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Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi =... Nov 2021Objective To investigate the effect of lncRNA CRNDE on proliferation, apoptosis, and cell cycle of U937 cells and its mechanism. Methods The expression level of CRNDE in...
Objective To investigate the effect of lncRNA CRNDE on proliferation, apoptosis, and cell cycle of U937 cells and its mechanism. Methods The expression level of CRNDE in bone marrow cells of AML patients was analyzed by GEPIA database; the mRNA expression levels of miR-136-5p, CRNDE, and minichromosome maintenance 5(MCM5) in AML cell lines were detected by quantitative real-time PCR (qRT-PCR). The lentiviral vector with CRNDE knocked down was constructed and transfected into U937 cells which were randomized into CRNDE knockdown group (sh-CRNDE group) and negative control group (sh-NC group); miR-136-5p mimic and miR-136-5p inhibitor were transfected respectively to overexpress and knock down miR-136-5p in U937 cells which were randomized into miR-136-5p-mimic group, NC-mimic group, miR-136-5p-inhibitor group, and NC-inhibitor group. The effect of CRNDE and miR-136-5p on proliferation was detected by CCK-8 assay and cell counting assay, and the effect of them on cell cycle and apoptosis was detected by flow cytometry. The mRNA expressions of miR-136-5p, CRNDE, and MCM5 were detected by qRT-PCR, and the protein expressions of MCM5, Bcl2, cyclin D1, and cyclin A2 were detected by Western blotting. Results CRNDE was highly expressed in the bone marrow and cell lines of AML patients. Knockdown of CRNDE upregulated miR-136-5p, inhibited the MCM5 mRNA and protein expressions and the cell proliferation, promoted the cell apoptosis, and blocked the cell cycle in G1 phase. Overexpression of miR-136-5p also inhibited the expression of MCM5 at both mRNA and protein levels, while knockdown of miR-136-5p reversed those effects. Conclusion CRNDE promotes the proliferation and inhibits the apoptosis of U937 cells by downregulating miR-136-5p and upregulating MCM5.
Topics: Apoptosis; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Humans; MicroRNAs; RNA, Long Noncoding; U937 Cells
PubMed: 34809738
DOI: No ID Found -
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 -
Journal of Immunology Research 2021We recently showed that both nontypeable (NTHi) and its surface plasminogen- (Plg-) binding proteins interact with lipoprotein(a) (Lp(a)) in a lysine-dependent manner....
We recently showed that both nontypeable (NTHi) and its surface plasminogen- (Plg-) binding proteins interact with lipoprotein(a) (Lp(a)) in a lysine-dependent manner. Because Lp(a) can be taken up by macrophages, we postulated that it serves as an opsonin to enhance phagocytosis of NTHi by macrophages. Based on colony-forming unit (CFU) counts, Lp(a) was found to increase U937 macrophage-mediated phagocytosis of NTHi49247 and NTHi49766 by 34% and 43%, respectively, after 120 min. In contrast, Lp(a) did not enhance phagocytosis of BL21 or JM109, which were unable to bind to Lp(a). As with U937 macrophages, Lp(a) was capable of increasing phagocytosis of NTHi49247 by peripheral blood mononuclear cell-derived macrophages. Opsonic phagocytosis by Lp(a) was inhibited by the addition of recombinant kringle IV type 10 (rKIV), a lysine-binding competitor; moreover, Lp(a) did not increase phagocytosis of NTHi by U937 macrophages that were pretreated with a monoclonal antibody against the scavenger receptor CD36. Taken together, our observation suggests that Lp(a) might serve as a lysine-binding opsonin to assist macrophages in rapid recognition and phagocytosis of NTHi.
Topics: CD36 Antigens; Cell Line; Cell Line, Tumor; Escherichia coli; Haemophilus Infections; Haemophilus influenzae; Humans; Leukocytes, Mononuclear; Lipoprotein(a); Macrophages; Opsonin Proteins; Phagocytosis; U937 Cells
PubMed: 34765679
DOI: 10.1155/2021/2185568 -
The American Journal of Surgical... Oct 2021Blast evaluation in patients with acute monocytic leukemias (AMoL) is notoriously difficult due to the lack of reliable surface markers and cytologic subtleties on the...
