Construction Of MIRNA-451 Expression Plasmid And Its Inhibitory Effect On The Expression Of INOS Gene

Objective To elucidate the effect of miRNA-451 on the expression of inducible nitric oxide synthase (iNOS) gene in the mesangial cells of mice.Methods Two template DNA sequences were designed based on mmu-miR-451 sequence in miRBase database. The mmu-miR-451 expression plasmid and a control plasmid, named pGenesil-1-miRNA-451 and pGeneil-control respectively, were generated by the cloning of annealed oligonucleotides into pGenesil-1, then transfected into mesangial cells, which were stably selected by G418 to establish mesangial cell lines stablely expressing pGenesil-1-miRNA-451 and pGenesil-control. The living cells were counted by MTT assay and cell growth curves were drew to analyze the cell proliferation. The iNOS mRNA level was assessed by RT-PCR. The expression of iNOS protein was determined by Western blot and immunocytochemistry.Results The recombinant vectors were verified by sequencing. The cell growth curves indicated the proliferation of cells transfected with pGenesil-1-miRNA-451 were inhibited significantly, compared with that of cells transfected with pGenesil-control and cells without treatments. RT-PCR analysis showed the levels of iNOS mRNA were almost unchanged in the cells with or without treatments. Western blot and immunocytochemistry demonstrated a significant decrease of iNOS protein levels in the cells transfected with pGenesil-1-miRNA-451, but not in the cells transfected with pGenesil-control, when compared with mesangial cells.Conclusion miRNA-451 repressed the expression of iNOS gene translationly, and significantly inhibited cell growth.