Projekt vychází z naší dosavadní výzkumné práce, ve které jsme tlumením genové exprese pomocí siRNA analyzovali možnost ovlivnění funkce genů, jejichž změny aktivity byly spojeny s různými onko-hematologickými procesy. Na rozdíl od použití siRNA je tentoprojekt zaměřen na analýzu miRNA, která dovoluje i sledování dereprese genové funkce blokováním endogenních miRNA. Součástí projektu je 1) profilování miRNA čipovou technikou na buňkách buněčných linií a různých buněčných typech izolovaných z periferníkrve zdravých osob a 2) sledování změn genové aktivity po blokaci vybraných miRNA s použitím anti-miRNA a to jak na úrovni transkripce tak translace. V práci budou použity buněčné linie K562, KU182, MOLM7, HL60 a HEL a celkové leukocyty získané z periferní krve zdravých dobrovolníků obojího pohlaví a různých věkových kategorií, případně monocyty a granulocyty a izolované buněčné typy CD34+, CD14+, CD3+. Sledovány budou v hemopoetických buňkách exprimované miRNA: miR15, miR16, miR142, miR155 a miR223.; The project is based on our previous research study, in which using siRNA-mediated gene silencing we analyzed possible function modulation of the genes whose dysregulation was associated with various onco-hematological processes. In contrast to siRNAs, this project is focused on miRNA analyses that also allow to observe de-repression of gene function using miRNA inhibitors. The project includes: 1) miRNA expression profiling in cell lines and in peripheral blood cells of healthy persons using microarrays; 2) analyses of gene activity changes after inhibition of selected miRNAs using Anti-miRNAs at both transcriptional and translational level. In this study we will use cell lines K562, KU812, MOLM7, HL60, HEL and total peripheral blood leukocytes obtained from healthy donors, eventually monocytes and granulocytes or cell subtypes CD34+, CD14+, CD3+ will be used. Expression of miR15, miR16, miR142, miR155 and miR223 will be monitored in hematopoiteic cells.

• MeSH
• exprese genu MeSH
• genetická transkripce MeSH
• mikro RNA MeSH
• regulace genové exprese MeSH
• transformované buněčné linie MeSH

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• biologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR

MicroRNAs (miRNAs), important regulators of cellular processes, show specific expression signatures in different blood cell lineages and stages of hematopoietic stem cell (HSC) differentiation, indicating their role in the control of hematopoiesis. Because neonatal blood displays various features of immaturity, we might expect differential miRNA regulation. Herein, we determined miRNA expression profiles of umbilical cord blood (UCB) cell lineages and compared them to those of bone marrow (BM) and peripheral blood (PB) cell counterparts. Further, we determined mRNA expression profiles using whole-genome microarrays. An approach combining bioinformatic prediction of miRNA targets with mRNA expression profiling was used to search for putative targets of miRNAs with potential functions in UCB. We pointed out several differentially expressed miRNAs and associated their expression with the target transcript levels. miR-148a expression was suppressed in HSCs and its level inversely correlated with the previously verified target, DNA methyltransferase 3B, suggesting dependence of de novo DNA methylation in HSCs on miR-148a. Prolonged cell survival of UCB HSCs may be associated with low expression of miR-143 and miR-145 and up-regulation of their downstream targets (high expression of c-MYC and miR-17-92 and following repression of TGFBR2). In HSCs, we monitored significant up-regulation of eight miRNAs, which were previously verified as regulators of HOX genes. Further, miR-146b may be associated with immaturity of neonatal immune system because it is strongly up-regulated in UCB granulocytes and T lymphocytes compared to PB cell counterparts. Comparative analysis revealed 13 miRNAs significantly altered between UCB and BM CD34(+) cells. In UCB CD34(+) cells, we monitored up-regulation of miR-520h, promoting differentiation of HSCs into progenitor cells, and reduction of miR-214, whose expression might support HSC survival. In conclusion, UCB cells show specific miRNA expression patterns, indicating different regulation in these cells.

• MeSH
• antigeny CD34 metabolismus   MeSH
• buněčný rodokmen genetika   MeSH
• dospělí MeSH
• fetální krev cytologie   metabolismus   MeSH
• hematopoetické kmenové buňky metabolismus   MeSH
• krevní buňky metabolismus   fyziologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mikro RNA genetika   metabolismus   MeSH
• novorozenec MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
• senioři MeSH
• shluková analýza MeSH
• stanovení celkové genové exprese MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• novorozenec MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• validační studie MeSH

Erythropoiesis is a multistep process regulated at the molecular level by intrinsic and extrinsic factors including microRNAs (miRNAs). We previously identified aberrant expression of miR-451 and miR-150 in polycythemia vera (PV) erythroid differentiating cells. To address the functional relevance of these miRNAs in erythroid differentiation, we employed synthetic mimics and inhibitors of miR-451 and miR-150 in erythroid differentiating K562 cells. We observed that miR-451 up-regulation and miR-150 down-regulation are associated with progression of erythroid maturation in K562 cells. Further, enforced expression of miR-451 promoted erythroid differentiation. Inhibition of miR-150 reduced hemoglobinization of K562 cells. Microarray data suggested potential targets regulated by miR-451: UBE2H, ARPP-19; and by miR-150: MS4A3, AGA, PTPRR. Our results demonstrate that miR-451 is involved in the regulation of erythroid differentiation and functions as an enhancer of differentiation. These data support the concept that aberrant expression of miRNAs may contribute to abnormal erythropoiesis such as that of PV.

