A novel mechanism of Ha-ras oncogene action: regulation of fibronectin mRNA levels by a nuclear posttranscriptional event

1994 ◽  
Vol 14 (5) ◽  
pp. 3085-3093
Author(s):  
L A Chandler ◽  
C P Ehretsmann ◽  
S Bourgeois
Keyword(s):  
Posttranscriptional Regulation ◽  
Tail Length ◽  
Common Mechanism ◽  
Osteosarcoma Cell ◽  
Mrna Levels ◽  
Ras Oncogene ◽  
Down Regulation ◽  
Regulate Gene Expression ◽  
Protein Levels ◽  
Human Osteosarcoma Cell

Although loss of cell surface fibronectin (FN) is a hallmark of many oncogenically transformed cells, the mechanisms responsible for this phenomenon remain poorly understood. The present study utilized the nontumorigenic human osteosarcoma cell line TE-85 to investigate the effects of induced Ha-ras oncogene expression on FN biosynthesis. TE-85 cells were stably transfected with metallothionein-Ha-ras fusion genes, and the effects of metal-induced ras expression on FN biosynthesis were determined. Induction of the ras oncogene, but not proto-oncogene, was accompanied by a decrease in total FN mRNA and protein levels. Transfection experiments indicated that these oncogene effects were not due to reduced FN promoter activity, suggesting that a posttranscriptional mechanism was involved. The most common mechanism of posttranscriptional regulation affects cytoplasmic mRNA stability. However, in this study the down-regulation of FN was identified as a nuclear event. A component of the ras effect was due to a mechanism affecting accumulation of processed nuclear FN RNA. Mechanisms that would generate such an effect include altered RNA processing and altered stability of the processed message in the nucleus. There was no effect of ras on FN mRNA poly(A) tail length or site of polyadenylation. There was also no evidence for altered splicing at the ED-B domain of FN mRNA. This demonstration of nuclear posttranscriptional down-regulation of FN by the Ha-ras oncogene identifies a new level at which ras oncoproteins can regulate gene expression and thus contribute to development of the malignant phenotype.

scholarly journals A novel mechanism of Ha-ras oncogene action: regulation of fibronectin mRNA levels by a nuclear posttranscriptional event.

1994 ◽  
Vol 14 (5) ◽  
pp. 3085-3093 ◽  
Author(s):  
L A Chandler ◽  
C P Ehretsmann ◽  
S Bourgeois
Keyword(s):  
Posttranscriptional Regulation ◽  
Tail Length ◽  
Common Mechanism ◽  
Osteosarcoma Cell ◽  
Mrna Levels ◽  
Ras Oncogene ◽  
Down Regulation ◽  
Regulate Gene Expression ◽  
Protein Levels ◽  
Human Osteosarcoma Cell

Although loss of cell surface fibronectin (FN) is a hallmark of many oncogenically transformed cells, the mechanisms responsible for this phenomenon remain poorly understood. The present study utilized the nontumorigenic human osteosarcoma cell line TE-85 to investigate the effects of induced Ha-ras oncogene expression on FN biosynthesis. TE-85 cells were stably transfected with metallothionein-Ha-ras fusion genes, and the effects of metal-induced ras expression on FN biosynthesis were determined. Induction of the ras oncogene, but not proto-oncogene, was accompanied by a decrease in total FN mRNA and protein levels. Transfection experiments indicated that these oncogene effects were not due to reduced FN promoter activity, suggesting that a posttranscriptional mechanism was involved. The most common mechanism of posttranscriptional regulation affects cytoplasmic mRNA stability. However, in this study the down-regulation of FN was identified as a nuclear event. A component of the ras effect was due to a mechanism affecting accumulation of processed nuclear FN RNA. Mechanisms that would generate such an effect include altered RNA processing and altered stability of the processed message in the nucleus. There was no effect of ras on FN mRNA poly(A) tail length or site of polyadenylation. There was also no evidence for altered splicing at the ED-B domain of FN mRNA. This demonstration of nuclear posttranscriptional down-regulation of FN by the Ha-ras oncogene identifies a new level at which ras oncoproteins can regulate gene expression and thus contribute to development of the malignant phenotype.

