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European Review For Medical and... Apr 2019The aim of this study was to elucidate the biological function of long non-coding RNA (lncRNA) HOTTIP (HOXA transcript at the distal tip) in the development of acute...
OBJECTIVE
The aim of this study was to elucidate the biological function of long non-coding RNA (lncRNA) HOTTIP (HOXA transcript at the distal tip) in the development of acute myeloid leukemia (AML), and to investigate the potential mechanism.
PATIENTS AND METHODS
Relative expression levels of HOTTIP, microRNA-608 and DDA1 in AML patients were determined by quantitative Real-time polymerase chain reaction (qRT-PCR). Meanwhile, the expressions of these genes in AML cell lines were detected as well. The regulatory effects of HOTTIP, microRNA-608 and DDA1 on the proliferative ability and cell cycle progression of AML cells were examined by cell counting kit-8 (CCK-8) and flow cytometry, respectively. Dual-luciferase reporter gene assay was performed to confirm the binding condition of microRNA-608 to HOTTIP and DDA1. Finally, the specific role of HOTTIP/microRNA-608/DDA1 axis in the development of AML was verified through a series of rescue experiments.
RESULTS
HOTTIP was highly expressed in AML-M5 patients than normal controls. No significant difference in HOTTIP expression was found between patients with other subtypes of AML (M0, M1, M2, M3, M4 and M6) and normal controls. HOTTIP expression was significantly up-regulated in AML cell lines U-937 and THP-1. Up-regulation of HOTTIP remarkably promoted the proliferative potential and cell cycle progression of AML cells. Dual-luciferase reporter gene indicated that HOTTIP could bind to microRNA-608, which was lowly expressed in AML-M5 patients. Overexpression of microRNA-608 significantly inhibited the proliferative ability and cell cycle progression of U-937 and THP-1 cells. More importantly, microRNA-608 could partially reverse the regulatory effect of HOTTIP on AML cells. Meanwhile, DDA1 was verified as the target of microRNA-608. Subsequent experiments elucidated that DDA1 significantly accelerated the proliferation and cell cycle of AML cells. Furthermore, DDA1 could reverse the inhibitory effect of microRNA-608 on proliferative ability and cell cycle progression of AML cells.
CONCLUSIONS
HOTTIP accelerated the proliferative ability and cell cycle of AML cells via up-regulating DDA1 expression by sponging microRNA-608.
Topics: Cell Cycle; Cell Proliferation; DNA-Binding Proteins; Disease Progression; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Myeloid, Acute; MicroRNAs; RNA, Long Noncoding; U937 Cells
PubMed: 31002141
DOI: 10.26355/eurrev_201904_17569 -
Analytical Biochemistry Nov 2016The binding and activation of macrophages by microscopic aggregates of oxLDL in the intima of the arteries may be an important step towards atherosclerosis leading to...
The binding and activation of macrophages by microscopic aggregates of oxLDL in the intima of the arteries may be an important step towards atherosclerosis leading to heart attack and stroke. Microbeads coated with oxLDL were used to activate, capture and isolate the oxLDL receptor complex from the surface of live cells. Analysis of the resulting tryptic peptides by liquid chromatography and tandem mass spectrometry revealed the Spleen Tyrosine Kinase (SYK), and many of SYK's known interaction network including Fc receptors (FCGR2A, FCER1G and FCGR1A) Toll receptor 4 (TLR4), receptor kinases like EGFRs, as well as RNA binding and metabolism proteins. High-intensity precursor ions (∼9*E3 to 2*E5 counts) were correlated to peptides and specific phosphopeptides from long isoform of SYK (SYK-L) by the SEQUEST, OMSSA and X!TANDEM algorithms. Peptides or phosphopeptides from SYK were observed with the oxLDL-microbeads. Pharmacological inhibitors of SYK activity significantly reduced the engulfment of oxLDL microbeads in the presence of serum factors, but had little effect on IgG phagocytosis. Anti SYK siRNA regulated oxLD engulfment in the context of serum factors and or SYK-L siRNA significantly inhibited engulfment of oxLDL microbeads, but not IgG microbeads.
