Modulation of Mouse Coagulation Gene Transcription following Acute In Vivo Delivery of Synthetic Small Interfering RNAs Targeting HNF4α and C/EBPα

Hepatocyte nuclear factor 4 alpha (HNF4 alpha) and CCAAT/enhancer-binding protein alpha (C/EBP alpha) are important for the transcriptional control of coagulation factors. To determine in vivo the direct role of HNF4 alpha and C/EBP alpha in control of genes encoding coagulation factors, a synthetic small interfering (si) RNA approach was used that enabled strong reduction of mouse hepatic HNF4 alpha and C/EBP alpha under conditions that minimized target-related secondary effects. For both HNF4 alpha and C/EBP alpha, intravenous injection of specific synthetic siRNAs (siHNF4 alpha and siC/EBP alpha) resulted in more than 75% reduction in their liver transcript and protein levels 2 days post-injection. For siHNF4 alpha, 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 HNF4 alpha target gene mRNA levels were also observed at 5 days post-siRNA injection, despite a limited level of HNF4 alpha knockdown at this time point. Compared to HNF4 alpha, C/EBP alpha knockdown had a modest impact on genes encoding coagulation factors. A strong reduction in C/EBP alpha 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/EBP alpha knockdown. We conclude that in the mouse, HNF4 alpha has a direct and essential regulatory role for multiple hepatic coagulation genes, while a role for C/EBP alpha 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.