Pax5 Activates Oncogenic Transcription Factors of the Ets Family by Repressing the mir-15a/16-1 microRNA Cluster.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 346-346
Author(s):  
Elaine Y. Chung ◽  
Diana Cozma ◽  
Duonan Yu ◽  
Michael Dews ◽  
Erik A. Wentzel ◽  
...  
Keyword(s):  
Transcription Factors ◽  
B Cell ◽  
Western Blotting ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Mrna Levels ◽  
Down Regulation ◽  
Protein Levels ◽  
Ets Family ◽  
Post Transcriptional Regulation

Abstract We have recently demonstrated that Pax5 promotes B-lymphomagenesis by upregulating key components of B-cell receptor signaling [Cozma et al, J Clin Inv, 117 (8), 2007]. Gene regulation by Pax5 often involves complex formation with other oncogenic transcription factors of the Ets family, namely Myb and Ets1. We determined that expression of these proteins themselves depends on the presence of Pax5, as seen in human diffuse large B-cell lymphomas with Pax5 knockdown and murine lymphomas with epigenetic silencing of Pax5 [Yu et al, Blood, 101:1950–1955, 2003; Johnson et al, Nat Immunol, 5:853–861, 2004]. Upon reconstitution with the Pax5 gene, Myb and Ets1 levels increase sharply. This occurs with little increase in steady-state mRNA levels, suggesting post-transcriptional regulation, possibly by microRNAs. To test this hypothesis, we compared miRNA profiles of Pax5-deficieint and sufficient cells and discovered that several miRNAs are indeed repressed by Pax5. Among them is the miR-15a/16-1 cluster whose predicted targets include both Myb and Ets1. Consistent with this prediction, forced expression of miR-15a/16 brings down Myb and Ets1 protein levels. This is accompanied by impaired Pax5 function and overall suppression of B-lymphomagenesis. Thus, Ets family members (along with previously identified bcl-2) are key targets of the miR-15a/16 locus, a tumor suppressor in chronic lymphocytic leukemia. Interplay between Pax5, Myb/Ets1, and miR-15a/16-1. (A) Upregulation of Myb and Ets 1 in tumors over-expressing Pax5ER fusion, as compared to control GFP-only neoplasms. (B) Down-regulation of Myb and Ets1 in Pax5 tumors engineered to over-express the miR-15a/16-1 cluster. All panels depict Western blotting. Interplay between Pax5, Myb/Ets1, and miR-15a/16-1. (A) Upregulation of Myb and Ets 1 in tumors over-expressing Pax5ER fusion, as compared to control GFP-only neoplasms. (B) Down-regulation of Myb and Ets1 in Pax5 tumors engineered to over-express the miR-15a/16-1 cluster. All panels depict Western blotting.

STAT3 Silencing as a Novel Therapeutic Strategy for Activated B Cell-Type Diffuse Large B-Cell Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 926-926
Author(s):  
Anna Scuto ◽  
Maciej Kujawski ◽  
Claudia Kowolik ◽  
Hua Yu ◽  
Stephen Forman ◽  
...  
Keyword(s):  
B Cell ◽  
Target Genes ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Mrna Levels ◽  
Down Regulation ◽  
Protein Levels ◽  
Stat3 Signaling ◽  
Marrow Stroma ◽  
And Control

