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siRNA对人食管癌EC9706细胞真核起始因子4E表达的影响[J]. 肿瘤防治研究, 2009, 36(12): 1003-1007. DOI: 10.3971/j.issn.1000-8578.2009.12.004
引用本文: siRNA对人食管癌EC9706细胞真核起始因子4E表达的影响[J]. 肿瘤防治研究, 2009, 36(12): 1003-1007. DOI: 10.3971/j.issn.1000-8578.2009.12.004
Effect of siRNA on Expression of Eukaryotic Initiation Factor 4E in Human Esophageal Carcinoma EC9706 Cells[J]. Cancer Research on Prevention and Treatment, 2009, 36(12): 1003-1007. DOI: 10.3971/j.issn.1000-8578.2009.12.004
Citation: Effect of siRNA on Expression of Eukaryotic Initiation Factor 4E in Human Esophageal Carcinoma EC9706 Cells[J]. Cancer Research on Prevention and Treatment, 2009, 36(12): 1003-1007. DOI: 10.3971/j.issn.1000-8578.2009.12.004

siRNA对人食管癌EC9706细胞真核起始因子4E表达的影响

Effect of siRNA on Expression of Eukaryotic Initiation Factor 4E in Human Esophageal Carcinoma EC9706 Cells

  • 摘要: 目的 探讨siRNA对人食管癌EC9706细胞中eIF4E表达的影响。 方法 针对GeneBank中eIF4E基因的不同靶点,设计并合成两条特异siRNA及一条无义siRNA寡核苷酸模板,利用体外转录法合成siRNA,在脂质体介导下以150ng/μl、200ng/μl和250ng/μl三种浓度转染EC9706细胞,分别培养24h、48h、72h,同时设正常对照组。采用免疫细胞化学及原位杂交技术检测转染前后EC9706细胞中eIF4E蛋白和mRNA的表达;应用流式细胞技术检测转染前后EC9706细胞周期的变化情况。 结果 转染siRNA后细胞形态发生明显改变。不同浓度的两条特异siRNA转染细胞后,eIF4E蛋白及mRNA表达量均较正常对照组降低,并且具有显著的剂量依赖性(P<0.05),但两转染组之间相比,差异无统计学意义(P>0.05);无义siRNA未表现出对eIF4E基因表达的抑制效应(P>0.05);两条特异siRNA转染组与无义siRNA转染组相比,差异有统计学意义(P<0.05)。三种不同浓度siRNA转染细胞72h后与无义转染组及正常对照组相比,G0/G1期细胞比例显著增加,S期细胞比例相对减少,差异有统计学意义(P<0.05)。 结论 特异性siRNA能有效抑制人食管癌EC9706细胞中eIF4E基因的表达,抑制细胞增殖。

     

    Abstract: Objective To investigate the effects of small interfering RNA(siRNA) on the expression of eukaryotic initiation factor 4E(eIF4E) gene in human esophageal carcinoma EC9706 cells. Methods By the sequence of eIF4E gene in GeneBank, the oligonucleotide templates encoding two specific siRNAs and a nonsense siRNA of eIF4E gene were designed and synthesized,and then all the siRNAs were synthesized by in vitro transcription. EC9706 cells were transfected by each siRNA at the concentration of 150ng/μl,200ng/μl and 250ng/μl for 24h,48h and 72h respectively. The normal group was set as control at the same time. The expression of eIF4E protein and mRNA before and after transfection were detected by using immunocytochemistry and in situ hybridization. Changes of cell cycle in EC9706 cells before and after transfection were detected by application of flow cytometry. Results It was observed that EC9706 cells had morphological changes after the transfection. Compared with normal group,there was a significant decrease of the eIF4E protein and mRNA level in every specific siRNA transfection group of different concentration meanwhile a significant dose-dependent manner(P<0.05),was also observed. However there was no significant difference between the two transfection groups(P>0.05). Nonsense siRNA did not show inhibitory effect on the expression of eIF4E gene(P>0.05). The two specific siRNA can obviously depress the expression of eIF4E compared with the nonsense siRNA. The difference has statistical significance(P<0.05).After the transfection of specific siRNA for 72h, the proportion of G0/G1 phase cells increased significantly, while the proportion of S-phase cells reduced. The difference has statistical significance(P<0.05). Conclusion Specific siRNA can effectively inhibit the expression of eIF4E gene in human esophageal carcinoma EC9706 cells.

     

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