scholarly journals Ovine herpesvirus-2-encoded microRNAs target virus genes involved in virus latency

2014 ◽  
Vol 95 (2) ◽  
pp. 472-480 ◽  
Author(s):  
Aayesha Riaz ◽  
Inga Dry ◽  
Claire S. Levy ◽  
John Hopkins ◽  
Finn Grey ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Lymphocyte Proliferation ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Malignant Catarrhal Fever ◽  
Virus Latency ◽  
Cell Cycle Inhibition ◽  
Host Genes ◽  
Virus Biology

Herpesviruses encode microRNAs (miRNAs) that target both virus and host genes; however, their role in herpesvirus biology is understood poorly. We identified previously eight miRNAs encoded by ovine herpesvirus-2 (OvHV-2), the causative agent of malignant catarrhal fever (MCF), and have now investigated the role of these miRNAs in regulating expression of OvHV-2 genes that play important roles in virus biology. ORF20 (cell cycle inhibition), ORF50 (reactivation) and ORF73 (latency maintenance) each contain predicted targets for several OvHV-2 miRNAs. Co-transfection of miRNA mimics with luciferase reporter constructs containing the predicted targets showed the 5′ UTRs of ORF20 and ORF73 contain functional targets for ovhv-miR-2 and ovhv2-miR-8, respectively, and the 3′ UTR of ORF50 contains a functional target for ovhv2-miR-5. Transfection of BJ1035 cells (an OvHV-2-infected bovine T-cell line) with the relevant miRNA mimic resulted in a significant decrease in ORF50 and a smaller but non-significant decrease in ORF20. However, we were unable to demonstrate a decrease in ORF73. MCF is a disease of dysregulated lymphocyte proliferation; miRNA inhibition of ORF20 expression may play a role in this aberrant lymphocyte proliferation. The proteins encoded by ORF50 and ORF73 play opposing roles in latency. It has been hypothesized that miRNA-induced inhibition of virus genes acts to ensure that fluctuations in virus mRNA levels do not result in reactivation under conditions that are unfavourable for viral replication and our data supported this hypothesis.

scholarly journals Inhibition of human lymphocyte proliferation by monoclonal antibody to transferrin receptor

Blood ◽  
1983 ◽  
Vol 62 (4) ◽  
pp. 821-826 ◽  
Author(s):  
J Mendelsohn ◽  
I Trowbridge ◽  
J Castagnola
Keyword(s):  
Cell Cycle ◽  
Monoclonal Antibody ◽  
Lymphocyte Proliferation ◽  
Human Lymphocytes ◽  
Nitrilotriacetic Acid ◽  
S Phase ◽  
Inhibitory Effects ◽  
Mitogenic Response ◽  
Cell Cycle Inhibition ◽  
Partial Reversal

Abstract A monoclonal antibody, 42/6, which blocks the binding of transferrin to its receptor on the cell membrane, inhibits proliferation of human lymphocytes stimulated by phytohemagglutinin. Anti-receptor antibody B3/25, which does not block transferrin binding, does not alter the mitogenic response. Addition of soluble iron, in the form of ferric nitrilotriacetic acid, results in partial reversal of inhibition. Lymphocytes in the quiescent phase of the cell cycle at the time of 42/6 antibody addition are unable to traverse S phase, whereas cells actively proliferating when antibody is added are sensitive to its inhibitory effects throughout all phases of the cell cycle. Inhibition is static rather than cidal, since it can be reversed by removal of antibody after up to 48 hr of exposure.

scholarly journals Cyclin G2 Regulates Adipogenesis through PPARγ Coactivation

Endocrinology ◽  
2010 ◽  
Vol 151 (11) ◽  
pp. 5247-5254 ◽  
Author(s):  
Victor Aguilar ◽  
Jean-Sébastien Annicotte ◽  
Xavier Escote ◽  
Joan Vendrell ◽  
Dominique Langin ◽  
...  
Keyword(s):  
Adipocyte Differentiation ◽  
Luciferase Reporter ◽  
Peroxisome Proliferator ◽  
Mrna Levels ◽  
Cell Cycle Regulators ◽  
Luciferase Reporter Gene ◽  
Peroxisome Proliferator Activated Receptor ◽  
Cyclin G2 ◽  
Regulatory Effects

