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Delineating aggressiveness of acute myeloid leukemia in a mouse model carrying mutations of Spil (PU.1) and Trp53.
Bašová, Petra
PU.1 downregulation within haematopoietic stem and progenitor cells (HSPCs) is the primary mechanism for the development of acute myeloid leukaemia (AML) in mice with homozygous deletion of the upstream regulatory element (URE) of PU.1 gene. p53 is a well known tumor suppressor that is often mutated in human haematologic malignancies including AML and adds to their aggressiveness; however its genetic deletion does not cause AML in mouse. Deletion of p53 in the PU.1ure/ure mice (PU.1ure/ure p53-/- ) results in more aggressive AML with shortened overall survival. PU.1ure/ure p53-/- progenitors express significantly lower PU.1 levels. In addition to URE deletion we searched for other mechanisms that in absence of p53 contribute to decreased PU.1 levels in PU.1ure/ure p53-/- mice. We found involvement of Myb and miR-155 in downregulation of PU.1 in aggressive murine AML. Upon inhibition of either Myb or miR-155 in vitro the AML progenitors restore PU.1 levels and lose leukaemic cell growth similarly to PU.1 rescue. The MYB/miR-155/PU.1 axis is a target of p53 and is activated early after p53 loss as indicated by transient p53 knockdown. Furthermore, deregulation of both MYB and miR-155 coupled with PU.1 downregulation was observed in human AML, suggesting that MYB/miR-155/PU.1 mechanism may be involved...
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Role onkogenní mikroRNA-155 a proto-onkogenu MYB u chronické lymfatické leukémie
Vargová, Karina ; Stopka, Tomáš (advisor) ; Móciková, Heidi (referee) ; Trka, Jan (referee)
(EN) Chronic lymphocytic leukemia (B-CLL) represents a disease of mature-like B-cells. Due to failed apoptosis but also due to enhanced proliferative signals, the leukemic B-cells accumulate in the peripheral blood, bone marrow, lymph nodes and spleen. The clinical course of B-CLL is very heterogeneous; in some patients B-CLL progresses very rapidly into an aggressive form. Such patients need therapy sooner while in other patients with indolent B-CLL the onset of therapy takes years. Several standard prognostic and disease progression markers are used for disease staging and monitoring, however a reliable marker that will suggest when to start therapy is unknown. Expression of small, non-coding microRNAs is often deregulated and represent important prognostic markers in variety of cancers including leukemia. Hence in our study we concentrated to miR-155, an important molecule regulating differentiation of hematopoietic cells, inflammation process and antibody production. Its aberrant expression was described in Hodgkin`s as well as in non-Hodgkin`s lymphoma, including indolent lymphoproliferations like B- CLL. Our results confirmed elevated levels of both, primary miR-155 transcript and mature form of miR-155 in our B-CLL patient samples (N=239). The aberrant expression of miR-155 in B-CLL samples...
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Delineating aggressiveness of acute myeloid leukemia in a mouse model carrying mutations of Spil (PU.1) and Trp53.
Bašová, Petra ; Stopka, Tomáš (advisor) ; Machová Poláková, Kateřina (referee) ; Zuna, Jan (referee)
PU.1 downregulation within haematopoietic stem and progenitor cells (HSPCs) is the primary mechanism for the development of acute myeloid leukaemia (AML) in mice with homozygous deletion of the upstream regulatory element (URE) of PU.1 gene. p53 is a well known tumor suppressor that is often mutated in human haematologic malignancies including AML and adds to their aggressiveness; however its genetic deletion does not cause AML in mouse. Deletion of p53 in the PU.1ure/ure mice (PU.1ure/ure p53-/- ) results in more aggressive AML with shortened overall survival. PU.1ure/ure p53-/- progenitors express significantly lower PU.1 levels. In addition to URE deletion we searched for other mechanisms that in absence of p53 contribute to decreased PU.1 levels in PU.1ure/ure p53-/- mice. We found involvement of Myb and miR-155 in downregulation of PU.1 in aggressive murine AML. Upon inhibition of either Myb or miR-155 in vitro the AML progenitors restore PU.1 levels and lose leukaemic cell growth similarly to PU.1 rescue. The MYB/miR-155/PU.1 axis is a target of p53 and is activated early after p53 loss as indicated by transient p53 knockdown. Furthermore, deregulation of both MYB and miR-155 coupled with PU.1 downregulation was observed in human AML, suggesting that MYB/miR-155/PU.1 mechanism may be involved...
