Chinese Journal of Tissue Engineering Research ›› 2012, Vol. 16 ›› Issue (2): 315-319.doi: 10.3969/j.issn.1673-8225.2012.02.029

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Construction and expressing activity of microRNA-125a plasmid expression vectors 

Zhang Tao, Li Dong, Li Hua, Gao Hui   

  1. Center of Tumor Diagnosis and Treatment, Chengdu Millitary General Hospital of Chinese PLA, Chengdu  610083, Sichuan Province, China
  • Received:2011-07-11 Revised:2011-10-18 Online:2012-01-08 Published:2012-01-08
  • About author:Zhang Tao★, Master, Chief physician, Center of Tumor Diagnosis and Treatment, Chengdu Millitary General Hospital of Chinese PLA, Chengdu 610083, Sichuan Province, China zhangato269@126.com

Abstract:

BACKGROUND: pSuper is a kind of eukaryotic expression vectors of non-coding RNA. It expresses the precursor of miRNA in plasma, and then the precursor is cut by Dicer enzyme to produce mature miRNA. This process completely imitates the formation process of endogenous miRNA. In this study, pSuper is utilized to construct an expression vector of miR-125a, which could inhibit liver cancer. It provides basis for the further investigation on antitumor mechanism.
OBJECTIVE: To construct the eukaryotic plasmid pSuper-miR-125a expressing miR-125a and to confirm the expressing efficiency and specificity of miR-125a in the plasmid.
METHODS: Target fragments in the precursor of miR-125a (Pre-miR-125a) were amplified by PCR. The fragments were cloned into pMD18-T transition plasmid using T-A cloning method. The target fragments were cut by restriction enzyme and then connected to pSuper plasmid to construct recombinant plasmid pSuper-miR-125a. The recombinant plasmid was transfected into Hela cells. The expressing efficiency and specificity of miR-125a in the recombinant plasmid was detected by real-time PCR.
RESULTS AND CONCLUSION: The fragments in miR-125a precursor (Pre-miR -125a) were successfully cloned into the promoter downstream of eukaryotic expression plasmid pSuper H1. The recombinant expression plasmid pSuper-miR-125a was constructed. The plasmid expresses miR-125a effectively and specifically after transfeced into Hela cells. These findings confirm that the eukaryotic expression plasmid pSuper is suitable for the expression research of miRNA.

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