Modulation of mouse coagulation gene transcription following acute in vivo delivery of synthetic small interfering RNAs targeting HNF4α and C/EBPα.
Modulation of mouse coagulation gene transcription following acute in vivo delivery of synthetic small interfering RNAs targeting HNF4α and C/EBPα.
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DOI：
10.1371/journal.pone.0038104
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
van Vlijmen BJ
中科院分区：
文献类型：
--
作者：
Safdar H;Cheung KL;Vos HL;Gonzalez FJ;Reitsma PH;Inoue Y;van Vlijmen BJ
肝细胞核因子4α（HNF 4 α）和CCAAT/增强子结合蛋白α（C/EBPα）在凝血因子的转录调控中起重要作用。为了在体内确定HNF 4 α和C/EBPα在控制编码凝血因子的基因中的直接作用，使用了一种合成的小干扰（si）RNA方法，该方法能够在最小化靶相关继发效应的条件下强烈降低小鼠肝脏HNF 4 α和C/EBPα。对于HNF 4 α和C/EBPα，静脉注射特异性合成siRNA（siHNF 4 α和siC/EBPα）导致注射后2天其肝脏转录物和蛋白质水平降低75%以上。对于siHNF 4 α，与注射对照siRNA的动物中的水平相比，这与凝血基因Hrg、Proz、Serpina 5、F11、F12、F13 b、Serpinf 2、F5和F9的转录水平显著和显著降低相一致（按效应大小顺序）。在siRNA注射后5天也观察到HNF 4 α靶基因mRNA水平显著降低，尽管在该时间点HNF 4 α敲低水平有限。与HNF 4 α相比，C/EBPα敲除对编码凝血因子的基因有适度的影响。C/EBPα转录物和蛋白质水平的强烈降低导致对照基因Pck 1和Fasn的转录物水平显著受影响，凝血基因Fba、Fbg和F5的适度下调。F5和F11是在长时间（5天）C/EBPα敲低后受到显著影响的唯一凝血基因。我们的结论是，在小鼠中，HNF 4 α对多个肝凝固基因具有直接和重要的调节作用，而C/EBPα的作用更为有限。此外，这项研究表明，合成的siRNA提供了一个简单和快速的手段，确定肝脏转录因子参与体内。
Hepatocyte nuclear factor 4α (HNF4α) and CCAAT/enhancer-binding protein α (C/EBPα) are important for the transcriptional control of coagulation factors. To determine in vivo the direct role of HNF4α and C/EBPα in control of genes encoding coagulation factors, a synthetic small interfering (si)RNA approach was used that enabled strong reduction of mouse hepatic HNF4α and C/EBPα under conditions that minimized target-related secondary effects. For both HNF4α and C/EBPα, intravenous injection of specific synthetic siRNAs (siHNF4α and siC/EBPα) resulted in more than 75% reduction in their liver transcript and protein levels 2 days post-injection. For siHNF4α, this coincided with marked and significantly reduced transcript levels of the coagulation genes Hrg, Proz, Serpina5, F11, F12, F13b, Serpinf2, F5, and F9 (in order of magnitude of effect) as compared to levels in control siRNA injected animals. Significant decreases in HNF4α target gene mRNA levels were also observed at 5 days post-siRNA injection, despite a limited level of HNF4α knockdown at this time point. Compared to HNF4α, C/EBPα knockdown had a modest impact on genes encoding coagulation factors. A strong reduction in C/EBPα transcript and protein levels resulted in significantly affected transcript levels of the control genes Pck1 and Fasn and a modest downregulation for coagulation genes Fba, Fbg and F5. F5 and F11 were the sole coagulation genes that were significantly affected upon prolonged (5 day) C/EBPα knockdown. We conclude that in the mouse, HNF4α has a direct and essential regulatory role for multiple hepatic coagulation genes, while a role for C/EBPα is more restricted. In addition, this study demonstrates that synthetic siRNA provides a simple and fast means for determining liver transcription factor involvement in vivo.
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