抑制Pygo2表达对U251细胞增殖的影响

目的 构建RNA干扰(RNAi)重组体抑制Pygo2的表达,并探讨其对脑胶质瘤U251细胞增殖的影响.方法 针对Pygo2 cDNA序列设计并合成一对特异性的含有"短发卡"的寡核苷酸序列及其随机对照序列,经退火后插入pSuper中构建重组体.经EcoRⅠ和HindⅢ双酶切鉴定和DNA测序后,用脂质体2000将其转染脑胶...

Effect of Pygo2 expression inhibition on proliferation of glioblastoma U251 cells

Objective To construct RNA interference recombinant to inhibit Pygo2 expression and explore its effect on proliferation of glioblastoma U251 cells.Methods A pair of specific short hairpin RNA(shRNA) and scrambled(scr) control shRNA were designed according to Pygo2 cDNA sequence,and then cloned into eukaryotic expression vector pSuper to construct recombinants.After EcoRⅠ and HindⅢ digestion identification and DNA sequencing,the recombinants were used to transfect glioblastoma U251 cells with lipofectamineTM 2000.RT-PCR and Western blotting were applied to detect the RNA interference effects of Pyro2 shRNA.Colony-forming assay and MTT assay,flow cytometry,and BrdU incorporation assay were employed to detect cell proliferation,cell cycle,and DNA synthesis,respectively.Results Double digestion and sequencing results verified that the inserted sequences were correct.Pygo2 shRNA remarkably inhibited the mRNA and protein expression of Pygo2 as well as the proliferation of U251 cells.Comparing with the colony formation rate in the scr shRNA group (78.9±6.8)%],that in the Pygo2 shRNA group (55.4±5.2)%] was significantly reduced(P<0.05).The BrdU incorporation rate in the control group (20.96±2.19)%] was significantly higher than that in the Pygo2 shRNA group (8.81±0.56)%](P<0.05),but it was similar to that in the scr shRNA group (20.35±1.73)%].Pygo2 shRNA decreased the U251 cell percentage in S phase and increased that in G1 phase obviously(P<0.01),inducing cell cycle arrest in G1 phase.Conclusion Down-regulation of Pygo2 expression can effectively inhibit DNA synthesis of U251 cells,induce cell cycle arrest in G1 phase,and inhibit cell proliferation.