miRNA-451表达质粒的构建及其对iNOS基因表达的抑制

目的:探讨miRNA-451对肾小球系膜细胞中诱导型一氧化氮合酶(Inducible nitric oxide synthase,iNOS)基因表达的影响.方法:依据miRBase数据库中mmu-miR-451序列,设计两条寡核苷酸片段,退火处理后克隆至pGenesil-1质粒,构建pGenesil-1-miRNA-451质粒及阴性对照质粒pGenesil-1-control.将pGenesil-l-miRNA-451及pGenesil-1-control质粒分别转染小鼠肾小球系膜细胞,经G418筛选,建立稳定表达pGenesil-1-miRNA-451及pGenesil-1-control的细胞株.采用MTT比色法检测活细胞数并绘制生长曲线;RT-PCR、Western blot及免疫细胞化学检测iNOS基因表达的差异.结果:经测序证实重组质粒构建成功,与未处理的小鼠肾小球系膜细胞和稳定表达pGenesil-1-control的细胞相比,在稳定表达pGenesil-1-miRNA-451的小鼠肾小球系膜细胞中,细胞增殖受到抑制,iNOS基因的蛋白表达明显受到抑制,而iNOS基因的mRNA水平无明显变化.结论:miRNA-451在翻译水平抑制了iNOS基因的表达,并对小鼠肾小球系膜细胞生长具有显著的抑制作用.

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-1-control respectively,were generated by the cloning of annealed oligonueleotides 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-1-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 level in the cells transfected with pGenesil-1-miRNA-451,but not in the cells transfected with pGenesil-1-control,when compared with mesangial cells.Conclusion:miRNA-451 repressed the expression of iNOS gene translafionly,and significantly inhibited cell growth.