Cell cycle regulators such as cyclins, cyclin-dependent kinases, or retinoblastoma protein play important roles in the differentiation of adipocytes. In the present paper, we investigated the role of cyclin G2 as a positive regulator of adipogenesis. Cyclin G2 is an unconventional cyclin which expression is up-regulated during growth inhibition or apoptosis. Using the 3T3-F442A cell line, we observed an up-regulation of cyclin G2 expression at protein and mRNA levels throughout the process of cell differentiation, with a further induction of adipogenesis when the protein is transiently overexpressed. We show here that the positive regulatory effects of cyclin G2 in adipocyte differentiation are mediated by direct binding of cyclin G2 to peroxisome proliferator-activated receptor γ (PPARγ), the key regulator of adipocyte differentiation. The role of cyclin G2 as a novel PPARγ coactivator was further demonstrated by chromatin immunoprecipitation assays, which showed that the protein is present in the PPARγ-responsive element of the promoter of aP2, which is a PPARγ target gene. Luciferase reporter gene assays, showed that cyclin G2 positively regulates the transcriptional activity of PPARγ. The role of cyclin G2 in adipogenesis is further underscored by its increased expression in mice fed a high-fat diet. Taken together, our results demonstrate a novel role for cyclin G2 in the regulation of adipogenesis.

scholarly journals The Role of miRNA-377 as a Tumor Suppressor in Lung Cancer by Negative Regulation of Genes Belonging to ErbB Signaling Pathway

2021 ◽  
Author(s):  
Saba Hashemi ◽  
Naghmeh Yari ◽  
Fatemeh Rahimi Jamnani ◽  
Reza Mahdian ◽  
Morteza Karimipoor ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Tumor Suppressor ◽  
Signaling Pathway ◽  
A549 Cells ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Erbb Signaling ◽  
Erbb Signaling Pathway ◽  
Human Nsclc

Abstract The ErbB signaling pathway plays important role in the pathogenesis of lung cancer. We explored the role of miRNA-377 as a tumor suppressor in NSCLC through silencing of some genes in the ErbB pathway.Targeting the effect of miRNA-377 on EGFR, MAPK1, ABL2, and PAK2 was evaluated. The expression levels of these genes and miRNA-377 were surveyed in NSCLC and normal human tissues, Calu-6, and A549 cells. Real-time PCR was used to figure out whether miRNA-377 could decrease the target genes mRNAs in transfected lung cancer cell lines. The effects of miRNA-377 on apoptosis cell and proliferation were analyzed. We showed that miRNA-377 targets EGFR, MAPK1, and PAK2 mRNAs in in-silico and luciferase reporter assay. The expression of miRNA-377 was significantly downregulated in human NSCLC tissues, Calu-6 and A549 cells compared to their controls. We observed a negative correlation between EGFR, MAPK1, PAK2, and miRNA-377 expression in human NSCLC tissues. A significant reduction in EGFR, MAPK1, and PAK2 mRNA levels was detected, following miRNA-377 transfection in Calu-6 and A549 cells. The higher levels of miRNA-377 in Calu-6, and A549 cells induced apoptosis and reduced proliferation, significantly. All these data reveal that miRNA-377 functions as a tumor suppressor in NSCLC and may serve as a potential therapeutic target for the treatment of NSCLC.

scholarly journals The Regulation of circRNA RNF13/miRNA-1224-5p Axis Promotes the Malignant Evolution in Acute Myeloid Leukemia

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Rong Zhang ◽  
Yingchun Li ◽  
Hongtao Wang ◽  
Ke Zhu ◽  
Guojun Zhang
Keyword(s):  
Cell Cycle ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Circular Rna ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Tenascin C ◽  
Qrt Pcr ◽  
Acute Myeloid

