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

胡燕; 廖珍媛; 李永菊; 陈超; 朱顺飞; 熊思东; 徐林

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

更新时间：2026-01-09

- Construction of pEGFP-C2-miR-126-sponge eukaryotic expression vector and its expression activity
- Journal of Army Medical University Vol. 36, Issue 1, Pages: 33-37(2014)
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  CLC： R394-33;R394.3;R735.7
- Published：2014
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胡燕,廖珍媛,李永菊,陈超,朱顺飞,熊思东,徐林.真核表达载体pEGFP-C2-miR-126-sponge的构建及其表达活性研究[J].陆军军医大学学报(原第三军医大学学报),2014,36(01):33-37.

　Hu Yan,Liao Zhenyuan,Li Yongju,Chen Chao,Zhu Shunfei,Xiong Sidong,Xu Lin.Construction of pEGFP-C2-miR-126-sponge eukaryotic expression vector and its expression activity[J].J Amry Med Univ (J Third Mil Med Univ),2014,36(01):33-37.
胡燕,廖珍媛,李永菊,陈超,朱顺飞,熊思东,徐林.真核表达载体pEGFP-C2-miR-126-sponge的构建及其表达活性研究[J].陆军军医大学学报(原第三军医大学学报),2014,36(01):33-37. DOI：

　Hu Yan,Liao Zhenyuan,Li Yongju,Chen Chao,Zhu Shunfei,Xiong Sidong,Xu Lin.Construction of pEGFP-C2-miR-126-sponge eukaryotic expression vector and its expression activity[J].J Amry Med Univ (J Third Mil Med Univ),2014,36(01):33-37. DOI：

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（

0.05）；MTT检测发现，p-miR-126-sp组中细胞增殖数目明显减少(

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（

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。

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 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 (

0.05). MTT assay showed that the proliferation of HepG2 cells was dramatically reduced in p-miR-126-sp transfected cells (P0.05); Furthermore

the number of both colony forming and migrating cells were also remarkably reduced in p-miR-126-sp transfected cells (

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.

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