真核表达载体pEGFP-C2-miR-126-sponge的构建及其表达活性研究

Construction of pEGFP-C2-miR-126-sponge eukaryotic expression vector and its expression activity

目的构建靶向miR-126的真核表达载体pEGFP-C2-miR-126-海绵体(sponge),并探讨其下调miR-126的表达对肝癌HepG2细胞体外生长的影响,为后续深入研究miR-126在肝癌发生中的作用提供前期实验基础。方法合成miR-126-sponge,构建pEGFP-C2-miR-126-sponge真核表达载体,体外瞬时转染肝癌细胞HepG2,荧光显微镜下观察EGFP的表达情况;Real-time PCR检测细胞中miR-126的表达水平;MTT法和克隆形成实验检测细胞的增殖变化;划痕实验观察细胞的体外迁移能力改变。结果成功构建pEGFP-C2-miR-126-sponge真核表达载体(命名为p-miR-126-sp);Real-time PCR结果显示,与对照组(p-Cont)相比,p-miR-126-sp组中miR-126表达水平显著降低(P<0.05);MTT检测发现,p-miR-126-sp组中细胞增殖数目明显减少(P<0.05);此外,p-miR-126-sp组的克隆形成数和细胞迁移数均明显减少(P<0.05)。结论 miR-126-sponge可显著下调miR-126的表达水平进而抑制肝癌细胞HepG2的体外增殖及迁移能力。

Objective To construct an eukaryotic expression vector of pEGFP-C2-miR-126-sponge targeting to miR-126 and investigate its effect on the expression of miR-126 and growth of hepatocellular carcinoma cell line HepG2,in order to provide preliminary experimental fundament for study on the role of miR-126 in the pathogenesis of the cancer. Methods The sequence of miR-126-sponge was designed and synthesized. The pEGFP-C2-miR-126-sponge eukaryotic expression vector was constructed and then transiently transfected into HepG2 cells in vitro. The proportion of EGFP-positive cells was observed under the fluorescence microscope. Then,the expression level of miR-126 was detected by real-time PCR. Moreover,the proliferation of HepG2 cells was observed by MTT assay and colony formation assay. Finally,the migration of HepG2 cells was also determined by Scratch assay. Results The pEGFP-C2-miR-126-sponge eukaryotic expression vector（ termed as p-miR-126-sp） was successfully constructed. Compared with control group（ p-Cont）,the expression level of miR-126 was significantly decreased in p-miR-126-sp transfected HepG2 cells（ P〈0. 05）. MTT assay showed that the proliferation of HepG2 cells was dramatically reduced in p-miR-126-sp transfected cells（ P〈0. 05）; Furthermore,the number of both colony forming and migrating cells were also remarkably reduced in p-miR-126-sp transfected cells（ P〈0. 05）. Conclusion The miR-126-sponge can significantly reduce the expression of miR-126 and inhibit the proliferation and migration of the HepG2 cells.