Topics: Chromatography, Liquid; Humans; Immunoglobulin G; Lipoproteins, LDL; Phagocytosis; Receptors, Fc; Receptors, Oxidized LDL; Syk Kinase; Toll-Like Receptor 4; U937 Cells
PubMed: 27510553
DOI: 10.1016/j.ab.2016.07.021 -
British Journal of Cancer Aug 2017Lurbinectedin is a novel anticancer agent currently undergoing late-stage (Phase II /III) clinical evaluation in platinum-resistant ovarian, BRCA1/2-mutated breast and...
BACKGROUND
Lurbinectedin is a novel anticancer agent currently undergoing late-stage (Phase II /III) clinical evaluation in platinum-resistant ovarian, BRCA1/2-mutated breast and small-cell lung cancer. Lurbinectedin is structurally related to trabectedin and it inhibits active transcription and the DNA repair machinery in tumour cells.
METHODS
In this study we investigated whether lurbinectedin has the ability to modulate the inflammatory microenvironment and the viability of myeloid cells in tumour-bearing mice.
RESULTS
Administration of lurbinectedin significantly and selectively decreased the number of circulating monocytes and, in tumour tissues, that of macrophages and vessels. Similar findings were observed when a lurbinectedin-resistant tumour variant was used, indicating a direct effect of lurbinectedin on the tumour microenviroment. In vitro, lurbinectedin induced caspase-8-dependent apoptosis of human purified monocytes, whereas at low doses it significantly inhibited the production of inflammatory/growth factors (CCL2, CXCL8 and VEGF) and dramatically impaired monocyte adhesion and migration ability. These findings were supported by the strong inhibition of genes of the Rho-GTPase family in lurbinectedin-treated monocytes.
CONCLUSIONS
The results illustrate that lurbinectedin affects at multiple levels the inflammatory microenvironment by acting on the viability and functional activity of mononuclear phagocytes. These peculiar effects, combined with its intrinsic activity against cancer cells, make lurbinectedin a compound of particular interest in oncology.
Topics: Animals; Antineoplastic Agents, Alkylating; Apoptosis; Carbolines; Caspase 8; Cell Adhesion; Cell Movement; Chemokine CCL2; Dioxoles; Down-Regulation; Female; Fibrosarcoma; Gene Expression; Gene Expression Profiling; HL-60 Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Interleukin-8; Leukocyte Count; Macrophages; Mice; Mice, Inbred C57BL; Monocytes; Neovascularization, Pathologic; Ovarian Neoplasms; Tetrahydroisoquinolines; Trabectedin; Tumor Microenvironment; U937 Cells; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays; rho GTP-Binding Proteins
PubMed: 28683469
DOI: 10.1038/bjc.2017.205 -
BMC Cancer Nov 2021Despite of the frequently reported Dnmt3a abormality in classical myeloproliferative neoplasms (cMPNs) patients, few research explores how the Dnmt3a is regulated by...
BACKGROUND
Despite of the frequently reported Dnmt3a abormality in classical myeloproliferative neoplasms (cMPNs) patients, few research explores how the Dnmt3a is regulated by Jak2 mutation. In this study, we have investigated how the Dnmt3a is regulated by Jak2 mutation and its effects on downstream signaling pathways in cMPNs.
METHODS
Specimens of Jak2 positive cMPN patients and normal controls were collected. Murine BaF3 cell line was used to construct cell models. Dual-Glo luciferase assays and chromatin immunoprecipitation (ChIP)-qPCR were performed to detect the impact of Stat5a on transcription activity of Dnmt3a. Soft agar colony formation assay and cell counting assay were performed to detect cell proliferation. BrdU staining and flow cytometry were used to investigate cell cycle distribution. Western blotting and quantitative reverse-transcription PCR (qPCR) were performed to detect the expression levels of genes.