Abstract Abstract 926 Among the non-Hodgkin's lymphomas, the diffuse large B cell lymphoma (DLBCL) represents the most frequent (30%) of the aggressive lymphomas. Persistent STAT3 signaling contributes to malignant progression in many diverse human tumors. IL-6 and IL-10 are major activators of STAT3 signaling and are important in the pathophysiology of DLBCL. STAT3 has been found to be persistently active in activated B cells (ABC), which are non-germinal center-derived DLBCL cells. We studied the consequences of STAT3 inhibition on multiple biological functions in two representative human cell lines of this group, Ly3 and Ly10 cells. For this purpose, we established stably transduced STAT3 shRNA-expressing lentivirus Ly3 cells, control lentivirus Ly3 cells, STAT3 shRNA-expressing lentivirus Ly10 cells and control lentivirus Ly10 cells. The stable expression of STAT3 shRNA results in 40-50% reduction of total STAT3 protein levels in the STAT3 shRNA lentivirus Ly3 cells compared to the control lentivirus cells. STAT3 down-regulation induced inhibition of cell proliferation (approximately 40%). Ly3 cells respond to IL-10 more than to IL-6 in terms of proliferation; both cytokines induced less proliferation in the STAT3 shRNA lentivirus Ly3 cells compared to the control lentivirus Ly3 cells. Similar results were obtained in Ly10 cells, which respond more to IL-6 than to IL-10 in terms of proliferation. We analyzed by quantitative real-time PCR the mRNA levels of different STAT3 target genes and observed significant reduction in mRNA levels of Mcl-1, Bcl-xL and Survivin in STAT3 shRNA lentivirus Ly3 cells, as well as significant reduction of Cyclin D2 and up-regulation of STAT1 in shRNA lentivirus Ly10 cells. Comparison of these gene expression profiles with data obtained from other B-cell lymphoma cell lines revealed that silencing of STAT3 resulted in down-regulation of different STAT3 target genes in a cell-dependent manner. We also observed that both STAT3 and control lentivirus Ly3 cells have the same protein levels of c-Myc; nevertheless STAT3 silencing resulted in inhibition of IL-10-inducible upregulation of c-Myc. We next investigated the effect of STAT3 inhibition on adhesion to bone marrow stroma and chemotaxis. STAT3 shRNA lentivirus Ly3 cells adhered less to the stroma layer than control cells, and the longer they were cocultured with the stroma cells in the presence of serum-free media the more they lost the ability to adhere. Moreover, STAT3 shRNA lentivirus Ly3 cells had decreased capacity to migrate toward SDF-1 alpha, an important factor that mediates proliferation, survival, chemotaxis, migration and adhesion into bone marrow stroma. Radiation, in combination with chemotherapy, is one of the therapies used for DLBCL patients. We therefore investigated whether STAT3 down-regulation sensitized Ly3 cells to radiation. Radiation induced a higher accumulation of phospho-H2A.X (first sentinel event following DNA damage such as DSBs) and apoptosis in STAT3 shRNA lentivirus cells compared to control cells. Moreover, IL-6 and IL-10 protected the STAT3 shRNA lentivirus Ly3 cells less than the control cells from the induction of phospho-H2A.X following radiation. We further investigated the effect of STAT3 silencing in animal models of Ly3 lymphoma (Nude or NOD-SCID mice). Tumors in control lentivirus Ly3-bearing mice grew robustly, whereas tumors in STAT3 shRNA lentivirus Ly3-bearing mice regressed 5 days after injection. This tumor regression was associated with Caspase-3-dependent apoptosis, significant reduction of STAT3 target genes at the protein level such as Mcl-1, c-Myc and Survivin (approximately 40% to 60% inhibition), and reduction of cytokine production such as IL-10, IL-15, Leptin and Thrombopoietin. Taken together, these results suggest that inhibition of STAT3 is a potential promising approach in the therapy of ABC-type DLBCL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The RNA-binding protein HuR regulates intestinal epithelial restitution by modulating Caveolin-1 gene expression

2020 ◽  
Author(s):  
Shan Cao ◽  
Lan Xiao ◽  
Junyao Wang ◽  
Guodong Chen ◽  
Yulan Liu
Keyword(s):  
Gene Expression ◽  
Posttranscriptional Regulation ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
Mrna Levels ◽  
Epithelial Repair ◽  
Caveolin 1 ◽  
Protein Levels ◽  
Ectopic Overexpression

