胃癌中靶向E2F1基因的miR-331真核表达载体的构建及功能  

Construction of a eukaryotic expression vector carrying the miR-331 gene

作　　者：程力[1] 李乐平[1] 张黎[1] 靖昌庆[1] 徐韬[1] 李辰生[1] 郭晓波[1] 

机构地区：[1]山东大学附属山东省立医院胃肠外科,山东省济南市250021

出　　处：《世界华人消化杂志》2011年第14期1451-1456,共6页World Chinese Journal of Digestology

摘　　要：目的:研究构建靶向E2F1基因的miR-331真核表达载体,评估其转染人胃癌细胞株SGC-7901细胞后对E2F1基因的干扰效果及其功能,探讨miR-331在胃癌中可能的作用机制.方法:将外源性重组真核表达载体pSuper/miR-331转染到人胃癌细胞株SGC-7901内,经G418筛选并建立高表达miR-331的稳定转染细胞株.稳定表达该miR-331的细胞为SGC-7901-pSuper/miR-331组,转染空质粒细胞及未处理细胞SGC-7901-pSuper组和SGC-7901组作为对照,采用实时荧光定量PCR验证miR-331在稳定转染细胞的表达,蛋白印迹检测其对E2F1基因表达的干扰效果和SGC-7901细胞的功能.结果:与SGC-7901-pSuper组相比,转染了pSu-per/miR-331高表达质粒的SGC-7901细胞中E2F1蛋白表达明显减少,降低了4.27倍(0.206±0.037vs0.879±0.082,P<0.05);与SGC-7901-pSuper组相比,转染了pSuper/miR-331高表达质粒的SGC-7901细胞的克隆形成明显减少,降低了3.80倍(1.863±0.098vs7.074±0.182,P<0.05),而SGC-7901-pSuper组与SGC-7901组组间无统计学意义.结论:miR-331真核表达载体构建和稳定表达胃癌细胞筛选成功,为继续深入的研究miR-331在胃癌中的功能奠定了基础.AIM：To explore the potential role of miR-331 in gastric carcinogenesis by constructing a eukaryotic expression vector carrying the miR-331 gene and investigating the impact of transfection with this vector on the expression of E2F1,a direct target of miR-331.METHODS：A recombinant plasmid carrying miR-331（pSuper/miR-331） was transfected into SGC-7901 cells by Lipofectin-mediated method.Cells stably expressing miR-331 were selected using G418.Untransfected SGC-7901 cells and those transfected with empty pSuper plasmid were used as controls.The expression of miR-331 was detected by TaqMan real-time PCR,while the expression of E2F1 protein was detected by Western blot.RESULTS：Compared with untransfected SGC-7901 cells,the expression level of E2F1 protein exhibited a 4.27-fold decrease in cells stably expressing miR-331（0.206 ± 0.037 vs 0.879 ± 0.082,P0.05）.The rate of colony formation on soft agar significantly decreased in cells stably transfected with the recombinant vector when compared to control cells（1.863 ± 0.098 vs 7.074 ± 0.182,P0.05）.CONCLUSION：A eukaryotic expression vector stably expressing miR-331 has been successfully constructed and can be used to study the functions of miR-331 in human gastric cancer.

关 键 词：胃癌 微小RNA-331 表达载体 SGC-7901细胞 

分 类 号：R735.2[医药卫生—肿瘤]