Dissecting the role of miR-9 in normal and malignant mast cell biology

 描述(申请人提供)：肥大细胞是广泛的生理和病理过程中的关键效应细胞，包括先天性免疫反应和过敏性疾病，慢性炎症性疾病，如心血管疾病和关节炎，以及肿瘤进展。激活的肥大细胞分泌一系列不同的因子，调节它们在炎症、免疫和组织重塑中的作用。然而，各种遗传因素影响肥大细胞生物学多个方面的确切机制尚未完全确定。MicroRNAs(MiRNAs)是一种调节基因表达的非编码小RNA，它们的失调与许多病理情况有关。我们实验室最近的工作发现miR-9的过度表达与侵袭性、转移性的犬肥大细胞瘤(MCT)有关，MCT是一种公认的自发性恶性肥大细胞疾病的模型。此外，使用慢病毒载体增强miR-9在正常和恶性肥大细胞中的高表达，而该miR-9的基础水平较低，促进了侵袭并增强了CMA1的表达，CMA1是一种肥大细胞特异性蛋白酶，参与组织重塑。为了更好地研究miR9在肥大细胞生物学中的作用，我们建立了一个组织特异性诱导miR-9表达的转基因模型，将这些小鼠与我们的合作者Stephen Galli博士(Stanford University)培育的羧基肽酶-3-Cre转基因小鼠进行杂交，并证实了该模型的功能，包括增强CPA3-Cre/miR-9双转基因肥大细胞的侵袭特性。因此，我们假设miR-9通过调节组织重塑和血管生成的因子来增强CMA1的表达并促进肥大细胞的侵袭。我们进一步假设，在正常肥大细胞中，miR-9在体内改变细胞运动并增强对抗原刺激的敏感性，在肥大细胞增多症小鼠模型中，miR-9有助于增强细胞侵袭和血管生成，并与更具侵袭性的生物学行为有关。为了验证这些假设，我们将完成以下特定目标：1)确定miR-9诱导正常肥大细胞CMA1表达和增强侵袭的分子机制；2)利用我们组织特异性miR-9表达的转基因小鼠模型，在体内评估miR-9对正常肥大细胞生物学的影响；以及3)在恶性肥大细胞疾病小鼠模型中，研究miR-9在促进肥大细胞侵袭、转移和肿瘤进展中的作用。总之，K01提案中概述的研究将提供对miR-9在体外和体内调节肥大细胞行为的分子机制的更完整的理解，特别是当它与诱导转移表型有关时。候选人：Joelle Fenger博士，注册兽医和董事会认证兽医肿瘤学家，目前正在完成她在俄亥俄州立大学比较和兽医学研究生研究项目的博士学位，这是一个双重住院医生/博士项目。在K01 SERCA获奖期间，Fenger博士将首先致力于完成她的博士论文研究和完成她的论文。在最后几年，她将被任命为研究助理教授，在此期间，她将获得更多技能，并发展精致的研究重点，作为她在最后一年过渡到独立学术科学家的基础。环境：Fenger博士的共同导师谢丽尔·伦敦博士和Guido Marcucci博士都是俄亥俄州立大学的教授和首席研究员，伦敦博士是兽医学院兽医生物科学系的教授，Marcucci博士是医学院内科的博士。伦敦博士在恶性肥大细胞疾病的生物学和转化肿瘤学方面拥有丰富的经验，马尔库奇博士在miRNA在血液系统恶性肿瘤中的作用和miRNAs的临床前治疗靶点方面拥有丰富的专业知识。作为获奖期间整体发展和培训计划的一部分，Fenger博士将接受一个由不同学科的顾问和合作者组成的小组的指导。她将在疾病中miRNA失调的更广泛领域发展技能，接受肥大细胞特定技术的正式实验室培训，以及老鼠病理生物学和老鼠疾病模型的高级培训。此外，在整个奖项期间，Fenger博士将在执行假设驱动的研究和建立合作研究伙伴关系方面得到指导，这些合作伙伴关系将为她过渡到独立研究人员的职业生涯做好准备。

 DESCRIPTION (provided by applicant): Mast cells are key effector cells in a wide variety of physiological and pathological processes, including innate immune responses and allergic disorders, chronic inflammatory diseases such as cardiovascular disease and arthritis, and tumor progression. Activated mast cells secrete a diverse array of factors that mediate their roles in inflammation, immunity, and tissue remodeling. However the exact mechanisms through which various genetic factors influence multiple aspects of mast cell biology have yet to be fully defined. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and their dysregulation is implicated in numerous pathologic conditions. Recent work by our laboratory found that miR-9 over-expression was associated with aggressive, metastatic canine mast cell tumors (MCT), a well-established model of spontaneous malignant mast cell disease. Furthermore, enforced high expression of miR-9 in normal and malignant mouse mast cells with low basal levels of this miR using lentiviral vectors promoted invasion and enhanced the expression of CMA1, a mast cell-specific protease involved in tissue remodeling. To better study the role of miR9 in mast cell biology, we have generated a transgenic model of tissue specific induced miR-9 expression, have crossed these mice with the carboxypeptidase-3-Cre transgenic mice generated by our collaborator Dr. Stephen Galli (Stanford University), and confirmed the functionality of this model including enhanced invasive properties of mast cells from the CPA3-Cre/miR-9 double transgenics. We therefore hypothesize that miR-9 