The integrity of the intestinal mucosal barrier protects hosts against pathological conditions. Early mucosal restitution after wounding refers to epithelial cell migration into a defect. The RNA-binding protein HuR plays an important role in the posttranscriptional regulation of gene expression and is involved in many aspects of cellular physiology. In the present study, we investigated the role of HuR in the regulation of cell migration through the posttranscriptional regulation of Caveolin-1 (Cav-1). Online software was used to identify Cav-1 mRNA as a potential target of HuR. The interaction of HuR with Cav-1 mRNA was investigated via ribonucleoprotein immunoprecipitation (RNP IP) assays and biotin pulldown analysis. HuR was found to bind specifically to the Cav-1 3’-UTR rather than the coding region or 5’-UTR. Transfection of cells with siHuR decreased both HuR protein levels and Cav-1 protein levels; conversely, ectopic overexpression of HuR via infection of cells with an adenoviral vector containing HuR cDNA (AdHuR) increased Cav-1 protein levels without disturbing Cav-1 mRNA levels. Thus, HuR enhanced Cav-1 expression in vitro by stimulating Cav-1 translation. Intestinal epithelium–specific HuR knockout in mice decreased Cav-1 protein levels without changing Cav-1 mRNA levels, consistent with the in vitro results. Decreasing the levels of HuR via siHuR transfection inhibited early epithelial repair, but this effect was reversed by ectopic overexpression of GFP-tagged Cav-1. These results indicate that posttranscriptional regulation of Cav-1 gene expression by HuR plays a critical role in the regulation of rapid epithelial repair after wounding.

Experimental Study on the Application of pHSP-PLk1-siRNA/DOX Complex in the Treatment of Osteosarcoma by Magnetotactic Bacterial Magnetosome

2019 ◽  
Vol 9 (9) ◽  
pp. 1286-1291
Author(s):  
YiPing Tian ◽  
XiaoLing Guo ◽  
RuiJun Cai ◽  
LanXia Li
Keyword(s):  
Magnetic Field ◽  
Control Group ◽  
Adhesion Assay ◽  
Osteosarcoma Cell ◽  
Blank Control Group ◽  
Protein Levels ◽  
Human Osteosarcoma Cell ◽  
Group A ◽  
Group B

The in vitro temperature experiment was used to determine the magnetic field strength, and the magnetic bodies were isolated and purified as carriers. In the previous study, pHSP-PLk1-siRNA/DOX complexes were constructed to target loose drugs. The subjects were arranged into 4 groups, namely magnetosome+drug group (group A), magnetosome+ blank control (group B), magnetosome+drug+magnetic field (group C), magnetosome+blank control+magnetic field (D) Group); co-incubated with human osteosarcoma cell line U2OS. The uptake rate and cell morphology of osteosarcoma cells were observed by laser confocal microscopy at 12, 24, 48 and 72 h. The cell cycle was observed by the aid of flow cytometry. The cells were detected by RT-PCR and Western blot. PLk1 mRNA and expression of protein levels, MTT assay for cell proliferation, adhesion assay and Transwell chamber for cell adhesion and invasion, and apoptosis kit for apoptosis.

scholarly journals The P-body component USP52/PAN2 is a novel regulator of HIF1A mRNA stability

2013 ◽  
Vol 451 (2) ◽  
pp. 185-194 ◽  
Author(s):  
John S. Bett ◽  
Adel F. M. Ibrahim ◽  
Amit K. Garg ◽  
Van Kelly ◽  
Patrick Pedrioli ◽  
...  
Keyword(s):  
Cancer Progression ◽  
Mrna Stability ◽  
Cellular Response ◽  
Hypoxia Inducible Factor ◽  
Tail Length ◽  
Mrna Levels ◽  
P Bodies ◽  
Protein Levels ◽  
Specific Protease ◽  
Processing Body

