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

真核表达载体pEGFP-C2-miR-126-sponge的构建及其表达活性研究 胡 燕1，廖珍媛1，李永菊1，陈 超1，朱顺飞1，熊思东2，徐 林1 （563000 贵州 遵义，遵义医学院免疫学教研室1； 215000 江苏苏州，苏州大学生物医学研究院2） [摘要] 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法检测细胞中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(P0.05)；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。 [关键词] miR-126-sponge；肝癌；HepG2细胞；细胞增殖 [中图法分类号] [文献标志码]A Construction of pEGFP-C2-miR-126-sponge eukaryotic expression vector and its expression activity Hu Yan1, Liao Zhenyuan1, Li Yongju1, Chen Chao1, Zhu Shunfei1, Xiong Sidong2, Xu Lin1 (Department of immunology, Zunyi Medical College, Zunyi，, Guozhou Province, 563000, China；; 2College of Biology Basic Medical Sciences, ，Suzhoochowou University，, Suzhou，, Jiangsu Province, 215000，, China) [Abstract] 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 HCC cell line HepG2 in vitro, and thus in order to provide preliminary experimental fundament for study successive researching on on the role of miR-126 in the pathogenesis of HCCthe 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 HCC 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 Rreal-time PCR. Moreover, the proliferation of HepG2 cells was observed by MTT assay and colo