Objective. To study the biological function of circular RNA RNF13 (circRNF13) in acute myeloid leukemia (AML) and its relationship with prognosis. Methods. We constructed stable AML cell lines with downregulated expression of circRNF13, and then, we explored the effect of downregulation of circRNF13 expression on the proliferation, migration, and invasion through qRT-PCR, MTT curve, colony formation, transwell migration and invasion experiment, cell cycle, apoptosis, Caspase 3/7 assay, and other experiments. We also studied the expression of C-myc and Tenascin-C by qRT-PCR to explore the role of circRNF13. Results. When the expression of circRNF13 was downregulated, the proliferation rate of AML cells decreased significantly, the cell cycle was blocked to G1 phase, and apoptosis rate increased significantly. C-myc related to cell proliferation decreased significantly at RNA level. Furthermore, when the expression of circRNF13 was downregulated, the migration and invasion ability of AML cells was significantly reduced, and the expression of Tenascin-C related to migration and invasion also decreased significantly. The luciferase reporter assay system confirmed that miRNA-1224-5p was the direct target of circRNF13. Conclusion. CircRNF13 inhibited the proliferation, migration, and invasion of AML cells by regulating the expression of miRNA-1224-5p. This study provides some clues for the diagnosis and treatment of AML.

MiR-134, Mediated by IRF1, Suppresses Tumorigenesis and Progression by Targeting VEGFA and MYCN in Osteosarcoma

2020 ◽  
Vol 20 (10) ◽  
pp. 1197-1208
Author(s):  
Zhuo Ma ◽  
Kai Li ◽  
Peng Chen ◽  
Qizheng Pan ◽  
Xuyang Li ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Mrna Levels ◽  
Invasion And Migration ◽  
And Migration

Background: Osteosarcoma (OS) is a prevalent primary bone malignancy and its distal metastasis remains the main cause of mortality in OS patients. MicroRNAs (miRNAs) play critical roles during cancer metastasis. Objective: Thus, elucidating the role of miRNA dysregulation in OS metastasis may provide novel therapeutic targets. Methods: The previous study found a low miR-134 expression level in the OS specimens compared with paracancer tissues. Overexpression of miR-134 stable cell lines was established. Cell viability assay, cell invasion and migration assay and apoptosis assay were performed to evaluate the role of miR-134 in OS in vitro. Results: We found that miR-134 overexpression inhibits cell proliferation, migration and invasion, and induces cell apoptosis in both MG63 and Saos-2 cell lines. Mechanistically, miR-134 targets the 3'-UTR of VEGFA and MYCN mRNA to silence its translation, which was confirmed by luciferase-reporter assay. The real-time PCR analysis illustrated that miR-134 overexpression decreases VEGFA and MYCN mRNA levels. Additionally, the overexpression of VEGFA or MYCN can partly attenuate the effects of miR-134 on OS cell migration and viability. Furthermore, the overexpression of miR-134 dramatically inhibits tumor growth in the human OS cell line xenograft mouse model in vivo. Moreover, bioinformatic and luciferase assays indicate that the expression of miR-134 is regulated by Interferon Regulatory Factor (IRF1), which binds to its promoter and activates miR-134 expression. Conclusion: Our study demonstrates that IRF1 is a key player in the transcriptional control of miR-134, and it inhibits cell proliferation, invasion and migration in vitro and in vivo via targeting VEGFA and MYCN.

Impact and mechanistic role of MicroRNA-1291 on pancreatic tumorigenesis.

2016 ◽  
Vol 34 (4_suppl) ◽  
pp. 243-243 ◽  
Author(s):  
Mei-Juan Tu ◽  
Yu-Zhuo Pan ◽  
Jing-Xin Qiu ◽  
Edward Jae-hoon Kim ◽  
Aiming Yu
Keyword(s):  
Cell Cycle ◽  
Cancer Biology ◽  
Critical Role ◽  
Experimental Studies ◽  
Mass Spectrometry Analysis ◽  
Ductal Adenocarcinoma ◽  
Luciferase Reporter ◽  
Spectrometry Analysis

