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Exosomes derived from cancer cells can affect various functions of mesenchymal stem cells (MSCs) via conveying microRNAs (miRs). miR-21 and miR-146a have been demonstrated to regulate MSC proliferation and transformation. Interleukin-6 (IL-6) secreted from transformed MSCs in turn favors the survival of multiple myeloma (MM) cells. However, the effects of MM exosomes on MSC functions remain largely unclear. In this study, we investigated the effects of OPM2 (a MM cell line) exosomes (OPM2-exo) on regulating the proliferation, cancer-associated fibroblast (CAF) transformation, and IL-6 secretion of MSCs and determined the role of miR-21 and miR-146a in these effects. We found that OPM2-exo harbored high levels of miR-21 and miR-146a and that OPM2-exo coculture significantly increased MSC proliferation with upregulation of miR-21 and miR-146a. Moreover, OPM2-exo induced CAF transformation of MSCs, which was evidenced by increased fibroblast-activated protein (FAP), α-smooth muscle actin (α-SMA), and stromal-derived factor 1 (SDF-1) expressions and IL-6 secretion. Inhibition of miR-21 or miR-146a reduced these effects of OPM2-exo on MSCs. In conclusion, MM could promote the proliferation, CAF transformation, and IL-6 secretion of MSCs partially through regulating miR21 and miR146a.
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OBJECTIVE: To screen aptamers binding CD33+/CD34- cells from patients with acute myeloblastic leukemia M2 subtype (AML-M2). METHODS: CD33+/CD34- cells from patients with AML-M2 were taken as targeted cells, CD33+/ CD34- cells from normal people were taken as anti-selecting cells, and aptamers in the single strand deoxyribonucleic acid (ssDNA) library were then selected repeatedly by cell-systematic evolution of ligands by exponential enrichment (C-SELEX) technology, and amplified by polymerase chain reaction (PCR) to generate sub-ssDNA library. During the experiment, PCR amplification with fluorescently labeled primer and flow cytometry were performed to analyze the aptamers'enrichment of sub-library, and the final round product of the sub-ssDNA library was cloned. After the sequencing, the primary and secondary structures of the aptamers were analyzed. RESULTS: Electrophoresis indicated that the product of PCR amplification for each round subssDNA library was able to see a clear DNA band in the agarose gel. After 13 rounds of screening, the fluorescence intensity of the sub-ssDNA library binding the cells ranged from 2.14% to 51.12%, reaching a steady state at the 13th round. A total of 30 clones were selected and sequenced, 22 of which contained 1 of the 4 conserved sequences of AAGTA, TATCT, AGATG and AAATT in their primary structure, but the remained eight aptamers contained none of the conserved sequence. Secondary structure analysis indicated that four stem-loops and loop simulation convex structures existed in the aptamers. CONCLUSION: C-SELEX technology can be used to screen the aptamers binding primary cells from patients with leukemia. The aptamers selected from the CD33+/CD34- cells from the patients of AML-M2 subtype might be used for the diagnosis and treatment for leukemia.
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Antígenos de Diferenciación Mielomonocítica/genética , Aptámeros de Nucleótidos/metabolismo , ADN de Cadena Simple/genética , Leucemia Mieloide Aguda/genética , Adolescente , Adulto , Antígenos CD34/genética , Antígenos CD34/inmunología , Antígenos de Diferenciación Mielomonocítica/inmunología , Aptámeros de Nucleótidos/genética , Femenino , Humanos , Leucemia Mieloide Aguda/inmunología , Masculino , Persona de Mediana Edad , Técnica SELEX de Producción de Aptámeros , Lectina 3 Similar a Ig de Unión al Ácido Siálico/genética , Lectina 3 Similar a Ig de Unión al Ácido Siálico/inmunología , Adulto JovenRESUMEN
OBJECTIVE: To screen and analyze CD34(+) cell specific microRNAs (miRNAs) from the patients with acute myelogenous leukemia (AML) and their expression. METHODS: CD34(+) cells were sorted from AML patients or the mobilized peripheral blood of the donors of hematopoietic stem cell transplantation (normal control subjects) and followed by the extraction of the cell total RNAs. The differentially expressed microRNAs (miRNAs, miR) were selected after hybridizing with miRNA microarray, real time polymerase chain reaction (real-time PCR) was subsequently applied to confirm the expression of the selected miRs, and PCR products were further cloned and sequenced to check their specificity. RESULTS: Of the differentially expressed miRNAs, 191 were found to be at least one-fold change in the CD34(+) cells between the AML patients and the normal control subjects. Of the 191 miRNAs, the expression difference of 94 was significant (P < 0.05). Among these 94 miRNAs, the expression of 44 miRNAs was increased and the other 50 miRNAs was decreased in the CD34(+) cells from the bone marrow of AML patients compared with the CD34(+) cells from the mobilized peripheral blood of the normal control subjects. Real time PCR verified that the expression level of miR-10a and miR-220c in the CD34(+) cells from the bone marrow of AML patients was 19.6% and 19.0% of that of CD34(+) cells from mobilized peripheral blood of the normal control subjects. DNA sequencing and BLAST DNA database searching results indicated that the PCR products were really miR-10a and miR-220c. CONCLUSION: A variety of differentially expressed-miRNAs are existed between AML and normal control subjects CD34(+) cells, the expression of miR-10a and miR-220c was significantly down-regulated in the CD34(+) cells from the bone marrow of AML patients.
