Department of Genetic Medicine and Development, University of Geneva Faculty of Medicine, Geneva, Switzerland.
Blood. 2010 Oct 7;116(14):2608-15. doi: 10.1182/blood-2010-02-268011. Epub 2010 Jun 22.
Elevated levels of fibrinogen are associated with increased risk of cardiovascular disease, whereas low fibrinogen can lead to a bleeding disorder. We investigated whether microRNAs (miRNAs), known to act as post-transcriptional regulators of gene expression, regulate fibrinogen production. Using transfection of a library of 470 annotated human miRNA precursor molecules in HuH7 hepatoma cells and quantitative measurements of fibrinogen production, we identified 23 miRNAs with down-regulating (up to 64% decrease) and 4 with up-regulating effects (up to 129% increase) on fibrinogen production. Among the down-regulating miRNAs, we investigated the mechanism of action of 3 hsa-miR-29 family members and hsa-miR-409-3p. Overexpression of hsa-miR-29 members led to decreased steady-state levels of all fibrinogen gene (FGA, FGB, and FGG) transcripts in HuH7 cells. Luciferase reporter gene assays demonstrated that this was independent of miRNA-fibrinogen 3'-untranslated region interactions. In contrast, overexpression of hsa-miR-409-3p specifically lowered fibrinogen Bβ mRNA levels, and this effect was dependent on a target site in the fibrinogen Bβ mRNA 3'-untranslated region. This study adds to the known mechanisms that control fibrinogen production, points toward a potential cause of variable circulating fibrinogen levels, and demonstrates that a screening approach can identify miRNAs that regulate clinically important proteins.
纤维蛋白原水平升高与心血管疾病风险增加有关，而纤维蛋白原水平降低则可能导致出血性疾病。我们研究了是否已知可作为基因表达转录后调控因子的 microRNAs（miRNAs）调节纤维蛋白原的产生。通过在 HuH7 肝癌细胞中转染 470 个注释的人 miRNA 前体分子文库，并对纤维蛋白原的产生进行定量测量，我们鉴定出 23 个下调（最多降低 64%）和 4 个上调（最多增加 129%）纤维蛋白原产生的 miRNA。在下调的 miRNA 中，我们研究了 3 个 hsa-miR-29 家族成员和 hsa-miR-409-3p 的作用机制。hsa-miR-29 成员的过表达导致 HuH7 细胞中所有纤维蛋白原基因（FGA、FGB 和 FGG）转录本的稳态水平降低。荧光素酶报告基因检测表明，这与 miRNA-纤维蛋白原 3'-非翻译区相互作用无关。相比之下，hsa-miR-409-3p 的过表达特异性降低纤维蛋白原 Bβ mRNA 水平，并且这种作用依赖于纤维蛋白原 Bβ mRNA 3'-非翻译区中的靶位点。本研究增加了控制纤维蛋白原产生的已知机制，指出了可变循环纤维蛋白原水平的潜在原因，并证明了筛选方法可以鉴定调节临床重要蛋白的 miRNA。
