A Foundational Study for Normal F8-Containing Mouse Models for the miRNA Regulation of Hemophilia A: Identification and Analysis of Mouse miRNAs that Downregulate the Murine F8 Gene

Abstract

Hemophilia A (HA) is associated with defects in the F8 gene, encoding coagulation factor VIII (FVIII). Our previous studies show that F8-targeting micro RNAs (miRNAs), a group of small RNAs involved in gene regulation, can downregulate F8 expression causing HA in individuals with normal F8-genotypes and increased HA severity in patients with mutations in F8. Understanding the mechanistic underpinnings of human genetic diseases caused or modulated by miRNAs require a small animal model, such as a mouse model. Here, we report a foundational study to develop such a model system. We identified the mouse 3′untranslated region (3′UTR) on murine F8-mRNA (muF8-mRNA) that can bind to murine miRNAs. We then selected three miRNAs for evaluation: miR-208a, miR-351 and miR-125a. We first demonstrate that these three miRNAs directly target the 3′UTR of muF8-mRNA and reduce the expression of a reporter gene (luciferase) mRNA fused to the muF8-3′ UTR in mammalian cells. Furthermore, in mouse cells that endogenously express the F8 gene and produce FVIII protein, the ectopic expression of these miRNAs downregulated F8-mRNA and FVIII protein. These results provide proof-of-concept and reagents as a foundation for using a normal F8-containing mouse as a model for the miRNA regulation of normal F8 in causing or aggravating the genetic disease HA.

Hsa-miR-5581-3p and hsa-miR-542-3p target the F8 gene in hemophilia A without F8 mutations

“…12 They also found that murine miRNAs, including miR-208a, miR-351 and miR-125a, could target the 3'UTR of F8 mRNA in mouse models and mammalian cells. 13 The above evidence strongly supports the involvement of miRNAs in the pathogenesis of HA.…”

RNA sequencing suggests that non‐coding RNAs play a role in the development of acquired haemophilia

“…As presented in Figure 3, both miRNAs had higher expression in the acquired haemophilia samples compared to healthy and mild haemophilia, while in the comparison with other groups the expression was less intense. Moreover, the interaction of miRNAs and 3'UTR of F8 gene was evaluated in murine models, the study conducted by Jankowska reported miR‐208a, miR‐351 and miR‐125a as modulators of F8 gene by their interaction with 3'UTR of F8 gene, 27 furthermore similar study reported 52 miRNAs that interact with F7 , F8 and other haemostatic‐associated genes 28 …”