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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 design and construct miRNAs targeting Snai1 and finally to identify effective interference plasmids and screen stable transfected gastric cancer cell line SGC-7901. Methods: Four pairs of Snai1 pcDNATM6.2-GW / EmGFPmiR microRNAs and one pair of null control microRNA interference plasmids were designed and constructed. The interference plasmids were transfected into gastric cancer cell line SGC-7901 with Roche BD transfection reagent, and the transfection efficiency was confirmed by observing the green fluorescence by inverted fluorescence microscope. SGC-7901 cells were treated with different concentrations of blasticidin, and the concentration of blasticidin in SGC-7901 cells was obtained. The effect of 4 pairs of interference plasmids and negative control plasmids on the expression of snai1 protein was detected by Western blot. Results: Sequencing showed that the Snai1 interference sequence and the reading frame were completely correct. The SGC-7901 cell line transiently transfected with the interference plasmids observed an up to 85% green fluorescence under an inverted fluorescence microscope. The screening concentration of blasticidin for SGC-7901 cells was 5 μg / ml. Western blot results showed that the interference sequence Mi-1 had a strong interference effect on Snai1. CONCLUSION: Snai1 was successfully constructed and eukaryotic expression vector was successfully constructed, and both plasmids and stable transfectants were screened out at the same time, which laid the foundation for further study on the role of Snai1 in gastric cancer.