干扰RNA沉默tpd52基因对胶质瘤细胞增殖及凋亡的影响

Effect of silencing tpd52 gene by RNA interference on proliferation and apoptosis of glioma cells

目的探究沉默tpd52基因表达后对胶质瘤U87细胞增殖、周期及凋亡的影响。方法将携带着shRNA-tpd52(抑制tpd52的核苷酸序列)、shRNA-NC(阴性对照的核苷酸序列)的慢病毒体外转染胶质瘤细胞系U87。在转染48 h后荧光显微镜下观察表达GFP细胞数量,采用荧光定量PCR、Western blot分别检测TPD52 mRNA及蛋白表达量,MTT检测细胞增殖能力,流式细胞术检测细胞周期分布,Annexin V和PI双染流式细胞术检测细胞凋亡。结果 U87细胞转染率>90%,与shRNA-NC组及空白对照组相比较,shRNA-tpd52组细胞TPD52 mRNA及蛋白表达量明显减少(P<0.01),而且细胞增殖能力明显下降,差异有统计学意义(P<0.05);细胞周期被阻滞在G0/G1期、细胞凋亡比例明显增加(P<0.05)。结论沉默胶质瘤细胞中tpd52基因表达后,可以有效抑制细胞增殖,阻滞细胞周期,促进细胞凋亡。

Obiective To explored the effect of silencing tumor protein D52（tpd52） gene by RNA interference on proliferation and apoptosis in glioma cel Is. Methods The lentivirus,expressing shRNA-tpd52（the nucleotide sequence of inhibiting tpd52） and shRNA-NC（the nucleotide sequence of negative control） were infected into human U87 glioma cells in vitro. In 48 h after transfection,the counts of expression GFP cells was measured by fluorescence microscope,and the expression level of TPD52 mRNA and protein were detected by real-time fluorescence quantitative PCR and Western blot analysis. MTT assay was performed to evaluated cells proliferation. Cell apoptosis and cycle were detected by the flow cytometry with Annexin V and PI. Results The transfection rate of U87 cells was more than 90%. In contrast with the shRNA-NC group and the blank control group,the expression of TPD52 mRNA and protein of shRNA-tpd52 group were decreased significantly（P〈0. 01）,and the ability of U87 cells proliferation was markedly inhibited（P〈0. 05）. It simultaneously inhibited the transition from G0 phase to G1 phase and promoted cell apoptosis of U87 cells（P〈0. 05）. Conclusion Silence of TPD52 gene expression might inhibit proliferation and colony formation,arrest cell cycle and enhance apoptosisof glioma cells.