enhances CMA1 expression and promotes invasion in mast cells through the modulation of factors responsible for tissue remodeling and angiogenesis. We further hypothesize that in normal mast cells, miR-9 alters cell motility and enhances sensitivity to antigen stimulation in vivo, and thatin mouse models of mastocytosis, miR-9 contributes to enhanced cell invasion and angiogenesis and is associated with a more aggressive biological behavior. To test these hypotheses, we will complete the following specific aims: 1) Identify the molecular mechanisms responsible for miR-9-induced CMA1 expression and enhanced invasion in normal mast cells; 2) Assess the effects of miR-9 on normal mast cell biology in vivo utilizing our transgenic mouse model of tissue specific miR-9 expression; and 3) Investigate the contribution of miR-9 in promoting mast cell invasion, metastasis, and tumor progression in mouse models of malignant mast cell disease. In summary, the studies outlined in this K01 proposal will provide a more complete understanding of the molecular mechanisms through which miR-9 regulates mast cell behavior both in vitro and in vivo, particularly as it relates to induction of the metastatic phenotype. The Candidate: Dr Joelle Fenger, a licensed veterinarian and Board Certified Veterinary Medical Oncologist, is currently completing her PhD in the Graduate Studies Program in Comparative and Veterinary Medicine at The Ohio State University in the context of a dual Residency/PhD program. During the K01 SERCA award period, Dr. Fenger's efforts will first be dedicated to completing her PhD thesis studies and completing her dissertation. Over the final years, she will be appointed as a Research Assistant Professor during which time she will gain further skill sets and develop a refined research focus that will serve as the foundation for her transition to an independent academic scientist in the final year of the award. The Environment: Dr. Fenger's co-mentors, Dr. Cheryl London and Dr. Guido Marcucci are both Professors and Principal Investigators at OSU; Dr. London in the Department of Veterinary Biosciences, College of Veterinary Medicine, and Dr. Marcucci in the Department of Internal Medicine, College of Medicine. Dr. London has extensive experience in the biology of malignant mast cell disease and translational oncology, and Dr. Marcucci has substantial expertise in the role of miRNA in hematologic malignancies and preclinical therapeutic targeting of miRNAs. As part of the overall development and training plan during the period of the award, Dr. Fenger will receiving mentorship from a diverse, inter-disciplinary group of advisors and collaborators. She will develop skill sets in the broader areas of miRNA dysregulation in disease, receive formal laboratory training in mast-cell specific techniques, and advanced training in mouse pathobiology and mouse models of disease. Additionally, throughout the duration of the award, Dr. Fenger will be mentored in executing hypothesis-driven research and establishing collaborative research partnerships that will prepare her as she transitions to a career as an independent researcher.