Blast evaluation in patients with acute monocytic leukemias (AMoL) is notoriously difficult due to the lack of reliable surface markers and cytologic subtleties on the aspirate smears. While blasts of most nonmonocytic acute leukemias express CD34, available immunohistochemical antibodies to monocytic blasts also mark normal background mature monocytes. We searched for a potential biomarker candidate by surveying specific gene expression profiles of monocyte progenitors. Our investigations led us to IRF8, which is a lineage-specific transcription factor critical for the production of monocytic and dendritic cell progenitors. In this study, we tested and validated a monoclonal antibody to IRF8 as a novel immunohistochemical stain for trephine core biopsies of human bone marrow. We assessed the expression of IRF8 in 90 cases of AMoL, including posttherapy staging bone marrows, 23 cases of chronic myelomonocytic leukemia, 26 cases of other acute myeloid leukemia subtypes, and 18 normal control marrows. In AMoL, there was high correlation of IRF8-positive cells to aspirate blast count (R=0.95). Comparison of IRF8 staining to aspirate blast percentage in chronic myelomonocytic leukemia also showed good correlation (R=0.86). In contrast, IRF8-positive cells did not correlate with blast count in other subtypes of acute myeloid leukemia (R=0.56) and staining was <5% in all normal control marrows, even those with reactive monocytosis. We found that IRF8 was also weakly reactive in B cells and hematogones, with the latter accounting for rare cases of discrepancies. When IRF8 was used to categorize cases as AMoL, positive for residual leukemia or negative, the sensitivity was 98%, specificity was 82%, positive predictive value was 86%, and negative predictive value was 98%. These results demonstrate that IRF8 may serve as a clinically useful immunostain to diagnose and track AMoLs on bone marrow core biopsies. This can be particularly impactful in the setting of poor aspiration and focal blast increase. In the era of new targeted therapies that have been reported to induce monocytic outgrowths of leukemia, a marker for malignant monoblasts may prove even more critical.
Topics: Aged; Biomarkers, Tumor; Biopsy; Bone Marrow Examination; Female; Humans; Immunohistochemistry; Interferon Regulatory Factors; Leukemia, Monocytic, Acute; Male; Middle Aged; Monocyte-Macrophage Precursor Cells; Predictive Value of Tests; Proof of Concept Study; Reproducibility of Results
PubMed: 34172624
DOI: 10.1097/PAS.0000000000001765 -
International Immunopharmacology Jul 2021Chronic obstructive pulmonary disease is characterized by chronic inflammation of the airway and lungs. Accumulating evidence has suggested that erythromycin (EM) plays...
Chronic obstructive pulmonary disease is characterized by chronic inflammation of the airway and lungs. Accumulating evidence has suggested that erythromycin (EM) plays a protective role against cigarette smoke-induced oxidative stress and the inflammatory response. However, the underlying mechanisms remain relatively unclear. The present study aimed to investigate the role of EM in inhibiting cigarette smoke-induced inflammation in human macrophages and its potential mechanism. A Cell Counting Kit-8 assay was used to determine the optimum concentration of EM and cigarette smoke extract (CSE) and it was found that 0.1 and 1% CSE and 0.1, 1.0 and 10 μg/ml EM exerted no significant effect on the cell proliferation activity, whereas 2 and 3% CSE exerted a significant inhibitory effect over the cell proliferation activity. We observed that 10 μmol/ml GW9662 (A PPARγ antagonist) and the presence of 1% CSE could promote the expression and activation of NF-κB p65. And this increased the expression of IL-6, IL-8 and reactive oxygen species (ROS). At the same time, 10 μmol/ml GW9662 and 1% CSE was found to inhibit the expression and activation of peroxisome proliferator activated receptors γ (PPARγ); However, 1 μg/ml EM was discovered to reverse these effects. Co-immunoprecipitation subsequently discovered an interaction between PPARγ and NF-κB p65. In conclusion, the present study suggested that EM may reduce the damage of PPARγ by inhibiting oxidative stress and reducing the expression of ROS and finally relieving cigarette smoke-induced inflammation through the PPARγ/NF-κB signaling pathway in macrophages.
Topics: Cell Proliferation; Erythromycin; Humans; Inflammation; Interleukin-6; Interleukin-8; Macrophages; NF-kappa B; PPAR gamma; Reactive Oxygen Species; Signal Transduction; Smoke; Tobacco Products; Transcription Factor RelA; U937 Cells
PubMed: 34162143
DOI: 10.1016/j.intimp.2021.107775 -
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 -
Journal of Extracellular Vesicles Apr 2021We compared four orthogonal technologies for sizing, counting, and phenotyping of extracellular vesicles (EVs) and synthetic particles. The platforms were:...