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Células Madre Hematopoyéticas , Leucemia Mieloide Aguda/genética , MicroARNs/metabolismo , Antígenos CD34/metabolismo , Femenino , Humanos , Leucemia Mieloide Aguda/metabolismo , Masculino , MicroARNs/genética , Persona de Mediana Edad , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
A little is known about the specific marker on the surface of acute leukemia cells, leading to the lack of the specific diagnosis method for acute leukemia. Therefore, in this study, cell-systematic evolution of ligands by exponential enrichment (cSELEX) was performed to screen the aptamers binding to CD33(+)/CD34(+) cells from the patients with acute myeloblastic leukemia (AML) of M(2) subtype (AML-M2) so as to provide the basis for finding the specific marker on the surface of AML-M(2) CD33(+)/CD34(+) cells. Firstly, AML-M2 CD33(+)/CD34(+) cells were sorted and used as targeted cells, and normal CD33(+)/CD34(+)cells were used as counter-targeted cells; the aptamers binding to CD33(+)/CD34(+) cells from patients with AML-M2 were screened from the single strand deoxyribonucleic acid (ssDNA) library by cSELEX. Subsequently, each aptamer structure was analyzed after cloning and sequencing. The results indicated that after 13 round of screenings, the enrichment of aptamers in the ssDNA library was ranged from 0.7% to 52.9%, and reached steady state at 13th round screening. Sequence analysis for 30 aptamers showed that most of the aptamers born one of the three conserved sequences of CCCCT, CTCTC, and CTCAC. Secondary structure analysis indicated that three different secondary structures existed in these aptamers. It is concluded that the aptamers binding to the AML-M(2) CD33(+)/CD34(+) cells are successfully screened, which lay the basis for further looking for the specific marker on the surface of AML-M2 CD33(+)/CD34(+) cells, and the molecular diagnosis of the AML-M2 leukemia.
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Antígenos CD34/genética , Antígenos CD/genética , Antígenos de Diferenciación Mielomonocítica/genética , Aptámeros de Nucleótidos/metabolismo , Leucemia Mieloide Aguda/genética , Antígenos CD/inmunología , Antígenos CD34/inmunología , Antígenos de Diferenciación Mielomonocítica/inmunología , Biomarcadores , Citometría de Flujo , Humanos , Inmunofenotipificación , Leucemia Mieloide Aguda/inmunología , Conformación de Ácido Nucleico , Técnica SELEX de Producción de Aptámeros , Lectina 3 Similar a Ig de Unión al Ácido SiálicoRESUMEN
Polycomb repressive complex 2 (PRC2), which mediates trimethylation of lysine 27 on histone H3 (K27me3), plays an important role in many types of stem cell differentiation. Here, we try to reveal how PRC2, PRC2-mediated repressive histone marker H3K27me3, and active histone marker histone H4 acetylation (acH4) regulate the CD11b transcription during alltrans retinoic acid (ATRA)-induced HL-60 leukemia cell differentiation. By using quantitative real-time polymerase chain reaction (qPCR) and western blot analysis, we found that the mRNA and protein expression levels of two members of PRC2 were decreased during ATRA-induced HL-60 differentiation, respectively. When treated with ATRA for 72 h, the EZH2 and SUZ12 mRNA levels were decreased to 35% and 38% of the control group, respectively. At the same time, the granulocytic mature surface marker CD11b expression was increased significantly at mRNA level detected by qPCR and protein level detected by flow cytometry. By using chromatin immunoprecipitation assay, we compared the local changes in SUZ12 binding and PRC2-mediated H3K27me3 at the promoter of CD11b during ATRA-induced HL-60 differentiation. Both the levels of SUZ12 binding and PRC2-mediated H3K27me3 at the promoter of CD11b were decreased for 4.1 and 3.8 folds, respectively. And we also found the increase in the acH4 level up to 4 folds after 72 h of ATRA treatment. These results suggested that the histone modification including PRC2-mediated repressive histone marker H3K27me3 and active histone marker acH4 may involve in CD11b transcription during HL-60 leukemia cells reprogramming to terminal differentiation.