HIF1A (hypoxia-inducible factor 1α) is the master regulator of the cellular response to hypoxia and is implicated in cancer progression. Whereas the regulation of HIF1A protein in response to oxygen is well characterized, less is known about the fate of HIF1A mRNA. In the present study, we have identified the pseudo-DUB (deubiquitinating enzyme)/deadenylase USP52 (ubiquitin-specific protease 52)/PAN2 [poly(A) nuclease 2] as an important regulator of the HIF1A-mediated hypoxic response. Depletion of USP52 reduced HIF1A mRNA and protein levels and resulted in reduced expression of HIF1A-regulated hypoxic targets due to a 3′-UTR (untranslated region)-dependent poly(A)-tail-length-independent destabilization in HIF1A mRNA. MS analysis revealed an association of USP52 with several P-body (processing body) components and we confirmed further that USP52 protein and HIF1A mRNA co-localized with cytoplasmic P-bodies. Importantly, P-body dispersal by knockdown of GW182 or LSM1 resulted in a reduction of HIF1A mRNA levels. These data uncover a novel role for P-bodies in regulating HIF1A mRNA stability, and demonstrate that USP52 is a key component of P-bodies required to prevent HIF1A mRNA degradation.

scholarly journals IIAEK Targets Intestinal Alkaline Phosphatase (IAP) to Improve Cholesterol Metabolism with a Specific Activation of IAP and Down-Regulation of ABCA1

2020 ◽  
Author(s):  
Asahi Takeuchi ◽  
Kentaro Hisamatsu ◽  
Natsuki Okumura ◽  
Yuki Sugimitsu ◽  
Emiko Yanase ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Molecular Mechanisms ◽  
Cholesterol Metabolism ◽  
Molecular Probe ◽  
Small Intestinal Mucosa ◽  
Mrna Levels ◽  
Intestinal Alkaline Phosphatase ◽  
Down Regulation ◽  
Protein Levels ◽  
Abca1 Mrna

IIAEK (Ile-Ile-Ala-Glu-Lys, lactostatin) is a novel pentapeptide from bovine milk β-lactoglobulin which lowers cholesterol levels. However, the molecular mechanisms underlying the suppression of intestinal cholesterol absorption by IIAEK are unknown. Therefore, we evaluated the effects of IIAEK on intestinal cholesterol metabolism in Caco-2 cells in a human intestinal model. We found that IIAEK significantly reduced the expression of intestinal cholesterol metabolism-associated genes, particularly that of the ATP-binding cassette transporter A1 (ABCA1) protein. Subsequently, we chemically synthesized a novel molecular probe, IIXEK, which can visualize a complex of target proteins interacting with photoaffinity-labeled IIAEK by fluorescent substances. Photoaffinity labeling and MS analysis with IIXEK for the rat small intestinal mucosa and intestinal lipid raft fractions of Caco-2 cells, we identified intestinal alkaline phosphatase (IAP) as a specific molecule interacting with IIAEK and discovered IIAEK common binding amino acid sequence, GFYLFVEGGR. Transfection of IAP siRNA counteracted the decrease in ABCA1 mRNA levels in Caco-2 cells. IIAEK significantly increased IAP mRNA and protein levels, and significantly decreased ABCA1 mRNA and protein levels in Caco-2 cells. In conclusion, we found that IIAEK targets IAP to improve cholesterol metabolism via a novel signaling pathway with a specific activation of IAP and down-regulation of intestinal ABCA1.

RNF38 inhibits osteosarcoma cell proliferation by binding to CRY1

2021 ◽  
pp. 1-7
Author(s):  
Jian Zhou ◽  
Zhen-yu Tang ◽  
Xiao-liang Sun
Keyword(s):  
Cell Proliferation ◽  
Western Blotting ◽  
Colony Formation ◽  
Akt Pathway ◽  
Osteosarcoma Cell ◽  
Mrna Levels ◽  
Osteosarcoma Cells ◽  
Protein Levels ◽  
Qrt Pcr

