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.
Highlights
Hemophilia A (HA) is an X-linked bleeding disorder mainly caused by defects in, or a deficiency of, blood coagulation factor VIII
As a prerequisite to developing a suitable small animal model such as a mouse, in this report, we first identified the 3 untranslated region (3 UTR) of murine F8-miRNAs on F8 gene expression (mRNA) and identified and characterized murine micro RNAs (miRNAs) that can bind to the murine F8 gene (muF8)-mRNA and dysregulate factor VIII (FVIII) expression, so that, based on these foundational studies, a normal F8-containing mouse could serve as an animal model for this line of investigations
Like in humans, the muF8 gene comprises of 26 exons, which encode a polypeptide chain of 2319 amino acids (NP_032003) vs. 2351aa in humans (NP_000123)) and exhibit a similar domain structure [9]
Summary
Hemophilia A (HA) is an X-linked bleeding disorder mainly caused by defects in, or a deficiency of, blood coagulation factor VIII. We have previously demonstrated the microRNA-based posttranscriptional downregulation of F8 [4,5,6] and that this mechanism is observed in HA patients with no defects in the F8 gene. Our previous studies for the first time provided experimental evidence by using HA patient samples that micro RNAs (miRNAs) can downregulate F8 gene expression and this phenomenon was observed in HA subjects with no mutations in the F8 gene [5]. As a prerequisite to developing a suitable small animal model such as a mouse, in this report, we first identified the 3 UTR of murine F8-mRNA and identified and characterized murine miRNAs that can bind to the muF8-mRNA and dysregulate FVIII expression, so that, based on these foundational studies, a normal F8-containing mouse could serve as an animal model for this line of investigations
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