RESULTS
Firstly, the results of western blotting and qPCR revealed that compared with the control samples, Dnmt3a is downregulated in Jak2 positive samples. Then we explored the mechanism behind it and found that Dnmt3a is a downstream target of Stat5a, the transcription and translation of Dnmt3a is suppressed by the binding of aberrantly activated Stat5a with Dnmt3a promoter in Jak2 positive samples. We further revealed the region approximately 800 bp upstream of the first exon of the Dnmt3a promoter, which includes a gamma-activated sequence (GAS) motif of Stat5a, is the specific site that Stat5a binds to. Soft agar colony formation assay, cell counting assay, and BrdU staining and flow cytometry assay found that Dnmt3a in Jak2-BaF3 cells significantly affected the cell proliferation capacity and cell cycle distribution by suppressing Cdkn1a via miR-17-5p/Cdkn1a axis and mediated G0/G1 arrest.
CONCLUSIONS
Transcription and translation of Dnmt3a is downregulated by the binding of Stat5a with Dnmt3a promoter in Jak2 cells. The GAS motif at promoter of Dnmt3a is the exact site where the Stat5a binds to. Dnmt3a conducted G0/G1 arrest through regulating miR-17-5p/Cdkn1a axis. The axis of Stat5a/Dnmt3a/miR-17-5p/Cdkn1a potentially provides a treatment target for cMPNs.
Topics: Aminopyridines; Animals; Binding Sites; Blotting, Western; Case-Control Studies; Cell Count; Cell Line; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; DNA Methyltransferase 3A; Down-Regulation; Exons; G1 Phase Cell Cycle Checkpoints; Humans; Imidazoles; Janus Kinase 2; K562 Cells; Mice; MicroRNAs; Monocytes; Mutation; Myeloproliferative Disorders; Promoter Regions, Genetic; Pyrazoles; Pyridazines; STAT5 Transcription Factor; Signal Transduction; Transcription, Genetic; Tumor Stem Cell Assay; Tumor Suppressor Proteins; U937 Cells
PubMed: 34773997
DOI: 10.1186/s12885-021-08915-0 -
Bioscience Reports Jun 2021Acute leukemia is a hematological malignant tumor. Long non-coding RNA urothelial cancer-associated 1 (UCA1) is involved in the chemo-resistance of diverse cancers, but...
Acute leukemia is a hematological malignant tumor. Long non-coding RNA urothelial cancer-associated 1 (UCA1) is involved in the chemo-resistance of diverse cancers, but it is unclear whether UCA1 is associated with the sensitivity of acute leukemia cells to daunorubicin (DNR). DNR (100 nM) was selected for functional analysis. The viability, cell cycle progression, apoptosis, and invasion of treated acute leukemia cells (HL-60 and U-937) were evaluated by cell counting kit-8 (CCK-8) assay, flow cytometry assay, or transwell assay. Protein levels were detected with Western blot analysis. Expression patterns of UCA1 and miR-613 were assessed by quantitative real-time polymerase chain reaction (qRT-PCR). The relationship between UCA1 and microRNA-613 (miR-613) was verified by dual-luciferase reporter assay. We observed that UCA1 expression was elevated in HL-60 and U-937cells. DNR constrained viability, cell cycle progression, invasion, and facilitated apoptosis of HL-60 and U-937 cells in a dose-dependent manner, but these impacts mediated by DNR were reverted after UCA1 overexpression. MiR-613 was down-regulated in HL-60 and U-937 cells, and UCA1 was verified as a miR-613 sponge. MiR-613 inhibitor reversed DNR treatment-mediated effects on viability, cell cycle progression, apoptosis, and invasion of HL-60 and U-937 cells, but these impacts mediated by miR-613 inhibitor were counteracted after UCA1 inhibition. Notably, the inactivation of the PI3K/AKT pathway caused by DNR treatment was reversed after miR-613 inhibitor introduction, but this influence mediated by miR-613 inhibitor was offset after UCA1 knockdown. In conclusion, UCA1 up-regulation facilitated the resistance of acute leukemia cells to DNR via the PI3K/AKT pathway by sponging miR-613.