The PI3K/AKT pathway plays an important role in the development of osteosarcoma. RNF38 interferes with activation of the AKT pathway. Cryptochrome1 (CRY1) inhibits osteosarcoma proliferation through the AKT pathway. We aimed to clarify whether RNF38 affects the proliferation of osteosarcoma cells by regulating the PI3K/AKT pathway through its interaction with CRY1. The mRNA levels of RNF38 were determined using qRT-PCR. Protein levels of RNF38, p-p70S6, p70S6, +p-AKT, AKT, p-mTOR, mTOR, and CRY1 were detected by western blotting. The proliferation of osteosarcoma cells was detected using CCK-8 and colony formation assays. The interaction between CRY1 and RNF38 was detected by co-immunoprecipitation and GST pull-down assays. RNF38 expression was higher in Saos-2 and U20S cells than in hFOB cells. Overexpression of RNF38 promoted the proliferation of osteosarcoma cells, the number of colonies, and p-AKT and p-mTOR levels, suggesting that overexpression of RNF38 activated the PI3K/AKT pathway. In addition, RNF38 directly binds to the N-terminal of CRY1. The simultaneous knockdown of RNF38 and CRY1 restored the level of p-AKT, which was reduced by RNF38 knockdown alone. RNF38 affects the proliferation of osteosarcoma cells by regulating the PI3K/AKT pathway through its interaction with CRY1.

scholarly journals Down-regulation of Sphingosine Kinase-1 by DNA Damage

2004 ◽  
Vol 279 (19) ◽  
pp. 20546-20554 ◽  
Author(s):  
Tarek A. Taha ◽  
Walid Osta ◽  
Lina Kozhaya ◽  
Jacek Bielawski ◽  
Korey R. Johnson ◽  
...  
Keyword(s):  
Cell Death ◽  
Sphingosine Kinase ◽  
Genotoxic Stress ◽  
Cysteine Proteases ◽  
Mrna Levels ◽  
Sphingosine Kinase 1 ◽  
Γ Irradiation ◽  
Down Regulation ◽  
Protein Levels ◽  
Lysosomal Protease

Sphingosine kinase 1 (SK1), a key enzyme in sphingosine 1-phosphate (S1P) synthesis, regulates various aspects of cell behavior, including cell survival and proliferation. DNA damaging anti-neoplastic agents have been shown to induce p53, ceramide levels, and apoptosis; however, the effects of anti-neoplastic agents on SK have not been assessed. In this study, we investigated the effects of a DNA damaging agent, actinomycin D (Act D), on the function of sphingosine kinase (SK1). Act D caused a reduction in the protein levels of SK1, as indicated by Western blot analysis, with a concomitant decrease in SK activity. The down-regulation was post-transcriptional, because the mRNA levels of SK1 remained unchanged. Similar decreases in SK1 protein were observed with other DNA damaging agents such as doxorubicin, etoposide, and γ-irradiation. ZVAD, the pancaspase inhibitor, and Bcl-2 annulled the effect of Act D on SK1, demonstrating a role for cysteine proteases downstream of Bcl-2 in the down-regulation of SK1. Inhibition of caspases 3, 6, 7, and 9 only partially reversed Act D-induced SK1 loss. Inhibition of cathepsin B, a lysosomal protease, produced a significant reversal of SK1 decline by Act D, suggesting that a multitude of ZVAD-sensitive cysteine proteases downstream of Bcl-2 mediated the SK1 decrease. When p53 up-regulation after Act D treatment was inhibited, SK1 down-regulation was rescued, demonstrating p53 dependence of SK1 modulation. Treatment of cells with S1P, the product of SK1, partially inhibited Act D-induced cell death, raising the possibility that a decrease in SK1 may be in part necessary for cell death to occur. Furthermore, the knockdown of SK1 by small interfering RNA in MCF-7 cells resulted in a significant reduction in cell viability. These studies demonstrate that SK1 is down-regulated by genotoxic stress, and that basal SK1 function may be necessary for the maintenance of tumor cell growth.

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