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RNA IP Seq analysis of Cnot7 in 4T1 mammary tumor cells

ABSTRACT: Native RNA immunoprecipitation of Cnot7-bound transcripts. Abstract: Accumulating evidence supports the role of an aberrant transcriptome as a driver of tumor cell metastatic potential. Deadenylation is a general regulatory node for post-transcriptional control by microRNAs and other determinants of RNA stability. We show here that CCR4-NOT subunit CNOT7 is a deadenylase-dependent driver of tumor cell autonomous metastatic potential. Metastasis promotion by CNOT7 is dependent on contact with CNOT1 and TOB1. We further show TOB1 independently drives metastasis. RNA-immunoprecipitation and integrated transcriptome wide analyses reveal that CNOT7-regulated transcripts are enriched for a tripartite 3’UTR motif bound by RNA-binding proteins known to complex with CNOT7, TOB1, and CNOT1. Collectively, our data support a model of CNOT7, TOB1, CNOT1, and RNA-binding proteins collectively exerting post-transcriptional control on a metastasis suppressive transcriptional program to drive tumor cell metastasis. 48 total samples, 4-5 biological replicates, two forms of control: input samples and vector controls

ORGANISM(S): Mus musculus

SUBMITTER: Ying Hu

PROVIDER: E-GEOD-73366 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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RNA IP Seq analysis of Cnot7 in 4T1 mammary tumor cells

Project description:Native RNA immunoprecipitation of Cnot7-bound transcripts. Abstract: Accumulating evidence supports the role of an aberrant transcriptome as a driver of tumor cell metastatic potential. Deadenylation is a general regulatory node for post-transcriptional control by microRNAs and other determinants of RNA stability. We show here that CCR4-NOT subunit CNOT7 is a deadenylase-dependent driver of tumor cell autonomous metastatic potential. Metastasis promotion by CNOT7 is dependent on contact with CNOT1 and TOB1. We further show TOB1 independently drives metastasis. RNA-immunoprecipitation and integrated transcriptome wide analyses reveal that CNOT7-regulated transcripts are enriched for a tripartite 3’UTR motif bound by RNA-binding proteins known to complex with CNOT7, TOB1, and CNOT1. Collectively, our data support a model of CNOT7, TOB1, CNOT1, and RNA-binding proteins collectively exerting post-transcriptional control on a metastasis suppressive transcriptional program to drive tumor cell metastasis.

2015-09-23 | GSE73366 | GEO

DNA barcoding reveals ongoing immunoediting of clonal cancer populations during metastatic progression and in response to immunotherapy

Project description:Cancers evade the immune system in order to grow or metastasise through the process of cancer immunoediting. While checkpoint inhibitor therapy has been effective for reactivating tumour immunity in some cancers, many solid cancers, including breast cancer, remain largely non-responsive. Understanding the way non-responsive cancers evolve to evade immunity, what resistance pathways are activated and whether this occurs at the clonal level will improve immunotherapeutic design. We tracked cancer cell clones during the immunoediting process and determined clonal transcriptional profiles that allow immune evasion in murine mammary tumour growth in response to immunotherapy with anti-PD1 and anti-CTLA4. Clonal diversity was significantly restricted by immunotherapy treatment at both the primary and metastatic sites. These findings demonstrate that immunoediting selects for pre-existing breast cancer cell populations, that immunoediting is not a static process and is ongoing during metastasis and immunotherapy treatment. Isolation of immunotherapy resistant clones revealed unique and overlapping transcriptional signatures. The overlapping gene signature was predictive of poor survival in basal-like breast cancer patient cohorts. Some of these overlapping genes have existing small molecules which can be used to potentially improve immunotherapy response.

2021-09-01 | E-MTAB-10027 | biostudies-arrayexpress

4T1 breast tumors: SPARC knock down primary tumors vs. Control primary tumors vs. Lung metastasis foci

Project description:Comparative analysis of the transcriptome of primary tumors generated from 4T1 cells transduced with a lentiviral vector expressing a siRNA against murine SPARC (4T1-C18), primary tumors generated from 4T1 cells transduced with a lentiviral vector expressing a scramble sequence (4T1-SCR) or lung metastasis foci from 4T1-SCR tumor-bearing mice (4T1-SCR MTTS). Three experimental conditions, 4T1-C18, 4T1-SCR and 4T1-SCR MTTS. Biological replicates: 4 4T1-C18, 4 4T1-SCR, 4 4T1-SCR MTTS independently grown in different mice. 2 days-old tumors and 30 days old lung foci. One replicate per array. All microarrays were processed the same day

2016-06-29 | E-GEOD-83831 | biostudies-arrayexpress

Tumour mutations in long noncoding RNAs enhance cell fitness

Project description:Tumour DNA contains thousands of somatic single nucleotide variants (SNVs) in non-protein-coding elements, yet their functional significance remains poorly understood. Amongst the most highly mutated elements are long noncoding RNAs (lncRNAs), functional transcripts with known roles in carcinogenesis. To search for driver mutations in lncRNAs, we apply an integrative driver discovery algorithm to SNVs from 2583 primary tumours and 3527 metastases to reveal 54 potential “driver lncRNAs”. Our algorithm confirms a particularly high mutation rate in the iconic cancer lncRNA, NEAT1, which has been ascribed by recent studies to passenger effects. We directly test the functionality of NEAT1 SNVs using in cellulo mutagenesis, identifying discrete regions where mutations reproducibly increase cell proliferation in diverse cell backgrounds, both cancerous and normal. In particular, mutations in the 5’ region alter ribonucleoprotein assembly and boost the population of subnuclear paraspeckles, thus mechanistically linking mutations to cellular proliferation. We then used RNA-pull down followed by mass spectrometry to identify the protein interactor changing between the wild type and mutant form of NEAT1.

