沉默Prohibitin基因对人结直肠癌细胞株HCT116增殖和凋亡的影响

Effects of prohibitin gene silence on proliferation and apoptosis of colorectal cancer HCT116 cells

目的 siRNA介导抗增殖蛋白Prohibitin（PHB）基因沉默对人结直肠癌细胞株HCT116增殖和凋亡的影响。方法 通过脂质体LipofectamineTM2000在HCT116细胞中转染PHB-siRNA,以Western blot来筛选siRNA片段。设立阴性对照NC组和高效沉默PHB138,PHB539siRNA片段为实验组,重新转染HCT116,通过平板克隆形成实验、CCK-8检测细胞增殖能力;AnnexinⅤ-FITC细胞凋亡试剂流式细胞术、Caspase-3/Caspase-9活性检测等方法检测细胞凋亡;AnnexinⅤ-FITC和Hoechst染色荧光显微镜观察。结果 Western blot结果显示siRNA片段PHB138、PHB539能明显抑制PHB蛋白表达;转染后HCT116平板克隆形成率分别PHB138（31.1±4.0）%、PHB539（12.4±1.2）%、NC（43.4±2.1）%,差异有统计学意义（P〈0.001）;CCK8实验发现转染后1~5d,实验组与对照组比较,细胞增殖未收到显著影响（P〉0.05）。在转染第7天,PHB539siRNA能显著抑制细胞增殖（P〈0.01）;Caspase-3活性度为PHB138（1.50±0.39）、PHB539（1.81±0.41）,Caspase-9活性度PHB138（2.02±0.43）、PHB539（2.81±0.51）,差异有统计学意义（P〈0.05）;AnnexinⅤ-FITC和Hoechst染色均在荧光显微镜下观测有明显的凋亡现象,且凋亡比例和强度比阴性对照强。结论靶向PHB-siRNA干扰可以有效地沉默结直肠癌细胞HCT116PHB基因,下调PHB蛋白水平,从而抑制结直肠癌细胞HCT116增殖、促进凋亡。

Objective To investigate the effects of small interfering RNA （siRNA） mediated silencing of prohibitin （PHB） gene expression on the proliferation and apoptosis of colorectal cancer HCT1l6 ceils. Methods Colorectal cancer HCT116 cells and three normal intestinal specimens were evaluated by Western blotting to detect the expression level of PHB protein. Three siRNA sequences targeting PHB （PHB138, PHB173, and PHB539） and one negativecontrol （NC） were designed to be transfected into the cultured cancer cells by using Invitrogen＇s LipofectamineTM 2000. The two siRNAs with the highest si- lencing efficiencies were selected for use in experimental analysis of proliferation （by the CCK-8 assay, plate colony formation as- say）, cell cycle （by flow cytometry）, and apoptosis （by fl0w cytometry and Caspase-3 and Caspase-9 activity assays）. In addi- tion, the classic apoptotic morphology was observed by annexin V-propidium iodide and Hoechst staining under a fluorescence microscope. Results All three PHB targeting siRNAs significantly inhibited PHB protein expression and cell growth of HCT116, and PHB138 and PHB539 were more effective than PHB173. HCT116 plate colony formation rate was significantly decreased upon PHB silencing, PHB138 was （31.1±4.0）%, PHB539 was （12.4±1. 2）% and NC was （43.4±2： 1）% （P〈 0. 001）. In addition, both caspase-3 and caspase-9 activities were significantly enhanced in the PHB silenced cells. Conclusion siRNA mediated silencing of PHB inhibits proliferation and promotes Apoptosis of colorectal cancer HCTll6 cells. It suggests a key role of PHB in the development of colorectal cancer HCT116 cells.