Construction of pcDNA-DEST47-miR-29b and its regulatory effects on expression of VEGF

Objective To construct the recombinant expression vector of has-miR-29b and to explore its effect on expression of VEGF.Methods Bioinformatics was used to analyze the potential binding sites of has-miR-29b and VEGF-3'UTR.The precursor of miR-29b was amplified by PCR and the PCR products were cloned into digestion pcDNA-DEST47 vector to construct pcDNA-DEST47-miR-29b expression vector.The effect of miR-29b interaction with the 3'-UTR of VEGF on luciferase activity was detected with a dual 1uciferase assay system and the expression level of VEGF protein affected by miR-29b was detected by Western blotting.Results The luciferase activity was significantly lower in cells co-transfected with psiCHECK2-VEGF luciferase reporter plasmid and pcDNA-DEST47-miR-29b compared with empty vector group and Lu-VEGF group(P 0.01).The luciferase activity was not inhibited in the other types of miRNA(Let-7g),whereas miR-29b could negatively regulate the luciferase activity by interacting with the 3'-UTR of VEGF.VEGF protein expression was significantly inhibited in Jurkat cells transfected with pcDNA-DEST47-miR-29b plasmid.Conclusion pcDNA-DEST47-miR-29b plasmid were successfully constructed; miR-29b targetedly regulates the expression of VEGF.