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	抑制Pygo2表达对U251细胞增殖的影响; Effect of Pygo2 expression inhibition on proliferation of glioblastoma U251 cells
	陈玉英 ; 王海东 ; 王占祥 ; 谭国伟 ; 刘希尧 ; 沈上杭
	2011
关键词	Pygo2 胶质瘤 细胞增殖 短发夹RNA Pygo2 glioma cell proliferation shRNA
英文摘要	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将其转染脑胶质瘤u251细胞,采用rT-PCr和WESTErn blOT检测PygO2 SHrnA对u251细胞PygO2 MrnA和蛋白的干扰效果,应用克隆形成实验和MTT法检测细胞的增殖情况,流式细胞术检测细胞周期,brdu掺入法检测dnA合成。结果双酶切和测序鉴定证实插入序列完全正确;PygO2 SHrnA显著抑制了其MrnA和蛋白的表达,MTT分析其细胞增殖也被显著抑制(P<0.01)。与SCr SHrnA组的克隆形成数(78.9±6.8)%相比,PygO2 SHrnA组克隆数(55.4±5.2)%显著减少(P<0.05)。与对照组的brdu掺入率(20.96±2.19)%相比,PygO2 SHrnA组的brdu掺入率(8.81±0.56)%显著减少(P<0.05),而SCr SHrnA组的brdu掺入率是(20.35±1.73)%无显著变化。而且,PygO2 SHrnA使u251细胞处于S期百分比显著减少、g1期显著增加(P<0.01),致细胞周期阻滞于g1期。结论成功构建了抑制PygO2表达的重组载体,下调PygO2表达能有效地抑制脑胶质瘤u251细胞dnA合成,使细胞阻滞于g1期,抑制细胞增殖。; 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.; 厦门市科技局基金(3502z20089001);福建省自然科学基金(2009D002);中国博士后基金(20080440728);重庆市教委科技基金(KJ100504)----
语种	zh_CN
内容类型	期刊论文
源URL	[http://dspace.xmu.edu.cn/handle/2288/122831]
专题	信息技术－已发表论文
推荐引用方式 GB/T 7714	陈玉英,王海东,王占祥,等. 抑制Pygo2表达对U251细胞增殖的影响, Effect of Pygo2 expression inhibition on proliferation of glioblastoma U251 cells[J],2011.
APA	陈玉英,王海东,王占祥,谭国伟,刘希尧,&沈上杭.(2011).抑制Pygo2表达对U251细胞增殖的影响..
MLA	陈玉英,et al."抑制Pygo2表达对U251细胞增殖的影响".(2011).

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