Project 4: MicroRNA targeting of normal and leukemia stem-progenitor cells (Civin) MicroRNAs bind to 3'untranslated region sites in target mRNAs to down-regulate translation to protein. Since microRNAs do not base-pair exactly with their target mRNAs, they can block protein translation of many mRNAs and serve as powerful switches to regulate cell functions. To extend our long-term studies on regulation of hematopoiesis and stem cell biology, we profiled microRNA expression in hematopoietic stemprogenitor cells (HSPCs). We combined this data with human HSPC mRNA expression results and microRNA-mRNA target predictions into a novel database which predicted that certain of the HSPCexpressed microRNAs (HE-microRNAs) targeted several mRNAs critical to hematopoiesis. On this informatic basis, we formulated a model for microRNA control of hematopoiesis in which many genes specifying hematopoietic differentiation are expressed by early HSPCs, but held in check by HE-microRNAs. For several target mRNAs, we then demonstrated experimentally that translation is actually decreased by microRNAs. Mir-155 potently reduced myeloid and erythroid colony formation of normal human HSPCs, and mir-16 selectively inhibited erythropoiesis. Since the cells that we studied include rare stem cells and various stages of progenitors, we propose in Aim 1, to expand our microRNA profiles of HSPCs to more highly purified subsets of primary human and mouse HSPCs and primary human acute myeloid leukemia (AMI) cells.
In Aim 2, we will determine if selected individual microRNAs experimentally inhibit development of primary HSPCs, as our model predicts, and if these microRNAs affect AMI stem cells and leukemogenesis. We will determine the proteins whose synthesis is inhibited by each functionally-active microRNA and thereby the molecular mechanisms of the hematopoietic effects. At this point in our studies, at least 2 of these microRNAs, mir-16 and mir-155 appear to be new regulators of normal hematopoietic and cancer stem cells. Relevance: We suggest that a major barrier impeding cure of the many cancer patients whom we cannot cure today is our failure to effectively attack and eliminate cancer stem cells. Specifically, we believe that development of more effective therapies in leukemia hinges on fuller understanding of the regulation of rare normal and leukemic hematopoietic stem cells. The studies in this project investigate the effects of new regulatory molecules called microRNAs, which appear to play previously unexpected, potent roles in control of blood formation and leukemia. Understanding the actions of these hematopoietic-regulatory microRNAs may provide new targets, for both expansion of normal stem cells and leukemia therapy.
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