Objective : To construct microRNA - 378 ( miR - 378 ) eukaryotic expression vector pcDNATM6. 2 Gw/ + EmGFP - miR378. Methods : Synthesis the two pre - miR - 378 single - stranded oligos from miRBase. Afer annealing to generate double - stranded oligonucleotides, it was cloned into pcDNATM 6. 2 - GW/ ± EmGFP - miR followed hy enzyme digestion and sequence analysis. Then the human gastric cancer cell line SGC - 7901 was transfected hy pcDNATM 6. 2 - GW/ ± EmGFP - miR378 , and the expression of miR - 378 was detected by real - time quantitative PCR ( RT - qRCP ). Results : pcDNATM 6. 2 - GW/ + EmGFP - miR378 was identified by enzyme digestion and sequence analysis. RT - qRCP showed that it could up - regulate the expression of miR - 378 in SGC 7901. Conculsion : The miR - 378 eukaryotic expression vector has been successfully constucted, providing a foundation for further study on the effect of miR - 378 0n human gastric cancer.%目的:构建microRNA-378(miR-378)真核表达质粒pcDNATM6.2-GW/±EmGFP-miR378.方法:根据miRBase提供的序列合成miR378前体序列,退火形成双链后,插入真核表达质粒pcDNATM6.2-GW/±EmGFP-miR,构建 pcDNATM 6.2-GW/±EmGFP-miR378.进行酶切和测序鉴定后,转染人胃癌细胞株SGC-7901,应用实时定量PCR检测转染后miR378的表达量.结果:pcDNATM 6.2-GW/±EmGFP-miR378的酶切、测序表明与设计一致,实时定量PCR证实转染pcDNATM 6.2-GW/±EmGFP -miR378能够上调miR378的表达水平.结论:成功构建了miR378真核表达质粒pcDNATM 6.2-GW/±EmGFP-miR378,为进一步探讨miR378在胃癌中的作用奠定了基础.
展开▼
