Hsa-mir-216a真核表达载体的构建及其在胃癌细胞SGC7901/DDP中的表达

Construction of hsa-mir-216a eukaryotic expression vector and its expression in gastric cancer cells SGC7901/DDP

目的:构建人mir-216a真核表达载体,实现其在胃癌细胞SGC7901/DDP中的表达,为进一步研究mir-216a的功能打下基础。方法:依据miRBase数据库中pre-mir-216a序列设计引物;PCR扩增pre-mir-216a基因并将其克隆至pGenesil-1载体;将pGenesil-1-hsa-mir-216a质粒转染SGC7901/DDP细胞,RT-PCR及实时定量RCR法检测成熟mir-216a在SGC7901/DDP细胞中的表达。结果:pGenesil-1-hsa-mir-216a质粒经酶切及测序鉴定正确;pGenesil-1-hsa-mir-216a质粒转染进SGC7901/DDP细胞后,RT-PCR检测mir-216a成熟体表达明显增强,实时定量PCR检测S/D/mir-216a组mir-216a成熟体表达较S/D/HK组上调(63.530±0.159)倍(t=1.201,P<0.01)。结论:成功构建了pGenesil-1-hsa-mir-216a真核表达质粒,并在胃癌细胞SGC/DDP中高效表达。

Objective： To construct a recombinant eukaryotic expression vector of hsa-mir-216a and to express it in gastric cancer SGC7901/DDP cells for detecting its effects.Methods： Primers were designed based on the pre-mir-216a sequence in miRBase database;the pre-mir-216a gene was amplified by PCR and cloned into pGenesil-1 vector.The gastric cancer SGC7901/DDP cells were transfected with pGenesil-1-hsa-mir-216a vector and the expression of mature miR-216a was examined in SGC7901/DDP cells by semi-quantitative RT-PCR and real-time PCR.Results： The results of DNA sequencing showed that sequences of pGenesil-1-hsa-mir-216a vector were correct;semi-quantitative RT-PCR and real-time PCR indicated that mature mir-216a was over-expressed in SGC7901/DDP cells after transfection of pGenesil-1-hsa-mir-216a vector,real-time PCR indicated that the expression of mature mir-216a in S/D/mir-216a cells was（63.530±0.159） folds that of S/D/HK cells（t=1.201,P0.01）.Conclusion： The eukaryotic expression vector of hsa-mir-216a was successfully constructed and effectively expressed in SGC7901/DDP cells.