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Up-regulation of microRNA miR-155 is reflected by low levels of its target mRNA encoding transcription factor PU.1 in primary tumors of human lymphomas
Hušková, Hana ; Stopka, Tomáš (advisor) ; Mráz, Marek (referee)
Lymphomas are heterogenous class of diseases characterized by proliferation of a malignant lymphocyte clone. MicroRNA miR-155 was found to be a key molecule in immune response, namely in inflammation and germinal reaction of B cells. On the other hand, miR-155 can drive lymphoproliferation in mouse and its levels were found to be elevated in certain lymphoma types in human. MiR-155 down-regulates expression of its target gene PU.1, a hematopoietic transcription factor important for B cell differentiation. Expression of the gene encoding miR-155, known as MIR155HG, is controled by several transcription factors, among them MYB, a member of an oncogenic E-box protein family. Levels of MYB itself are controled by microRNA miR-150. In this study, we measured levels of miR-155, PU.1, MYB and miR-150 in lymph nodes of patients with chronic lymphocytic leukemia/small lymphocytic lymphoma (B-CLL/SLL, N=20), diffuse large B-cell lymphoma (DLBCL, N=24), follicular lymphoma (FL, N=29), Hodgkin lymphoma (HL, N=25), marginal zone lymphoma (MZL, N=13), and mantle cell lymphoma (MCL, N=10). We also measured levels of these molecules in lymph nodes with the finding of strong inflammation (N=4). We found that patients of all the diagnoses except of MCL display heterogeneously elevated levels of miR-155 and correspondingly...
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The role of transcription factors PU.1 a GATA-1 during leukemia differentiation.
Burda, Pavel ; Stopka, Tomáš (advisor) ; Kořínek, Vladimír (referee) ; Machová Poláková, Kateřina (referee)
Hematopoiesis is coordinated by a complex regulatory network of transcription factors among them PU.1 (Spi1, Sfpi1) and GATA-1 represent key molecules. GATA-1 and PU.1 bind each other on DNA to block each others transcriptional programs to prevent development of undesired lineage during hematopoietic commitment. Murine erythroleukemia (MEL) cells, transformed erythroid precursors that are blocked from completing the late stages of erythroid differentiation, co-express GATA-1 and PU.1 and as my and others data document, are able to respond to molecular removal (down-regulation) of PU.1 or addition (up-regulation) of GATA-1 by inducing terminal erythroid differentiation. We provide novel evidence that downregulation of GATA-1 or upregulation of PU.1 induces incompletely differentiation into cell cycle arrested monocytic-like cells. Furthermore, PU.1- dependent transcriptome is negatively regulated by GATA-1 in MEL cells, including CCAAT/enhancer binding protein alpha (Cebpa) and Core-binding factor, beta subunit (Cbfb) that encode additional key hematopoietic transcription factors. Chromatin immunoprecipitation and reporter assays identified PU.1 motif sequences near Cebpa and Cbfb that are co-occupied by PU.1 and GATA-1 in the leukemic blasts. Furthermore, transcriptional regulation of these loci by...
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Transcriptional regulation of miR-17-92 microRNA cluster during macrophage differentiation.
Rybářová, Jana ; Stopka, Tomáš (advisor) ; Pospíšek, Martin (referee)
miR-17-92 cluster (Oncomir1) encodes seven microRNAs (miRNA, miR) regulating many biological processes including proliferation, differentiation or apoptosis. Overexpression of microRNAs encoded by miR-17-92 cluster is found in a number of tumors including acute and chronic myeloid leukemias (Dixon-McIver et al., 2008; Li et al., 2008; Venturini et al., 2007). Myeloid progenitors express miR-17-92 cluster at a high level, while macrophage differentiation associates with its downregulation. Our laboratory found, that miR-17-92 cluster is repressed by transcription factor Early growth response 2 (Egr2) upon differentiation of primary myeloid PUER progenitors, induced with transcription factor PU.1. Aim of this thesis is to further test the abovementioned data by preparing a reporter vectors set, carrying various fragments of miR-17-92 putative promoter, which enables us to study regulation of transcription of miR-17-92 cluster. This task complicated by presence of increased GC content of the miR-17-92 promoter was successfully accomplished resulting in amplification of eight fragments containing the various parts of miR-17-92 promoter including region -3.3 to 0 kb relative to the start of miR-17-5p sequence, that were inserted into pGL3 reporter vector. Transfection of pGL3 reporter vector carrying...
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