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Extracellular microRNAs in hematological malignancies and their use for diagnosis and treatment monitoring
Šulcová, Dominika ; Pospíšil, Vít (advisor) ; Čermák, Vladimír (referee)
MicroRNAs are short non-coding RNAs that negatively regulate gene expression at post-transcriptional level by interfering with mRNA translation and stability. Recently, microRNAs were surprisingly found to be present in various body fluids including blood plasma and serum, cerebrospinal fluid, saliva, milk or urine. These extracellular microRNAs are resistant to RNases and stable in high temperature or pH. Extreme stability of extracellular microRNAs is caused by their association with protective protein complexes (mostly with Argonaute proteins). MicroRNAs are frequently deregulated in cancer and specific tumor- related microRNAs can be also detected in body fluids, indicating that extracellular microRNAs can be used as tumor specific markers. This Bachelor thesis reviews basic principles of microRNA function and biogenesis with focus on extracellular microRNAs and their role in intercellular communication, and it highlights the role of extracellular microRNAs in hematological malignancies and their possible use in diagnosis and treatment.
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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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Extracellular microRNAs and their role in pathologies especially in the field of gynecology and obstetrics.
Štěrbová, Monika ; Hromadníková, Ilona (advisor) ; Balušíková, Kamila (referee)
microRNAs (miRNAs) represent a relatively newly discovered group of RNA molecules and they serve to regulate gene expression. In spite of processes of differentiation, proliferation and apoptosis, miRNAs influence the whole biological systems, such as embryogenesis, oncogenesis, and immunity. There have been a number of experiments in recent years concerning diagnoses and predictions of complications during pregnancy, and tumour growth. Extracellular miRNA molecules participating in circulation of patients are used in the non-invasive diagnostics. RNA molecules usually get into the extracellular fluid during the apoptosis process. I chose four diseases, which extracellular miRNA have diagnostic potential - preeclampsia, intrauterine growth retardation, gestational diabetes mellitus and breast cancer - for my work. An aberrant expression of different levels of various extracellular miRNAs has been reported in these diseases but the clinical use of microRNAs in the diagnosis and prediction of those still requires further research and optimization. Keywords: breast cancer, extracellular nucleic acids, fetal growth retardation, gestational diabetes mellitus, microRNA, PCR, preeclampsia
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Messenger RNA stability and microRNA activity in mouse oocytes
Flemr, Matyáš ; Svoboda, Petr (advisor) ; Motlík, Jan (referee) ; Hampl, Aleš (referee)
The oocyte-to-zygote transition represents the only physiological event in mammalian life cycle, during which a differentiated cell is reprogrammed to become pluripotent. For its most part, the reprogramming relies on the accurate post-transcriptional control of maternally deposited mRNAs. Therefore, understanding the mechanisms of post-transcriptional regulation in the oocyte will help improve our knowledge of cell reprogramming. Short non- coding microRNAs have recently emerged as an important class of post-transcriptional regulators in a wide range of cellular and developmental processes. MicroRNAs repress their mRNA targets via recruitment of deadenylation and decapping complexes, which typically accumulate in cytoplasmic Processing bodies (P-bodies). The presented work uncovers an unexpected feature of the microRNA pathway which is found to be suppressed in fully-grown mouse oocytes and through the entire process of oocyte-to-zygote transition. This finding is consistent with the observation that microRNA-related P-bodies disassemble early during oocyte growth and are absent in fully-grown oocytes. Some of the proteins normally associated with P-bodies localize to the oocyte cortex. At the final stage of oocyte growth, these proteins, together with other RNA-binding factors, form subcortical...
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The role of microRNAs in lymphomas with a focus on miR-155
Hušková, Hana ; Stopka, Tomáš (advisor) ; Svoboda, Petr (referee)
MicroRNAs (miRNAs) are 19-25 nucleotide noncoding RNAs which regulate the expression of target mRNAs at both posttranscriptional and translational level. The physiological functions of miRNAs include development, differentiation, cell cycle regulation and apoptosis. miRNA deregulation has been found in various human diseases, including lymphoproliferative disorders. This Bachelor thesis provides introduction to delineate roles of miRNAs in normal hematopoiesis and cites recent publications on miRNAs in lymphomas with a focus on the role of miR-155. Key words microRNA, hematopoiesis, lymphoma, miR-155
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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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Specifická izolace microRNA pomocí magnetizovatelných mikročástic
Vlahová, Veronika
MicroRNAs are small non-coding RNA molecules with length of about 22 nt. These molecules participate on regulation of gene expression at the post-transcriptional level. They represent the largest group of regulators in the cell and therefore are also involved in all key processes such as proliferation, differentiation or apoptosis. Moreover, they participate in tumor transformation. These small molecules have a great potential to be diagnostic markers or assist in the treatment and prevention of diseases. This research was focused on the development of isolation method using magnetic particles with subsequent electrochemical detection of microRNA. Optimization steps were performed and then the entire method was successfully applied to real samples of HEK293 cells expressing increased levels of miR-124. The developed method proved to be sufficiently specific and applicable to the analysis of microRNA.
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