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Antígeno CD11b/genética , Diferenciación Celular/efectos de los fármacos , Histonas/metabolismo , Proteínas Represoras/metabolismo , Transcripción Genética/efectos de los fármacos , Tretinoina/farmacología , Acetilación , Antígeno CD11b/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteína Potenciadora del Homólogo Zeste 2 , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HL-60 , Humanos , Proteínas de Neoplasias , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Complejo Represivo Polycomb 2 , Proteínas del Grupo Polycomb , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Represoras/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Myeloperoxidase (MPO) plays a crucial role in the differentiation of acute lymphoblastic leukemia (ALL) and acute non-lymphoblastic leukemia (ANLL). In this report, we proposed the application of the enhanced chemiluminescent (ECL) technique to the determination of MPO activity in blasts of acute leukemia (AL). METHODS: Bone-marrow samples were obtained from 23 patients with AL (ALL, 5 cases; ANLL, 13 cases; AUL, 1 cases; mixed-lineage AL, 4 cases). Cells were incubated with a standard reaction mixture and chemiluminescence was measured. The mean peak light emission (PLE) was assessed. RESULTS: When the cut-off point of PLE was settled at 2483, which was set for the discrimination between ANLL and ALL (mean + 3 x SD of ALL samples, n=5), all cases of ALL were MPO-negative, and ten of the thirteen ANLL patients were MPO-positive, which was concordant with cytochemical staining. In addition, this technique was able to demonstrate MPO activity in 4 mixed-lineage AL cases which did not stain for MPO in cytochemistry preparations. CONCLUSIONS: Our ECL technique is simple, inexpensive, and easier to perform compared to other procedures used to measure MPO activity in AL.
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Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/patología , Mediciones Luminiscentes/métodos , Peroxidasa/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Adolescente , Adulto , Anciano , Niño , Diagnóstico Diferencial , Femenino , Células HL-60 , Humanos , Lactante , Leucemia Mieloide Aguda/diagnóstico , Masculino , Persona de Mediana Edad , Peroxidasa/análisis , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnósticoRESUMEN
The study was aimed to investigate the expression of VEGF mRNA and VEGF protein in HL-60 cells treated with diallyl disulfide (DADS), and to explore the antileukemic mechanism of DADS in respect of VEGF production. Semi-quantitative RT-PCR and ELISA were used to detect the expression of VEGF mRNA and secretion of VEGF protein in HL-60 cell lines treated by DADS respectively. The results showed that the expression of VEGF mRNA and secretion of VEGF protein were found in HL-60 cells. The expression of VEGF mRNA and secretion of VEGF protein in HL-60 cells could be down regulated by treatment with 0.625, 1.25, and 2.5 microg/mL DADS for 48 and 72 hours and the effects had a dose dependent relationship (r > 0.9, P < 0.01). The differences between DADS treated HL-60 cell groups and the control group were statistically significant (P < 0.01), there were also statistically significant differences among three DADS-treated HL-60 cell groups (P < 0.05). It is concluded that DADS effectively inhibits the proliferation of human leukemia cell line HL-60 cells; DADS exerts its antileukemic effects by reduction of the expression of VEGF mRNA and VEGF protein secretion.