沉默id1基因表达对食道癌细胞Eca-109生物学行为的影响  

Effects of RNAi-silenced id1 Gene on the Biology Behavior of Esophageal Cancer Cell Lines Eca-109

作　　者：裴斐[1] 张彤彤[1] 张娜娟[1] 杨超[1] 杨春蕾[1] 

机构地区：[1]四川大学生命科学院医学生物学和细胞生物学教研室,成都610064

出　　处：《四川大学学报（医学版）》2010年第3期382-385,共4页Journal of Sichuan University(Medical Sciences)

基　　金：四川省科技厅项目(05SG022-021-05)资助

摘　　要：目的探讨靶向沉默分化抑制因子1基因(id1)的短发夹RNA(shRNA)表达质粒载体对食道癌细胞生物学行为的影响。方法设计针对id1基因的shRNA序列,合成序列并克隆入pGFU6/neo质粒中。重组质粒转染食道癌细胞,RT-PCR检测id1基因mRNA的表达,Western blot检测Id1、p16及增殖细胞核抗原(PCNA)蛋白表达。台盼蓝染色、克隆形成实验检测细胞生长,流式细胞仪检测细胞增殖以及各周期分布。结果重组质粒经酶切鉴定和测序,证实表达干扰质粒构建成功。转染重组质粒的食道癌细胞id1 mRNA和蛋白的表达明显下降(P<0.05),食道癌细胞增殖受到抑制,G0/G1期细胞比例增加,而S期的细胞比例明显下降(P<0.05)。结论构建的id1 shRNA表达质粒能有效下调id1基因的表达和抑制食道癌细胞增殖。Objective To study the effect of shRNA expressing vector targeting to id1 gene on the biological behavior of the esophageal cancer cell.Methods The specific shRNA sequence was designed, synthesized and cloned into pGFU6/neo vector.Then,the resulting recombinants were transfected into Eca-109 cells using LipofetamineTM2000 following the instruction of the manufactures.The expression levels of id1 mRNA were analyzed by RT-PCR and the levels of Id1,p16 and PCNA protein were detected by Western blot assay.The growth of Eca-109 cells was analyzed using clone formation assay and trypan blue exclusion assay.Distribution of cell cycle and proliferation of Eca-109 cells were assessed by flow cytometry.Results The sequence of recombinant plasmid was confirmed by sequence analysis.The expression levels of id1 mRNA and Id1 protein significantly decreased in Eca-109 cells transfected with pGFU6/neo-Id1-1 vector compared to the negative control group（P 〈0.05）.The recombinant plasmid expressing id1 shRNA could effectively inhibit cell proliferation and induce cell cycle arrest in G0/G1 phase with a remarkably decrease of cells in S phase（P〈0.05）.Conclusion All the above data suggested that the expression of id1 gene was significantly inhibited in Eca-109 cells transfected with pGFU6/neo-id1-1 recombinant.Furthermore,the proliferation of id1 knockdown cells was depressed.

关 键 词：id1基因 RNA干扰 短发夹RNA 食道癌细胞 

分 类 号：R735.1[医药卫生—肿瘤]