We compared four orthogonal technologies for sizing, counting, and phenotyping of extracellular vesicles (EVs) and synthetic particles. The platforms were: single-particle interferometric reflectance imaging sensing (SP-IRIS) with fluorescence, nanoparticle tracking analysis (NTA) with fluorescence, microfluidic resistive pulse sensing (MRPS), and nanoflow cytometry measurement (NFCM). EVs from the human T lymphocyte line H9 (high CD81, low CD63) and the promonocytic line U937 (low CD81, high CD63) were separated from culture conditioned medium (CCM) by differential ultracentrifugation (dUC) or a combination of ultrafiltration (UF) and size exclusion chromatography (SEC) and characterized by transmission electron microscopy (TEM) and Western blot (WB). Mixtures of synthetic particles (silica and polystyrene spheres) with known sizes and/or concentrations were also tested. MRPS and NFCM returned similar particle counts, while NTA detected counts approximately one order of magnitude lower for EVs, but not for synthetic particles. SP-IRIS events could not be used to estimate particle concentrations. For sizing, SP-IRIS, MRPS, and NFCM returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of 60 nm. NTA detected a population of particles with a mode diameter greater than 100 nm. Additionally, SP-IRIS, MRPS, and NFCM were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. Finally, for tetraspanin phenotyping, the SP-IRIS platform in fluorescence mode was able to detect at least two markers on the same particle, while NFCM detected either CD81 or CD63. Based on the results of this study, we can draw conclusions about existing single-particle analysis capabilities that may be useful for EV biomarker development and mechanistic studies.
Topics: Biomarkers; Cell Line; Chromatography, Gel; Extracellular Vesicles; Humans; Microfluidics; Microscopy, Electron, Transmission; Nanoparticles; Particle Size; Polystyrenes; Single Molecule Imaging; Ultracentrifugation; Ultrafiltration
PubMed: 33850608
DOI: 10.1002/jev2.12079 -
Iranian Journal of Allergy, Asthma, and... Dec 2020The T-cell immunoglobulin and mucin-3 (TIM-3)/galectin-9 (Gal-9) autocrine loop is an indispensable signaling in acute myeloid leukemia (AML) cells, which induces their...
Oridonin Could Inhibit Inflammation and T-cell Immunoglobulin and Mucin-3/Galectin-9 (TIM-3/Gal-9) Autocrine Loop in the Acute Myeloid Leukemia Cell Line (U937) as Compared to Doxorubicin.
The T-cell immunoglobulin and mucin-3 (TIM-3)/galectin-9 (Gal-9) autocrine loop is an indispensable signaling in acute myeloid leukemia (AML) cells, which induces their self-renewal through activation of nuclear factor-kappa b (NF-kB) and β-catenin pathways. In this study, we evaluated the effects of oridonin and doxorubicin on the TIM-3/Gal-9 autocrine loop. We also evaluated oridonin anti-inflammatory and anti-cancer properties on U937 cells, as an AML cell line in comparison to doxorubicin as a common anthracycline drug for AML treatment. Cell counting kit-8 (CCK-8) was applied to evaluate the cytotoxicity of oridonin and doxorubicin on U937 cells and also to determine the impact of galectin-9 (Gal-9) on their proliferation. The effects of oridonin and doxorubicin on Gal-9, TIM-3, and interleukin-1β (IL-1β) gene expression were determined by real-time polymerase chain reaction (RT-PCR). The Gal-9 secretion level was measured by enzyme-linked immunosorbent assay (ELISA) and activation of NF-kB pathway was assessed by western blotting. In a dose-dependent manner, oridonin and doxorubicin were capable to eradicate U937 cells while Gal-9 expanded them. Following the treatment of U937 cells with oridonin, the expression of Gal-9, TIM-3, and IL-1β genes was down-regulated, and the Gal-9 secretion and NF-kB phosphorylation were diminished, whereas doxorubicin increased all of these factors. Doxorubicin is a common treatment agent in AML, but it may induce inflammation and up-regulate the TIM3/Gal-9 autocrine loop, consequently can enhance the possibility of disease relapse. Meanwhile, oridonin is capable to inhibit the essential signaling pathways in AML cells and reduce the inflammation and expansion of tumor cells and postpone AML recurrence.
