稳定eIF1过表达的鼻咽癌细胞株的构建及其功能研究

Study on construction and function of CNE1 cells stably over-expressing eIF1 gene

目的构建稳定真核翻译起始因子1(eIF1)过表达的鼻咽癌细胞株,研究其对鼻咽癌细胞增殖和迁徙活性的影响。方法采用pEGFP-C1真核表达系统,构建eIF1过表达载体,转染鼻咽癌CNE1细胞,进而获得稳定转染细胞株CNE1-eIF1及其对照细胞,以实时荧光定量PCR(qPCR)和蛋白质印迹法(Western blot)验证该细胞中eIF1的过表达情况。采用细胞增殖与迁徙实验分别检测CNE1-eIF1细胞增殖和迁徙活性。结果酶切电泳鉴定及测序检测显示pEGFP-C1-eIF1真核表达载体构建成功。稳定eIF1过表达的鼻咽癌CNE1细胞CNE1-eIF1的eIF1基因和蛋白的表达水平与空载质粒转染相比分别增加2.85倍和2.58倍(P<0.05),而其增殖和迁徙活性组分别下调55%和36%(P<0.05)。结论成功构建eIF1过表达鼻咽癌细胞株,eIF1过表达可显著下调鼻咽癌细胞的增殖和迁徙活性,提示其具有潜在的抑癌作用。

Objective To establish nasopharyngeal carcinoma cell line （CNE1） with eIF1 gene stable over-expression and to study its effects on the proliferation and migration of nasopharyngeal carcinoma cells. Methods EIF1 over-expression vector was constructed by adopting the pEGFPC1 eukaryotic expression system for transfecting nasopharYngeal carcinoma CNE1 cells. Thus the stably transfected EIFI-elF1 and its control cells were obtained. The over-expression situation of eIF1 in these cells was verified by real time fluorescence quantitative PCR（qPCR） and Western blot. The proliferation and migration activity of CNE1 eIF1 cells were tested by adopting the cell proliferation and migration tests. Results The enzyme digestion eleetrophoresis identification and sequencing showed that the pEGFPCI-eIF1 eukaryotic expression vector was successfully constructed. After mRNA and protein expression identification,compared with the, reloading plasmid transfection group,the eIF1 gene mRNA and protein expression levels in nasopharyngeal carcinoma cell line CNE1 stably over-expressing elF1 were up-regulated by 2.85 folds and 2.58 folds respectively （P〈0.05）, while,its proliferation and migration activities were down-regulated by 55 % and 36 % respective （P〈0.05）. Conclusion The nasopharyngeal carcinoma cell line over-expressing elF1 is successfully constructed, the eIF 1 over-expression could significantly down-regulate the proliferation and migration activities of nasopharyngeal carcinoma cells,suggesting that eIF1 has potential anti-tumor effect.