Topics: Antibiotics, Antineoplastic; Apoptosis; Cell Cycle; Cell Movement; Cell Proliferation; Daunorubicin; Drug Resistance, Neoplasm; Gene Expression Regulation, Leukemic; HL-60 Cells; Humans; Leukemia; MicroRNAs; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinase; Proto-Oncogene Proteins c-akt; RNA, Long Noncoding; Signal Transduction; U937 Cells
PubMed: 33969374
DOI: 10.1042/BSR20201389 -
Blood Feb 2006TEL2/ETV7 is highly homologous to the ETS transcription factor TEL/ETV6, a frequent target of chromosome translocation in human leukemia. Although both proteins are...
TEL2/ETV7 is highly homologous to the ETS transcription factor TEL/ETV6, a frequent target of chromosome translocation in human leukemia. Although both proteins are transcriptional inhibitors binding similar DNA recognition sequences, they have opposite biologic effects: TEL inhibits proliferation while TEL2 promotes it. In addition, forced expression of TEL2 but not TEL blocks vitamin D3-induced differentiation of U937 and HL60 myeloid cells. TEL2 is expressed in the hematopoietic system, and its expression is up-regulated in bone marrow samples of some patients with leukemia, suggesting a role in oncogenesis. Recently we also showed that TEL2 cooperates with Myc in B lymphomagenesis in mice. Here we show that forced expression of TEL2 alone in mouse bone marrow causes a myeloproliferative disease with a long latency period but with high penetrance. This suggested that secondary mutations are necessary for disease development. Treating mice receiving transplants with TEL2-expressing bone marrow with the chemical carcinogen N-ethyl-N-nitrosourea (ENU) resulted in significantly accelerated disease onset. Although the mice developed a GFP-positive myeloid disease with 30% of the mice showing elevated white blood counts, they all died of T-cell lymphoma, which was GFP negative. Together our data identify TEL2 as a bona fide oncogene, but leukemic transformation is dependent on secondary mutations.
Topics: Alkylating Agents; Animals; Bone Marrow; Bone Marrow Transplantation; Carcinogens; Cell Transformation, Neoplastic; Ethylnitrosourea; Female; Gene Expression; HL-60 Cells; Hematopoiesis; Humans; Lymphoma, T-Cell; Male; Mice; Proto-Oncogene Proteins c-ets; Repressor Proteins; Transcription Factors; Translocation, Genetic; U937 Cells; ETS Translocation Variant 6 Protein
PubMed: 16234363
DOI: 10.1182/blood-2005-03-1196 -
Virology May 1999The ability of HIV to match levels of viral mRNA to the activation state of the host cell may play a role in its ability to persist as well as to replicate. This linkage...
The ability of HIV to match levels of viral mRNA to the activation state of the host cell may play a role in its ability to persist as well as to replicate. This linkage depends on the function of the viral transcriptional regulatory protein, Tat, which increases the efficiency of RNA elongation (transcriptional processivity) in response to cellular activation. To quantify levels of Tat function in vivo, a quantitative competitive RT-PCR assay was developed that reflects levels of TAR leader fragments (nonprocessive transcripts) and viral mRNA (processive transcripts), indicating low or high levels of Tat function, respectively. The abundance of these RNA species was measured in peripheral blood mononuclear cells (PBMC) of 22 HIV-1-positive individuals (CD4(+) T cell counts 63-934/mm3) and in established cell line models of HIV constitutive replication (H9IIIB) and reversible latency (U1 and ACH-2). In PBMC, the level of total viral transcripts ranged over four orders of magnitude; however, nonprocessive transcription predominated: 70% of PBMC samples had a ratio of processive to total transcripts of <0.3 and none of the samples had 100% processivity. The cell line studies revealed that, even in activated H9IIIB cells, nonprocessive transcription dominates and that latently infected cells can have different transcriptional responses to activation. This is the first study that enumerates degrees of transcriptional processivity in the circulating mononuclear cell compartment and the results suggest that limitation of Tat function may be a common phenotype throughout the course of the disease.
