1 （normal group） was the normal cultured LoVo cells. Group 2 （positive experimental group） was the LoVo cells transfected with mTOR-RNAi plasmid vector. Group 3 （negative control group） was the LoVo cells transfected empty plasmid vector as negative control. Cell growth was assessed using MTT assay and tumor colony formation was detected. Cell cycle and apoptotic rate were analyzed via flow cytometry. Furthermore, the contents of adenosine triphosphate （ATP） in LoVo cells were measured by high performance liquid chromatography （HPLC）. Results The rates of cell proliferation in the positive experimental group were slower than those in the normal cultured group. The colon formations of LoVo cells in the normal cultured group,the positive experimental group and the negative control group were （26. 8 ± 4. 9）%, （11.8 ±2. 1）% and （21.3 ±4. 7）% respectively. Compared with those in the normal cultured group, the colon formations of LoVo cells in the positive experimental group were decreased significantly, and the ratios of cells in G1 phase were increased by 8.6% and that in S phase were decreased by 8. 0% and those in apoptosis were increased by 7. 0% （all,P〈0. 01）. The ATP contents in the normal cultured group, the positive experimental group and the negative control group were （2. 47 ±0. 20） nmol/10^6 , （1.62 ± 0. 18） nmol/10^6 and （2. 37 ± 0. 21 ） nmol/10^6 respectively. The ATP contents in the positive experimental group were lower than those in the normal cultured group （P〈0. 01）. Conclusion Our results suggested that selective knock-down of mTOR could suppress LoVo cell proliferation by cutting down energy metabo lism and inducing cell apoptosis.