• MeSH
• akutní erytroblastická leukemie genetika   patologie   MeSH
• buněčná diferenciace účinky léků   genetika   MeSH
• buňky K562 MeSH
• erytroidní buňky účinky léků   patologie   MeSH
• erytropoéza účinky léků   genetika   MeSH
• hemin farmakologie   MeSH
• lidé MeSH
• mikro RNA genetika   fyziologie   MeSH
• nádorové buněčné linie MeSH
• regulace genové exprese u leukemie účinky léků   MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
• stanovení celkové genové exprese MeSH
• transfekce MeSH
• upregulace účinky léků   fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

Polycythemia vera (PV) is an acquired myeloproliferative clonal disorder, characterized by augmented erythropoiesis. To better define PV pathogenesis, we performed an in vitro erythroid expansion from peripheral blood mononuclear cells of controls and PV patients and evaluated the cells for proliferation, apoptosis, erythroid differentiation, and morphology at the defined time points. PV erythroid progenitors exhibited increased proliferation at days 9-14 and accelerated maturation at days 7-14, with a larger S-phase population (40%) than controls (20%) at day 11; however, the proportion of apoptotic cells was comparable to controls. Previously, we have identified PV-specific dysregulation of several microRNAs (i.e. miR-150, 451, 222, 155, 378). We had analyzed expression profiles of selected target genes of these microRNAs based on in silico prediction and their known function pertinent to the observed PV-specific erythropoiesis differences. p27, cMYB and EPOR showed differential expression in PV erythroid progenitors at the specific stages of erythroid differentiation. In this study, we identified accelerated maturation and hyper-proliferation at early stages of PV erythropoiesis. We speculate that aberrant expression of p27, c-MYB, and EPOR may contribute to these abnormal features in PV erythropoiesis.

• MeSH
• buněčný cyklus MeSH
• erytroidní buňky cytologie   patologie   MeSH
• erytroidní prekurzorové buňky cytologie   patologie   MeSH
• erytropoéza MeSH
• geny myb genetika   MeSH
• inhibitor p27 cyklin-dependentní kinasy genetika   MeSH
• kultivované buňky MeSH
• lidé MeSH
• mikro RNA genetika   MeSH
• polycythaemia vera genetika   patologie   MeSH
• receptory erythropoetinu genetika   MeSH
• regulace genové exprese MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

BACKGROUND: Polycythemia vera is a clonal hematopoietic stem cell disorder in which the JAK2 V617F mutation is observed in >95% of patients, but an as yet unidentified process appears to initiate the clonal expansion of hematopoiesis. Because microRNA regulate hematopoietic differentiation, we hypothesized that dysregulated expression of microRNA may contribute to the pathophysiology of polycythemia vera. DESIGN AND METHODS: We performed gene expression profiling in five patients with polycythemia vera and in five controls using CombiMatrix MicroRNA CustomArray. ANOVA identified deregulated microRNA in polycythemia vera, and their expression was studied in a larger set of samples by quantitative reverse transcriptase polymerase chain reaction. The expression of these microRNA was also analyzed in other myeloproliferative disorders. RESULTS: We observed down-regulation of let-7a and up-regulation of miR-182 in polycythemia vera granulocytes, up-regulation of miR-143, miR-145 and miR-223 in polycythemia vera mononuclear cells, up-regulation of miR-26b in polycythemia vera platelets, and down-regulation of miR-30b, miR-30c and miR-150 in polycythemia vera reticulocytes. JAK2 V617F frequency was positively correlated with miR-143 expression and inversely correlated with let-7a, miR-30c, miR-342 and miR-150. Transcript level of predicted target genes was determined, and overexpression of IRAK2 was detected in all granulocytes from patients with myeloproliferative disorders and in polycythemia vera reticulocytes. Abnormally high HMGA2 microRNA was found in myelofibrosis granulocytes. CONCLUSIONS: Our study demonstrates that peripheral blood cells from patients with polycythemia vera have microRNA signatures distinct from those of controls. Our findings of aberrant microRNA expression underline the complexity of the molecular basis of polycythemia vera.

