Event
Bioengineering Seminar Series: Curt I. Civin, M.D.
Friday, April 17, 2009
11:00 a.m.-12:00 p.m.
Room 2110, Chemical and Nuclear Engineering Bldg.
Professor John Fisher
jpfisher@umd.edu
MicroRNA Maestros of Hematopoiesis and Leukemia: Different MicroRNAs Can Positively or Negatively Regulate Differentiation, Proliferation, Apoptosis, and Drug Resistance of Human Leukemia Cells and Normal Hematopoietic Cells
Presented by Curt I. Civin, M.D.
Professor
Stem Cell Biology & Regenerative Medicine
Applied Science Engineering
University of Maryland, Baltimore
Based on expression of microRNAs (miRs) and their predicted target messenger RNAs (mRNAs) in human CD34+ hematopoietic stem-progenitor cells (HSPCs), we hypothesized that certain HSPC-expressed microRNAs (HE-miRs) can down-regulate key hematopoietic proteins and thereby regulate hematopoiesis. As a first example, we found that miR-155 was expressed in human CD34+ HSPCs and in mouse Kit+Sca1+Lin- HSPCs (a subset enriched in early HSPCs). MiR-155 was known already to be overexpressed in several types of cancer, including many lymphomas and leukemias. To study the functional role of miR-155 in hematopoiesis and leukemias, we developed molecular tools to efficiently up- and down-regulate miR-155 in hematopoietic cells. To supplement enforced expression experiments using a miR-155/GFP dual promoter lentivector, we lipofected a synthetic 22-mer miR-155 sense oligonucleotide into cells. In functional studies, enforced miR-155 expression increased hematopoietic cell proliferation, in addition to inhibiting hematopoietic differentiation. For loss-of-function experiments, we designed an antisense locked nucleic acid (LNA)-containing antimiR-155 that potently bound to the complementary miR-155. Upon transfection into hematopoietic cells, this LNA antimiR-155 blocked miR-155-mediated inhibition of target mRNAs. Thus, modulation of miR-155 and the pathways it regulates may be useful both in ex vivo expansion of HSPCs and in leukemia treatment.
As a second example, we investigated the action of miR-27a, which appeared to have effects opposite to those of miR-155. In general, miR-27a was expressed at lower (or absent) levels in human leukemias, as compared to normal HSPCs. Lipofection of synthetic miR-27a or lentiviral expression of miR-27a decreased human leukemia cell proliferation. Drug-resistant human leukemia cell lines exhibited increased spontaneous apoptosis and became more susceptible to drug- and growth factor withdrawal-induced apoptosis upon enforced expression of miR-27a. Using luciferase assays, we showed that the anti-apoptotic molecules YWHAQ and PLK2 and the drug-resistance pump ABCC4 were targets of miR-27a. Leukemia cells with enforced miR-27a expression had reduced proliferation and decreased percentages of cells in the G1 cell cycle phase. Certain predicted miR-27a targets may explain this effect on cell cycling. Thus, based on its expression, functional effects, and targets, miR-27a may function as a tumor suppressor miR—lack of miR-27a expression in leukemias may contribute to development and/or progression of these cancers.
