Mutual Regulatory Loop between miR-155 and PU.1 Is a Candidate Pathogenesis Factor in CLL.

MicroRNAs are non-coding post-transcriptional regulators of gene expression often dysregulated in human cancers including hematological malignancies. MiR-155 is a putative oncogenic factor found overexpressed in chronic lymphocytic leukemia (CLL) cells. We have confirmed the increased levels of miR-155 in peripheral blood lymphocytes in 12 patients with chronic lymphocytic leukemia (CLL) compared to 11 normal controls, using quantitative PCR assay. Among the predicted high-scoring hematopoietic target genes of miR-155 is the ets-family transcription factor PU.1, a key regulator of myelo-lymphoid lineage development. Using gene expression profiling of CLL peripheral lymphocyte samples compared to normal controls, we have determined significant downregulation of the PU.1 mRNA and of additional numerous miR-155 targets previously reported to be required for myelo-lymphoid differentiation, and of a set of gene targets of PU.1-chromatin DNA interactions that we determined by chromatin immunoprecipitation analyses. Functional pathway analysis of the miR-155 targets downregulated in CLL patients suggest the suppression of T-cell receptor pathway, TGF-b/Bmp, MAPK, Jak/Stat and Wnt signaling and remodeling of actin cytoskeleton and cell-matrix interactions, as well as the regulation of numerous transcription regulators including c-FOS, c-MYC, MAFB and HDAC4. Recent reports suggested a role of miR-155 in lymphocytic differentiation, we have tested whether PU.1 may regulate expression of its own regulator miR-155. We used two cell systems expressing a transgene encoding PU.1 fused with the ligand binding domain of the estrogen receptor, ER):

1. murine erythroleukemia (MEL) cells (Rao 1997) and

2. PU.1 null hematopoietic progenitor cells stably transfected with PU.1-ER (Walsh 2002).

Using a miRNA microarray assay we have identified a set of 21 miRNAs that are modulated by PU.1. Indeed, miR-155 was among the upregulated targets alongside a known PU.1 miR target miR-233. The direct interaction between miR-155 promoter and PU.1 protein was confirmed by chromatin immunoprecipitation in MEL cells. In summary, our data suggests that increased miR-155 levels may contribute to CLL by inhibiting PU.1 function and suppressing PU.1-mediated programs and that in two independent conditional PU.1 cell systems PU.1 directly binds miR-155 promoter and upregulates its expression suggesting thus a mutual regulatory mechanism between PU.1 and miR-155.