Topics: Cell Line; Gene Expression Regulation, Viral; Gene Products, tat; HIV-1; Humans; Leukocytes, Mononuclear; Mitogens; RNA Processing, Post-Transcriptional; RNA, Messenger; RNA, Viral; T-Lymphocytes; Transcription, Genetic; U937 Cells; tat Gene Products, Human Immunodeficiency Virus
PubMed: 10329550
DOI: 10.1006/viro.1999.9647 -
Anticancer Research Mar 2024Chemotherapy drugs for leukemia, such as 5-azacytidine (Aza), have often various adverse effects. Hesperetin (Hes), a naturally occurring compound, is a potential...
BACKGROUND/AIM
Chemotherapy drugs for leukemia, such as 5-azacytidine (Aza), have often various adverse effects. Hesperetin (Hes), a naturally occurring compound, is a potential adjuvant agent for anticancer therapy. This study aimed to investigate the effect of an Aza-Hes combination on acute leukemia cell lines, which elucidates the role of combination treatment in leukemia progression.
MATERIALS AND METHODS
HL-60 and U937 cells were treated with Aza and Hes at various concentrations or their combination. Cell proliferation and apoptosis was evaluated using the Cell Counting Kit-8 assay and annexin V/propidium iodide staining, respectively. Cell cycle analysis was conducted using flow cytometry. The expression of apoptosis-related and cell cycle-related proteins in leukemia cells was analyzed through western blotting. The synergistic effect of the Aza and Hes agents was estimated using the Chou-Talalay method.
RESULTS
We observed that Aza or Hes monotherapy engendered a dose-dependent reduction in HL-60 and U937 cell viability. However, treatment with the Aza-Hes combination for 24 h synergistically inhibited U937 cell proliferation by inducing both apoptosis and S-phase cell cycle arrest. Furthermore, the Aza-Hes combination down-regulated p-ERK and p-c-Jun N-terminal kinase expression and up-regulated p-p38 expression.
CONCLUSION
Overall, our findings indicate that the Aza-Hes combination induces apoptosis and S-phase cell-cycle arrest through the mitogen-activated protein kinase pathway. In conclusion, the Aza-Hes combination is a potential antileukemia treatment.
Topics: Humans; Azacitidine; U937 Cells; Apoptosis; Cell Cycle; Cell Cycle Checkpoints; Cell Proliferation; Leukemia, Myeloid, Acute; Cell Cycle Proteins; Cell Line, Tumor; Hesperidin
PubMed: 38423637
DOI: 10.21873/anticanres.16898 -
Haematologica Dec 2014CXC chemokine receptor 4 (CXCR4) is an essential regulator for homing and maintenance of hematopoietic stem cells within the bone marrow niches. Analysis of clinical... (Comparative Study)
Comparative Study
CXC chemokine receptor 4 (CXCR4) is an essential regulator for homing and maintenance of hematopoietic stem cells within the bone marrow niches. Analysis of clinical implications of bone marrow CXCR4 expression in patients with acute myeloid leukemia showed not only higher CXCR4 expression was an independent poor prognostic factor, irrespective of age, white blood cell counts, cytogenetics, and mutation status of NPM1/FLT3-ITD and CEBPA, but also showed CXCR4 expression was inversely associated with mutations of CEBPA, a gene encoding transcription factor C/EBPα. Patients with wild-type CEBPA had significantly higher CXCR4 expression than those with mutated CEBPA. We hypothesized that CEBPA might influence the expression of CXCR4. To test this hypothesis, we first examined endogenous CXCR4 expression in 293T and K562 cells over-expressing wild-type C/EBPα p42 and demonstrated that CXCR4 levels were increased in these cells, whilst the expression of the N-terminal mutant, C/EBPα p30, diminished CXCR4 transcription. We further showed p42 was bound to the CXCR4 promoter by the chromatin immunoprecipitation assays. Induction of p42 in the inducible K562-C/EBPα cell lines increased the chemotactic migration. Moreover, decreased expression of C/EBPα by RNA interference decreased levels of CXCR4 protein expression in U937 cells, thereby abrogating CXCR4-mediated chemotaxis. Our results provide, for the first time, evidence that C/EBPα indeed regulates the activation of CXCR4, which is critical for the homing and engraftment of acute myeloid leukemia cells, while p30 mutant impairs CXCR4 expression.