243 Background: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death. Better understanding of pancreatic cancer biology and identification of new targets are highly warranted. MicroRNAs (miRs or miRNAs) play a critical role in the control of tumor progression via crosstalk with cancer signaling pathways. Our recent studies showed that miR-1291 improved chemosensitivity through targeting of efflux transporter ABCC1. This current study investigated the mechanistic role of miR-1291 in the suppression of pancreatic tumorigenesis. Methods: PANC-1 and AsPC-1 cell lines were stably transfected with miR-1291. Cell cycle status and apoptosis of stable miR-1291-expressing cells were tested against control cells using flow cytometry. Cells were injected subcutaneously into nude mice and tumorigenesis was measured in vivo. Proteomic studies were performed by two-dimensional difference gel electrophoresis, matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis. Computationally predicted miR-1291 targets were assessed by luciferase reporter assay and Western blot. Primary PDAC and control samples were tested for miR-1291 and target gene expression levels. Results: Our data showed that stable miR-1291-expressing PANC-1 and AsPC-1 cells both showed a significantly lower rate of proliferation than the control cells, which was associated with a cell cycle arrest and enhanced apoptosis. Furthermore, miR-1291 suppressed the tumorigenesis of PANC-1 cells in mouse models in vivo. Proteomic studies revealed the protein level of several cancer-related genes were downregulated by miR-1291, including a pancreatic tumor promoting protein AGR2 which was reduced ~10-fold. Through computational and experimental studies we further identified that FOXA2, a transcription factor governing AGR2 expression, was a direct target of miR-1291. In addition, we found a significant down-regulation of miR-1291 in a set of PDAC patient tumor samples overexpressing AGR2. Conclusions: These results indicate that miR-1291 suppresses pancreatic tumorigenesis via targeting of FOXA2-AGR regulatory pathway providing new insight supporting development of miR-1291-based therapy for PDAC.

Role of Survivin in Drug Resistant B-Cell Acute Lymphoblastic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 10-10
Author(s):  
Eugene Park ◽  
Enzi Jiang ◽  
Gregor von Levetzow ◽  
Cihangir Duy ◽  
Lars Klemm ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Drug Resistance ◽  
B Cell ◽  
Real Time Pcr ◽  
Mrna Levels ◽  
Drug Resistant ◽  
Novel Therapies ◽  
Protein Levels ◽  
Ic50 Value

Abstract Abstract 10 Survivin/BIRC5, an inhibitor of apoptosis (IAP) protein,is critical for the survival and proliferation of cancerous cells and has become the target of an increasing number of preclinical novel therapies against primarily solid tumors. Survivin is expressed in AML and ALL cells and has been implicted in leukemia relapse. Here we test the hypothesis that Survivin is critical to chemotherapeutic drug resistance in ALL. To test this hypothesis, we initially compared survivin mRNA levels in both patient-derived B-ALL cells and B-ALL cell lines versus normal B-cells in various stages of development using real-time PCR. ALL cells encompassing various cytogenetic subgroups showed significantly greater mRNA levels of Survivin versus normal B cell precursors ranging from 2 to greater than 20-fold versus controls. To determine whether Survivin contributes to drug resistance, we lentivirally overexpressed Survivin in primary B-ALL and B-ALL cell lines. Overexpression of Survivin attenuated the effect of Vincristine on ALL cell proliferation when compared to ALL cells transduced with empty vector controls. Vincristine IC50 value determination for the control was 0.1 nM, whereas Survivin overexpression resulted an IC50 value of 10 nM (p<0.01). Similarly, significantly higher concentrations of L-Asparaginase (>0.01 U/l vs ab >0.1; p<0.05) and Dexamethasone (0.1 nmol/l vs >10 nmol/l; p<0.013) were needed to achieve drug cytotoxicity. We conclude that Survivin overexpression decreases sensitivity of ALL cells to standard chemotherapy. Conversely, lentivirally-mediated survivin shRNA knockdown of the same cell type significantly sensitized a greater percentage of the leukemia to 10nM vincristine versus non-silencing controls. Survivin shRNA targeting resulted in a 30.2% greater affected population than controls, assayed using MTT (p<0.003). Overexpression or knockdown of Survivin was confirmed by Western Blot and real-time PCR. Of note, expression levels of Survivin is heterogeneous within the same ALL sample: Transduction of patient-derived ALL cells with a Survivin-GFP reporter construct revealed that a small subpopulation of Survivin-GFP high ALL exhibiting approximately 7-fold higher levels of endogenous Survivin than bulk leukemia cells. Consistent with cell cycle-dependent regulation of Survivin, Survivin-GFP high ALL were enriched in G2/M phase of the cell cycle. In addition, endogenous variations of Survivin protein levels were in the same range as changes in Survivin protein levels in overexpression and knockdown experiments, which indicates that these experiments tested physiologically occurring levels of Survivin. Next, we determined whether Survivin contributes to self-renewal of drug resistant ALL cells. Survivin overexpression of primary samples yielded three times more colonies, than controls in a primary plating CFU assay (p<0.019). Taken together, our data suggests that targeting Survivin in B-ALL may sensitize to chemotherapy and highlights the role of Survivin in drug resistant B-ALL as a target for novel therapies. Disclosures: No relevant conflicts of interest to declare.

