自身启动子调控miR-7真核表达载体的构建及其意义  

Construction of an eukaryotic expression vector encoding miRNA-7 with self promoter and its significance

作　　者：郭萌萌[1] 廖珍媛[1] 赵娟娟[1] 陶弋婧 胡燕[1] 陈超[1] 秦娜琳[1] 郑静[1] 徐林[1] 

机构地区：[1]遵义医学院免疫学教研室暨贵州省免疫学研究生教育创新基地,贵州遵义563099

出　　处：《遵义医学院学报》2015年第3期219-225,共7页Journal of Zunyi Medical University

基　　金：国家自然科学基金资助项目(NO:31370918);教育部新世纪优秀人才计划资助项目(NO:NCET-12-0661);贵州省国际合作项目(NO:10C315)

摘　　要：目的构建带有miR-7自身启动子的miR-7重组真核表达载体,研究其对人肺癌细胞增殖、迁移和凋亡的影响。方法预测miR-7启动子序列,设计含有该序列和miR-7前体序列目的片段的引物,PCR扩增后亚克隆入p GL3.0-Basic载体以构建miR-7自身启动子调控的真核表达载体p GL3.0-Basic-miR-7-promoter(命名为p-miR-7-pro);将所构建载体转染人肺癌95D细胞,Real-time PCR探针法检测95D细胞中miR-7的表达水平;MTT和克隆形成实验分别检测95D细胞的增殖和克隆形成能力;划痕法观察95D细胞体外迁移能力的变化;FACS检测95D细胞的凋亡情况。结果酶切和测序结果证明成功构建p-miR-7-pro真核表达载体,转染95D细胞后可有效表达miR-7;与对照组相比,p-miR-7-pro转染组95D细胞的体外增殖能力受到明显的抑制(0.60±0.03 vs 0.19±0.05,P<0.05);同时其迁移能力也显著降低(473±7 vs 99±2,P<0.05);而细胞凋亡则显著增加(9.233±0.586%vs 12.967±0.643%,P<0.05)。结论成功构建由自身启动子调控的miR-7真核表达载体,为后续研究miR-7在肺癌基因治疗中的作用提供重要实验基础。Objective To construct an eukaryotic expression vector encoding miRNA-7（miR-7） with its own promoter and explore its effects on the growth,migration and apoptosis of lung cancer cells.Methods The PCR primer was designed to amplify the promoter sequence and precursor sequence fragment of miR-7.The PCR products were subcloned into p GL3.0-Basic vector to construct the eukaryotic expression vector p GL3.0-Basic-miR-7-promoter（Named as p-miR-7-pro）.Then,the recombinant plasmid p-miR-7-pro was transiently transfected into human lung cancer cell line 95 D cells in vitro.The expression level of miR-7 in 95 D cells was detected by real-time PCR.The proliferation and colony formation ability of cells were detected by MTT assay and colony formation assay,respectively.The migration of cells in vitro was determined by scratch assay.The apoptosis of cells was investigated by flow cytometry analysis.Results Enzyme digestion and DNA sequencing validated the successful construction of p-miR-7-pro eukaryotic expression vector.The expression level of miR-7 in p-miR-7-pro transfected group was higher than that in control group.Moreover,compared with control group（p-Cont）,the proliferation and migration ability of cells in p-miR-7-pro-transfected group significantly decreased,respectively（0.60 ± 0.03 vs 0.19 ± 0.05,P 〈0.05）（473 ± 7 vs 99 ± 2,P 〈0.05）.Conversely,the apoptosis of 95 D cells significantly increased（9.233 ± 0.586 % vs 12.967 ±0.643%,P 〈0.05）.Conclusion The eukaryotic expression vector encoding miRNA-7 with self promoter is successfully constructed,which provides an important foundation for the study on the effects of miR-7 in gene therapy against lung cancer.

关 键 词：miR-7 真核表达 肺癌 95D细胞 增殖 迁移 凋亡 

分 类 号：R734.2[医药卫生—肿瘤]