• Deciphering the role of microRNA-708 in precursor B-cell acute lymphoblastic leukemia biology

      Doshi, Kshama Atul; Civin, Curt I. (2012)
      MicroRNAs (miRs) are known to play major roles in both normal hematopoietic differentiation, as well as hematopoietic malignancies. In this work, we report that miR-708; a little studied miR, is up-regulated in most B-cell acute lymphoblastic leukemia (B-ALL) cell lines and primary patient samples compared to hematopoietic stem-progenitor cells (HSPCs) and mature healthy B-cells. Our first hypothesis in this regard was that miR-708 has an oncogene-like function in B-ALL biology. To test this hypothesis, we performed loss/gain of function assays to examine effect of miR-708 modulation on B-ALL cell proliferation. While overexpression of miR-708 failed to alter cell proliferation rate, knock-down of this miR conferred a growth disadvantage on B-ALL cell proliferation. Our second hypothesis was that accumulation of non-functional immature B cells in B-ALL is due to deregulated expression of miR-708 during B cell development. To test this hypothesis, we overexpressed miR-708 in donor murine hematopoietic progenitor (Lin-) cells and monitored their hematopoietic differentiation in recipient mice following bone marrow transplantation. Post transplantation, we failed to observe any influence of miR-708 on hematopoietic differentiation of Lin- cells. In addition, we also observed a positive correlation that exists between expression pattern of miR-708 and its host gene OdZ4. Hence, we hypothesize that up-regulated miR-708 could be a passenger of up-regulated OdZ4 in B-ALL. To test this hypothesis, we are currently examining effects of OdZ4 knock-down on B-ALL cell proliferation. In future we also propose to examine if miR-708 cooperates with OdZ4 function in B-ALL. Future work also needs to address if miR-708 affects oncogenic processes, other than growth, that could be involved in B-ALL biology
    • The Role of Selected MicroRNAs in Hematopoiesis and Leukemia

      Tan, Yee Sun; Civin, Curt I. (2014)
      MicroRNAs (miRs) are short non-coding RNAs which regulate expression of mRNA targets, and are known to modulate many cellular processes including hematopoiesis and hematological malignancies. Expression profiling studies have routinely been used to identify candidate miRs important in hematopoiesis and leukemia, but it is often challenging to identify miRs that regulate a given cellular function because differential miR levels may not be indicative of a physiological role. Two strategies to address this problem of identifying miRs that are "drivers" of hematopoiesis or leukemias are evaluated in this study. In the first study, we identified miR-509 via a human genome-wide gain-of-function screen for miRs that inhibit growth of the NALM6 human B-ALL cell line. Growth inhibitory effects of miR-509 were validated in independent assays and two other B-ALL cell lines. MiR-509-transduced NALM6 cells had reduced numbers of cells in cell cycle S-phase and increased apoptosis. Using miR-target prediction algorithms and a filtering strategy, RAB5C mRNA predicted as a relevant target of miR-509. Enforced miR-509 expression in NALM6 cells reduced RAB5C levels, and RAB5C was demonstrated to be a direct target of miR-509. Knockdown of RAB5C in NALM6 cells recapitulated the growth inhibitory effects of miR-509. Co-expression of the RAB5C open reading frame without its 3' untranslated region blocked the growth-inhibitory effect mediated by miR-509. These findings establish RAB5C as a novel target of miR-509 and an important endogenous regulator of B-ALL cell growth, with potential as a therapeutic target. In the second study, the zebrafish, Danio rerio, was used as a model organism to evaluate the functional role of hematopoietic stem-progenitor cell (HSPC)-enriched miRs that are highly expressed in the earliest subsets of mouse HSPCs compared to progenitor cells, starting with miR-10a. Expression profiling of miR-10a levels indicated that this miR is expressed during early hematopoiesis in zebrafish. Preliminary findings suggest loss-of-function of miR-10a (and miR-10 family members) resulted in reduced numbers of hematopoietic stem cells, and future experiments include elucidating the targets of miR-10a which may be important in hematopoiesis.