The Interplay Between Microrna-223 and E2F1 Regulates Cell Cycle Control During Granulopoiesis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 255-255
Author(s):  
John Anto Pulikkan ◽  
Viola Dengler ◽  
Philomina Sona Peramangalam ◽  
Abdul A. Peer Zada ◽  
Carsten Müller tidow ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Chromatin Immunoprecipitation ◽  
Cell Cycle Progression ◽  
Conflicts Of Interest ◽  
Myeloid Cells ◽  
Granulocytic Differentiation ◽  
Cycle Progression ◽  
Protein Levels ◽  
Cell Cycle Inhibition

Abstract Abstract 255 Transcription factor CCAAT enhancer binding protein α (C/EBPα) functions as a master regulator of granulocyte development by co-ordinating cell cycle inhibition and differentiation. Recent findings demonstrate that deregulation of C/EBPα is a critical step in the development of acute myeloid leukemia (AML). Inhibition of E2F1, the key regulator of cell cycle progression by C/EBPα is essential for granulopoiesis and disruption of this function of C/EBPα leads to leukemia. The mechanism with which C/EBPα inhibits E2F1 in granulopoiesis is poorly understood. Recent advances in our understanding about microRNAs suggest that these molecules have profound impact in gene expression programmes. Also, deregulation of microRNAs has been shown as a hall mark of many cancers including leukemia. microRNA-223 (miR-223) is upregulated by C/EBPα during granulopoiesis. The pivotal role of miR-223 in granulopoiesis is shown by the finding that mice deficient for miR-223 display defects in granulopoiesis. In this study, we explored the role of miR-223 in the cell cycle inhibition function of C/EBPα. Computational analysis by using programmes such as Target Scan suggests that E2F1 is a putative target of miR-223. Luciferase assays using 3'UTR of E2F1 suggest E2F1 is a potential target of miR-223. Western blot analysis using bone marrow cells isolated from miR-223 null mice shows accumulation of E2F1 protein levels. Interestingly, E2F1 protein levels were downregulated during miR-223 overexpression in myeloid cells. Analysis of miR-223 by quantitative Real-Time RT-PCR in AML patient samples shows that miR-223 is downregulated in different subtypes of AML. Proliferation assays, cell cycle analysis and BrdU assays show that miR-223 functions as an inhibitor of myeloid cell cycle progression. Several studies have reported the ability of E2F1 to block granulocytic differentiation. We next analysed whether E2F1 is inhibiting myeloid differentiation through miR-223. Promoter assays show that E2F1 inhibits the miR-223 promoter activity. By using Chromatin immunoprecipitation assays, we found that E2F1 binds to miR-223 promoter in leukemia derived cell lines and this binding is reversed during granulocytic differentiation. We also observed that E2F1 is bound to the miR-223 promoter in blast cells isolated from AML patients as analysed by chromatin immunoprecipitation assays. In addition, we show that overexpression of E2F1 leads to down regulation of miR-223 levels in myeloid cells. All these data suggest that E2F1 functions as a transcriptional repressor of the miR-223 gene. Taken together, our data suggest that granulopoiesis is regulated by the interplay between miR-223 and E2F1 and deregulation of this interplay may lead to the development of AML. Overexpression of miR-223 could be a potential strategy in the treatment of AML patients in which E2F1 inhibition by C/EBPα is deregulated. Disclosures: No relevant conflicts of interest to declare.

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