Topics: Adolescent; Adult; Aged; Aged, 80 and over; Blotting, Western; Bone Marrow; CCAAT-Enhancer-Binding Proteins; Case-Control Studies; Chemotaxis; Cohort Studies; Female; Flow Cytometry; Follow-Up Studies; Gene Expression Regulation, Neoplastic; Hematopoietic Stem Cells; Humans; K562 Cells; Leukemia, Myeloid, Acute; Male; Middle Aged; Mutation; Neoplasm Recurrence, Local; Neoplasm Staging; Nucleophosmin; Prognosis; Promoter Regions, Genetic; RNA, Messenger; RNA, Small Interfering; Real-Time Polymerase Chain Reaction; Receptors, CXCR4; Reverse Transcriptase Polymerase Chain Reaction; Survival Rate; U937 Cells; Young Adult
PubMed: 25193961
DOI: 10.3324/haematol.2014.107821 -
Journal of Cellular Biochemistry Mar 2019Dysregulation of microRNAs is closely implicated in the initiation and progression of human cancers including acute myeloid leukemia (AML). Though miR-139-5p was...
Dysregulation of microRNAs is closely implicated in the initiation and progression of human cancers including acute myeloid leukemia (AML). Though miR-139-5p was reported to be a potent tumor suppressor in adult AML, its underlying molecular mechanism in AML remains to be further defined. Herein, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis were conducted to determine the expressions of miR-139-5p and tetraspanin3 (Tspan3) in AML patients and cells. Luciferase reporter assay, qRT-PCR, and Western blot analysis were carried out to detect the interaction between miR-139-5p and Tspan3. Cell proliferation, cell cycle distribution, invasion, and migration were evaluated by cell counting kit-8, flow cytometry, transwell invasion, and migration assays, respectively. Western blot analysis was conducted to determine phosphorylated-protein kinase B (Akt) and Akt levels. We found that a significant reduction in miR-139-5p expression and a prominent increase in Tspan3 expression were observed in AML patients and cells. Tspan3 was confirmed as a direct target of miR-139-5p and was negatively modulated by miR-139-5p. Rescue experiments showed that overexpression of miR-139-5p constrained cell proliferation, invasion and migration capabilities, and induced cell cycle arrest at the S phase in AML cells, which were partially reversed by Tspan3 overexpression. In addition, we found that miR-139-5p suppressed the phosphoinositide 3-kinase (PI3K)/Akt pathway in AML cells by targeting Tspan3. In conclusion, our study concluded that miR-139-5p suppressed the leukemogenesis in AML cells by targeting Tspan3 through inactivation of the PI3K/Akt pathway, providing a better understanding of AML progression.
Topics: Cell Proliferation; Genes, Tumor Suppressor; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; MicroRNAs; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; RNA, Neoplasm; Signal Transduction; THP-1 Cells; Tetraspanins; U937 Cells
PubMed: 30367526
DOI: 10.1002/jcb.27728