摘要
目的构建miR-17家族(miR-17、miR-20a、miR-20b、miR-93、miR-106a、miR-106b)真核表达载体,探究它们对人肝癌SMMC-7721细胞体外增殖的影响。方法通过Eco RⅠ和HindⅢ双酶切真核表达载体pc DNA6.2-GW/Em GFP-miR,得到大片段后与人工合成的miR-17家族成熟体5p的寡聚核苷酸连接,通过DNA测序、BLAST检验以及双酶切电泳方法验证是否成功构建miR-17家族的真核表达载体。将构建成功的载体,用Lipofectamine2000瞬时转染人肝癌SMMC-7721细胞,应用real-time PCR方法检测miR-17家族表达水平,用CCK8法检测细胞增殖能力的变化。结果双酶切真核表达载体pc DNA6.2-GW/Em GFP-miR,得到的大片段与人工合成的miR-17家族成熟体5p寡聚核苷酸连接,通过DNA测序、BLAST检验及双酶切电泳的方法验证成功构建了miR-17家族真核表达载体。瞬时转染人肝癌SMMC-7721细胞后miR-17家族表达水平有了显著增加,能够促进SMMC-7721细胞的体外增殖。结论成功构建miR-17家族真核表达载体,得到瞬时转染miR-17家族的人肝癌SMMC-7721细胞,并观察到miR-17家族过表达可以促进人肝癌细胞的增殖。
Objective To construct microRNA-17 family＇ s eukaryotic expression vectors and explore their effects on the proliieration ot human liver cancer cell SMMC-7721. Methods The pcDNA6. 2-GW/EmGFP-miR eukaryotic expression vector was digested using Hind HI and EcoR I, and the obtained large fragment was connected to miR-17 family 5p oligonuleotide, and then identified by DNA se- quencing,BLAST test and electrophoresis after double enzyme digestion. The miR-17 family＇s eukaryotic expression vectors were trans- fected into SMMC-7721 cells and their expression levels were determined by real-time PCR. The proliferation of SMMC-7721 cell was ex- amined by CCK8. Results The microRNA-17 family＇ s eukaryotic expression vectors were confirmed to be established successfully. The expression levels of miR-17 families in SMMC-7721 ceils transfected with eukaryotic expression vectors were increased significantly（P 〈 O. 05）. The proliferation of SMMC-7721 cells in vitro was also promoted. Conclusion The miR-17 family＇ s eukaryotic expression vec- tors are successfully constructed and human liver cancer cell SMMC-7721 instantaneously expressing miR-17 family is obtained, which preliminarily confirms that the overexpression of miR-17 family can promote the proliferation of human liver cancer cell.
出处
《山西医科大学学报》
CAS
2015年第6期523-527,607,共6页
Journal of Shanxi Medical University
基金
国家自然科学基金资助项目(31100921)
