Snai1 miRNA干扰质粒构建及胃癌细胞系稳定株筛选

Construction of Snai1 miRNA recombinant eukaryotic expression vectors and screening of stably transfected cell clone

目的:设计并构建针对Snai1的微小干扰核糖核酸(miRNA),最终鉴定出有效干扰质粒并筛选稳定转染的胃癌细胞株SGC-7901。方法:设计并构建4对Snai1的pcDNATM6.2-GW/EmGFPmiR microRNA及1对无效对照microRNA干扰质粒。将干扰质粒用罗氏BD转染试剂转染胃癌细胞株SGC-7901,通过倒置荧光显微镜观察绿色荧光确定转染效率。分别用不同浓度l的杀稻瘟菌素作用于SGC-7901细胞,得到杀稻瘟菌素对SGC-7901细胞的筛选浓度。Westernblot检测4对干扰质粒、阴性对照质粒对snai1蛋白水平表达的影响。结果:测序表明,Snai1干扰序列及读码框完全正确,干扰质粒瞬时转染的SGC-7901细胞系在倒置荧光显微镜下观察绿色荧光达85%以上。杀稻瘟菌素对于SGC-7901细胞的筛选浓度为5μg/ml。Westernblot结果显示,干扰序列Mi-1对Snai1有较强的干扰效果。结论:成功构建了Snai1干扰真核表达载体,同时筛选出有效干扰质粒及稳定转染株,为进一步研究Snai1在胃癌中的作用奠定了基础。

Objective：To construct recombinant eukatyotic expression vector of micro-RNA for Snai1gene,and screen the stably transfected gastric carcinoma cell clone SGC-7901.Methods：Microinterference ribonucleic acid（miRNA） nucletides of Snai1were syn-thesized and inserted into pcDNATM6.2-GW/EmGFPmiR vector,which were confirmed by sequencing,then the recombinant miRNA vectors were transfected into SGC-7901 by Roche BD.The transfection efficiency was observed under inverted fluorescence microscope.According to the fatal dose of blasticidin to SGC-7901,and the selection concentration of blasticidin to SGC-7901 cell.The protein ex-pression of Snai1was detected by Western blot.Results：Sequencing suggested that miRNA eukaryotic expression vectors targeting Snia1 possesse correct nucleotide sequence and read frame,and the express of the green fluorescent protein was over 85% when the transient transfected SGC-7901 cell line observed nuder inverted fluorescence microscope.The screening concentration of blasticidin to SGC-7901 cell was 5μg/ml.The results of Western blot showed that the sequence of Mi-1could more effectively knockdown the expres-sion level of Snai1 than the others.Conclusion：miRNA eukaryotic expression vectors targeting Snia1 were successfully constructed and the effectively interference miRNA were identified,and the stably transfected cell clone obtained may be useful for understanding the ef-fect of Snai1 in gastric cancer.