Abstract
Abstract Introduction: Our laboratory previously demonstrated that miR-9 promotes the invasive properties of normal and malignant mast cells and that overexpression of miR-9 is associated with spontaneous metastasis in canine mast cell tumors (MCTs), a well-established large animal model of malignant mast cell disease. The purpose of this study was to further investigate the biologic effect and mechanisms by which mir-9 enhances normal mast cell invasion. Methods: Bone marrow-derived mast cells (BMMCs) were generated from Cpa3-Cre; miR-9Δfl/Δfl, and Cpa3-Cre; miR-9Δfl/Δfl mice and Taqman miRNA assays were used to quantify mature miR-9 expression. BMMCs were evaluated for differences in proliferative capacity, the ability to migrate through Matrigel, sensitivity to degranulation, and the expression and release of growth factor expression and release following chemical stimulation. RNA sequencing was performed by the OSUCCC Genomics Resource and changes in protein and mRNA expression were validated with Western blotting and quantitative PCR. Results and Conclusions: We generated a transgenic mouse carrying a floxed STOP-miR-9 transgene (miR-9Δfl/Δfl) in which expression of miR-9 occurs only after Cre-mediated recombination and crossed these mice with Cpa3-Cre mice which express Cre recombinase under the control of the carboxypeptidase A3 (Cpa3) promoter, restricting expression of miR-9 to mast cells and basophils. Bone marrow-derived mast cells (BMMCs) derived from Cpa3-Cre;miR-9Δfl/Δfl mice significantly enhanced the invasive phenotype of mast cells. Moreover, the enforced expression of miR-9 in BMMCs markedly changed the production and release of cytokine and growth factors in response to chemical stimulation, including substantial up-regulation of IL-4, IL-13, and GM-CSF transcript and enhanced TGF-β1 release. RNA sequencing revealed a unique transcriptional profile associated with miR-9 over-expression in the Cpa3-Cre;miR-9Δfl/Δfl derived BMMCs and identified increased expression of several mast cell-restricted proteases involved in extracellular matrix and tissue remodeling including CMA1 and MCP-6. Furthermore, transduction of Cpa3-Cre;miR-9Δfl/Δfl derived BMMCs with CMA1 shRNA abrogated miR-9-dependent mast cell invasion. Studies are underway to investigate the regulatory pathway mediated by miR-9 that contributes to CMA1 up-regulation and promotes the invasive phenotype. In summary, these data provide evidence supporting a critical role for miR-9 in mast cell invasion and suggest that dysregulation of miR-9 may contribute to pathologic conditions involving mast cell-mediated tissue remodeling. Citation Format: Joelle M. Fenger, Feng Xu, Xaioli Zhang, Peter Y. Yu, Misty D. Bear, Shanice L. Reynolds, Michael L. Pennell, Vincenzo Coppola, William C. Kisseberth, Stephen J. Galli, Cheryl A. London. A novel Cpa3-Cre; miR-9fl/fl mouse reveals a functional role for miR-9 in promoting mast cell invasion via up-regulation of CMA1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3043. doi:10.1158/1538-7445.AM2017-3043
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