Modulation of mouse coagulation gene transcription following acute in vivo delivery of synthetic small interfering RNAs targeting HNF4α and C/EBPα.

Abstract

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.