Topics: Cell Line, Tumor; Diterpenes, Kaurane; Doxorubicin; Galectins; Hepatitis A Virus Cellular Receptor 2; Humans; Immunoglobulins; Inflammation; Leukemia, Myeloid, Acute; NF-kappa B; Signal Transduction; T-Lymphocytes; U937 Cells
PubMed: 33463129
DOI: 10.18502/ijaai.v19i6.4929 -
Scientific Reports Jul 2020Loss of estrogens at menopause is a major cause of osteoporosis and increased fracture risk. Estrogens protect against bone loss by decreasing osteoclast number through...
Loss of estrogens at menopause is a major cause of osteoporosis and increased fracture risk. Estrogens protect against bone loss by decreasing osteoclast number through direct actions on cells of the myeloid lineage. Here, we investigated the molecular mechanism of this effect. We report that 17β-estradiol (E) decreased osteoclast number by promoting the apoptosis of early osteoclast progenitors, but not mature osteoclasts. This effect was abrogated in cells lacking Bak/Bax-two pro-apoptotic members of the Bcl-2 family of proteins required for mitochondrial apoptotic death. FasL has been previously implicated in the pro-apoptotic actions of E. However, we show herein that FasL-deficient mice lose bone mass following ovariectomy indistinguishably from FasL-intact controls, indicating that FasL is not a major contributor to the anti-osteoclastogenic actions of estrogens. Instead, using microarray analysis we have elucidated that ERα-mediated estrogen signaling in osteoclast progenitors decreases "oxidative phosphorylation" and the expression of mitochondria complex I genes. Additionally, E decreased the activity of complex I and oxygen consumption rate. Similar to E, the complex I inhibitor Rotenone decreased osteoclastogenesis by promoting osteoclast progenitor apoptosis via Bak/Bax. These findings demonstrate that estrogens decrease osteoclast number by attenuating respiration, and thereby, promoting mitochondrial apoptotic death of early osteoclast progenitors.
Topics: Adenosine Triphosphate; Animals; Apoptosis; Biomarkers; Bone Density; Bone and Bones; Cell Count; Cell Differentiation; Cells, Cultured; Estrogens; Female; Gene Expression Regulation; Mice; Mice, Knockout; Mitochondria; Monocyte-Macrophage Precursor Cells; Osteoclasts; Osteogenesis; Oxidative Phosphorylation; Signal Transduction
PubMed: 32686739
DOI: 10.1038/s41598-020-68890-7 -
American Journal of Reproductive... Aug 2020The state of self-renewal and self-maintain of decidual macrophages would be important for immune homeostasis at the maternal-fetal interface. The roles of interleukin...
PROBLEM
The state of self-renewal and self-maintain of decidual macrophages would be important for immune homeostasis at the maternal-fetal interface. The roles of interleukin (IL)-24 derived from decidual stromal cells (DSCs) on decidual macrophages have not been explored.
METHOD OF STUDY
IL-24 expression in DSCs was interfered by lentivirus, and the transcription levels of IL-24 in DSCs were verified by real time (RT)-PCR. The levels of IL-24 receptors were determined by flow cytometry assays. The effect of recombination human IL-24 (rhIL-24) on the differentiation and apoptosis of macrophages was analyzed by flow cytometry in vitro. The viability of macrophages was detected by Cell Counting Kit-8 assays.
RESULTS
The growth of DSCs was not affected obviously only by IL-24 knockdown while the growth of knockdown DSCs was inhibited significantly after co-cultured with decidual macrophages. The levels of IL-24 receptors (IL-20R1 and IL-22R1) were moderately to highly expressed on decidual macrophages and human macrophage cell line U937. The differentiation of decidual macrophages treated by rhIL-24 or co-cultured with IL-24 knockdown DSCs was not affected. Both apoptosis and viability of U937 cells were promoted by rhIL-24. The ratio of Bcl-2/Bax was down-regulated and Ki-67 was up-regulated by IL-24 treatment. The expression of Bcl-2/Bax was up-regulated while Ki-67 was down-regulated in U937 cells after co-cultured by IL-24 knockdown DSCs.
CONCLUSION
IL-24 secreted by DSCs promotes the renewal and homeostasis of decidual macrophages possibly via down-regulating the ratio of Bcl-2/Bax and up-regulating of the expression of Ki-67 in early pregnancy.
Topics: Adult; Apoptosis; Cell Differentiation; Cell Self Renewal; Decidua; Female; Gene Knockdown Techniques; Homeostasis; Humans; Interleukins; Macrophages; Pregnancy; Proto-Oncogene Proteins c-bcl-2; Stromal Cells; U937 Cells; Young Adult; bcl-2-Associated X Protein
PubMed: 32356306
DOI: 10.1111/aji.13261