Construction of MiRNA Eukaryotic Expression Vector and its Stable Expression in Human Liver Cancer Cells

摘要

Recent research indicates that miR-26a is involved in various biological processes including cell cycle, apoptosis and Hepatocellular carcinoma (HCC). In present report, we demonstrate the construction of recombinant plasmid miR-26a expression vector and its stable expression of miR-26a in transfected human liver cancer cells (HepG2). The double strands oligo encoding the miR-26a was designed and synthesized to generate the mature miR-26a expression plasmid. Use of the vectors in mammalian cells permits visual detection of cells expressing the pre-miRNA through co-cistronic expression of EGFP. The positive clones were screened by restriction enzyme digestion and sequenced. The new expression vector of miR-26a was named pHsa-miR-26a. PHsa-miR-26a and its controls were transfected to HepG2 cells. Fluorescence detection displayed that fluorescence intensity of GFP was highest during 48 and 72 hours post-transfection, and qRT-PCR showed a significantly increase in miR-26a expression in the vector transfected cells compared with the expression in its controls. The recombinant plasmid expression vector of miR-26a was constructed successfully, which may facilitate further study of its function in the process of cell cycle and HCC.