• MeSH
• buněčná diferenciace MeSH
• buněčný rodokmen MeSH
• granulocyty cytologie   MeSH
• hematopoéza MeSH
• Janus kinasa 2 genetika   MeSH
• leukocyty mononukleární cytologie   MeSH
• lidé MeSH
• mikro RNA * metabolismus   MeSH
• mutace * MeSH
• polycythaemia vera * genetika   metabolismus   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• primární myelofibróza metabolismus   MeSH
• regulace genové exprese * MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
• stanovení celkové genové exprese * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

OBJECTIVES: MicroRNAs (miRNAs) play key roles in a wide variety of normal and pathological cellular processes. A number of studies identified hematopoietic-specific miRNAs that are necessary for correct function of blood cells. Out of our microarray data, we chose 13 miRNAs that showed differential expression in peripheral blood cells (miR-15b, miR-16, miR-24, miR-30c, miR-106b, miR-142-3p, miR-142-5p, miR-150, miR-155, miR-181, miR-223, miR-342, and miR-451) and examined their expression in separated hematopoietic cell lineages. METHODS: Using quantitative real-time polymerase chain reaction, we measured relative expression of the miRNAs in fractions of reticulocytes, platelets, granulocytes, monocytes, B- and T-lymphocytes as well as in several hematopoietic cell lines. RESULTS: We observed that miR-16 and miR-142-3p were highly expressed in all native cell lineages, miR-451 reached the maximal expression in reticulocytes, miR-223 in platelets, granulocytes and monocytes, and miR-150 in B- and T-lymphocytes. Hierarchical clustering analysis grouped the lineage samples according to their origin based on the expression of these miRNAs. To validate discrimination power of the miRNAs, we quantified expression of the 13 miRNAs in several immortalized cell lines. Although the cell lines showed miRNA expression patterns considerably different from those of native cell lineages, clustering analysis distinguished between myeloid, lymphoid and non-hematopoietic cells. CONCLUSIONS: In conclusion, the study reports the expression levels of 13 miRNAs in particular blood cell lineages as well as immortalized cell lines. We demonstrate that the expression profiles of these miRNAs may be used for discrimination of the hematopoietic cell lineages.

• MeSH
• buněčná diferenciace fyziologie   MeSH
• buňky K562 MeSH
• financování organizované MeSH
• HeLa buňky MeSH
• leukocyty cytologie   metabolismus   MeSH
• lidé MeSH
• lymfoidní progenitorové buňky cytologie   metabolismus   MeSH
• mikro RNA biosyntéza   MeSH
• prekurzorové buňky granulocytů cytologie   metabolismus   MeSH
• regulace genové exprese fyziologie   MeSH
• retikulocyty cytologie   metabolismus   MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů metody   MeSH
• stanovení celkové genové exprese metody   MeSH
• trombocyty cytologie   metabolismus   MeSH
• Check Tag
• lidé MeSH

OBJECTIVE: Polycythemia vera (PV) is a myeloproliferative disorder, arising from the acquired mutation(s) of a hematopoietic stem cell. The JAK2 V617F somatic mutation is found in most PV patients; however, it is not the disease-initiating mutation. Because microRNAs (miRNAs) play a regulatory role in hematopoiesis, we studied miRNA expressions in PV and normal erythropoiesis. METHODS: Peripheral blood mononuclear cells were cultured in a three-phase liquid system resulting in synchronized expansion of erythroid progenitors. Using gene-expression profiling by CombiMatrix MicroRNArray, we searched for PV-specific changes at days 1, 14, and 21. Twelve miRNA candidates were then reevaluated by quantitative real-time polymerase chain reaction in a larger number of samples obtained from progenitors at the same stage of differentiation. RESULTS: A significant difference in miR-150 expression was found in PV. In normal erythropoiesis, three expression patterns of miRNAs were observed: progressive downregulation of miR-150, miR-155, miR-221, miR-222; upregulation of miR-451, miR-16 at late stages of erythropoiesis; and biphasic regulation of miR-339, miR-378. The miR-451 appears to be erythroid-specific. CONCLUSIONS: We identified the miRNAs with regulated expression in erythropoiesis; one appeared to be PV-specific. Their miRNA expression levels define early, intermediate, and late stages of erythroid differentiation. The validity of our findings was confirmed in nonexpanded peripheral blood cells.

• MeSH
• buněčná diferenciace genetika   MeSH
• časové faktory MeSH
• erytroidní prekurzorové buňky MeSH
• erytropoéza * genetika   MeSH
• kultivované buňky MeSH
• leukocyty mononukleární MeSH
• lidé MeSH
• mikro RNA * genetika   MeSH
• polycythaemia vera * genetika   MeSH
• regulace genové exprese u nádorů * MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
• stanovení celkové genové exprese MeSH
• studie případů a kontrol MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• Research Support, N.I.H., Extramural MeSH