ABSTRACT
AIM: To observe the effect of microRNA-16 (miR-16) on the megakaryocytic differentiation of K562 cells, and to explore the potential mechanism.METHODS:miR-16 was over-expressed or silenced by transfection with miR-16 mimics or inhibitor in K562 cells.The level of miR-16 was detected by real-time PCR.The expression of CD41, CD42b and CD61, as megakaryocytic differentiation markers, was detected by flow cytometry.The effect of miR-16 on the expression of myeloblastosis oncogene ( MYB) was measured by Western blotting, and flow cytometry was performed to confirm whether the effect of miR-16 on expression of CD41, CD42b and CD61 was mediated by MYB.RESULTS:Transfection with miR-16 mimics dramatically elevated the level of miR-16 and the expression of CD41, CD42b and CD61 in the K562 cells.Transfection with miR-16 inhibitor decreased the level of miR-16 and the expression of CD41, CD42b and CD61 in the K562 cells (P<0.05).The expression of MYB was regulated by miR-16, and MYB silencing reversed the regulation of CD41, CD42b and CD61 induced by miR-16.CONCLUSION:miR-16 regulates the megakaryocytic dif-ferentiation of K562 cells by targeting MYB.
ABSTRACT
VASP (Vasodilator-stimulated phosphoprotein) e Zyxin são proteínas reguladoras de actina que controlam a adesão célula-célula. Zyxin dirige a montagem da actina através da interação e recrutamento da VASP a sítios específicos da adesão. A fosforilação da VASP ou da Zyxin altera suas atividades nas junções aderentes. PKA fosforila VASP em serina 157, regulando, assim, importantes funções celulares de VASP. VASP interage com ABL e é substrato da oncoproteína BCR-ABL. A presença da proteína BCR-ABL promove a oncogênese em pacientes com leucemia mieloide crônica (LMC) devido à ativação constitutiva da atividade tirosina quinase. Apesar de já descrita alteração da expressão de VASP e Zyxin em diferentes tumores epiteliais, o papel de VASP e Zyxin na LMC, na via de sinalização BCR-ABL e a participação destas proteínas na hematopoiese são desconhecidos. Desta maneira, demonstramos aqui ausência de p-VASP ser157 em células de medula óssea de pacientes com LMC, em contraste com a presença de p-VASP ser157 em doadores saudáveis. Pacientes com LMC em remissão, responsivos a inibidores de tirosina quinase, apresentam p-VASP ser157, enquanto os pacientes resistentes não expressam p-VASP ser157. Utilizando células K562 inibidas para VASP ou Zyxin, observamos que VASP e Zyxin modulam as proteínas anti-apoptóticas BCL-2 e BCL-XL da via de sinalização do BCR-ABL. Em adição, células K562 silenciadas para a VASP apresentam diminuição na atividade de FAK y925 e demonstramos que VASP interage com FAK. A expressão de VASP e Zyxin e de suas formas ativas aumenta durante a diferenciação megacariocítica e a inibição de VASP implica em diminuição na expressão do marcador CD61. Identificamos no presente estudo a participação de VASP e Zyxin na via do BCR-ABL, regulando a expressão de proteínas efetoras anti-apoptóticas e, também, na diferenciação megacariocítica...
VASP (vasodilator-stimulated phosphoprotein) and Zyxin are actin regulatory proteins that control cell-cell adhesion. Zyxin directs actin assembly by interacting and recruiting VASP to specific sites of adhesion. The phosphorylation of VASP and Zyxin modifies their activity in cell-cell junctions. PKA phosphorylates VASP at serine 157 regulating VASP cellular functions. VASP interacts with ABL and VASP is a substrate of BCR-ABL oncoprotein. The presence of BCR-ABL protein drives oncogenesis in patients with chronic myeloid leukemia (CML) due to a constitutive activation of tyrosine kinase activity. It has been described an altered expression of VASP and Zyxin in different types of tumor; however the function of VASP and Zyxin in CML, in BCR-ABL pathway and in hematopoiesis remains unknown. We describe here the absence of p-VASP ser157 in CML bone marrow cells, in contrast to p-VASP ser157 expression in healthy donors. Patients responsive to tyrosine kinase inhibitors present p-VASP ser157, while resistant patients do not have p-VASP ser157. In K562 cells we observed that VASP and Zyxin modulate anti-apoptotic proteins BCL-2 and BCL-XL. VASP depletion in K562 cells decreases FAK y925 activity and VASP interacts with FAK. Expression of VASP, p-VASP, Zyxin and p-Zyxin increases during megakaryocyte differentiation and VASP inhibition affects this differentiation through reduced CD61 expression in VASP depleted cells. We identify here the participation of VASP and Zyxin in BCR-ABL pathway affecting anti-apoptotic proteins and, also, in megakaryocyte differentiation. Then, the altered expression of VASP activity in CML patients may contribute to CML pathogenesis, affecting cellular differentiation or leukemic cell adhesion...
Subject(s)
Humans , Male , Female , Adult , Middle Aged , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Bone Marrow Cells , Cell Adhesion , ZyxinABSTRACT
PURPOSE: CD34+ cells from umbilical cord blood (UCB) were cultured with stromal cells and growth factors, and cell proliferation and megakaryocytic differentiation were observed. The purposes of this study are to find out the optimal culture condition for the megakaryocytic differentiation of CD34+ cells from UCB. METHODS: CD34+ cells of mobilized peripheral blood (PB) and UCB were cultured in IMDM at a concentration of 1x105 cells/mL for 11 days. Thrombopoietin (TPO) 5 ng/mL and 50 ng/mL, flt-3 ligand (FL) 50 ng/mL and stem cell factor (SCF) 50 ng/mL were used as growth factors. Stromal cells cultured from bone marrow (BM) mononuclear cells appeared as a single layer of fusiform adherent cells with expression of SH-2 and ASMA. RESULTS: When PB CD34+ cells were cultured with growth factors only, the cell number increased in TPO 5 ng/mL and 50 ng/mL, TPO and FL, TPO, FL and SCF group increased upto mean 2.0 (in TPO 5 ng/mL), 2.9 (in TPO 50 ng/mL), 2.4 (TPO and FL), 2.8 (TPO, FL and SCF) fold, and the expression of CD61a were mean 11.5%, 12.5%, 13.2% and 17.0%, respectively. When the stromal cells were added to the growth factors, the cell number increased upto mean 4.6, 4.9, 9.2 and 68.5 fold and CD61a were expressed in mean 43.1%, 48.4%, 35.6%, and 6.2% of cultured cells. For UCB CD34+ cells, the cell number were increased upto 7.5, 7.6, 8.2 and 23.6 fold with growth factors and stromal cells. Expression of CD61a were mean 26.0%, 34.3%, 31.5% and 23.5%, respectively. CONCLUSION: Stromal cell enhanced the cellular proliferation and megakaryocytic differentiation of UCB CD34+ cells. The combination of TPO, FL and SCF increased total cell number upto the highest value but the proportion of the CD61a+ cells were relatively low. The combination of TPO and FL induced both cellular proliferation and megakaryocytic differentiation in a large amount.