2023-07-20 | PXD034007 | Pride

Ago-RIP-Seq Analysis to Identify miR-375 Targets in Human Macrophages

Project description:miR-375 is not expression in human macrophages, however there is an enhanced accumulation of miR-375 in macrophages upon coculture with breast cancer cells. The target landscape of this miR in macrophages is not known. Ago-RIP-Seq was aimed to identify miR-375 targets in human macrophages.

2019-02-08 | E-MTAB-6943 | biostudies-arrayexpress

RIP-Chip analysis of Musashi (Msi1) in the U251, a human glioma cell line

Project description:Musashi1 (Msi1) is a highly conserved RNA binding protein that is required during the development of the nervous system. Msi1 has a role in neural stem cells, controlling the balance between self-renewal and differentiation. Msi1 has also been implicated in cancer, being highly expressed in multiple tumor types. In this study, we analyzed Msi1 expression in a large cohort of medulloblastoma samples and showed that Msi1 is highly expressed in tumor tissue compared to normal cerebellum and that high Msi1 expression is associated with a poor prognosis. Using a nude mouse xenograft model, we demonstrate that Msi1 is important for tumor growth. We then used RIP-chip (ribonucleoprotein immunoprecipitation followed by microarray analysis) to identify mRNA targets of Msi1 in medulloblastoma. In conclusion, our results suggest that Msi1 functions as a regulator of multiple processes in medulloblastoma formation and could become an important therapeutic target. RIP-Chip analysis to identify mRNA preferentially associated with Msi1 protein. RIP-Chip experiments were performed on two biologically replicated samples. A total of 8 microarrays were carried on using technical replicates of Msi1 antibody vs. prebleed serum for each dye orientation. We prepared two biological replicates for two different arrays. Each array consisted of 4 microarrays with 2 replicates for each dye orientation.

2012-04-11 | E-GEOD-37216 | biostudies-arrayexpress

RIP-Chip analysis of Musashi (Msi1) in the Daoy human medulloblastoma cell line.

Project description:RIP-chip analysis to identify mRNA preferentially associated with Msi1 protein. RIP-Chip experiments were performed on two biologically replicated samples transfected with the BAP-Msi1 construct and a control sample from cells transfected with the BAP-Control construct. A total of 8 microarrays were carried on using technical replicates of BAP-Msi1 vs. BAP-Control for each dye orientation.

2011-07-25 | E-GEOD-30904 | biostudies-arrayexpress

The nuclear receptor ERbeta engages AGO2 in regulation of gene transcription, RNA splicing and RISC loading. Part 2

Project description:The RNA-binding protein Argonaute 2 (AGO2) is a key effector of RNA-silencing pathways It exerts a pivotal role in microRNA maturation and activity, and can modulate chromatin remodeling, transcriptional gene regulation and RNA splicing. The Estrogen Receptor beta (ERβ) is endowed with oncosuppressive activities, antagonizing hormone-induced carcinogenesis and inhibiting growth and oncogenic functions in luminal-like breast cancers (BCs), where its expression correlates with a better prognosis of the disease. Applying interaction proteomics coupled to mass spectrometry (MS) to characterize nuclear factors cooperating with ERβ in gene regulation, we identify AGO2 as a novel partner of ERβ in human BC cells. ERβ-AGO2 association was confirmed in vitro and in vivo both in the nucleus and in cytoplasm and is shown to be RNA-mediated. ChIP-Seq demonstrates AGO2 association to a large number of ERβ binding sites, and total and nascent RNA-Seq in ERβ+ vs ERβ- cells, and before and after AGO2 knock-down in ERβ+ cells, reveals a widespread involvement of this factor in ERβ-mediated regulation of gene transcription rate and RNA splicing. Moreover, isolation and sequencing by RIP-Seq of ERβ-associated long and small RNAs in the cytoplasm suggests involvement of the nuclear receptor in RISC loading, indicating that it may able to control directly also mRNA translation efficiency and stability.These results demonstrate that AGO2 can act as a pleiotropic functional partner of ERβ, indicating that both factors are endowed with multiple roles in the control of key cellular functions

2016-12-31 | E-MTAB-4639 | biostudies-arrayexpress

Tissue Type-Specific Expression of the dsRNA-Binding Protein 76 and Genome-wide Elucidation of its Target mRNAs

Project description:RNA-binding proteins accompany all steps in the life of mRNAs and provide dynamic gene regulation functions for rapid adjustment to changing extra- or intracellular conditions. The association of RNA-binding proteins with their targets is regulated through changing sub-cellular distribution, post-translational modification or association with other proteins. Tissue type-specific isoform distribution and target mRNA binding patterns are major determinants of post-transcriptional gene regulation by RNA-binding proteins.We demonstrate that the dsRNA binding protein 76 (DRBP76), synonymous with nuclear factor 90, displays inherently distinct tissue type-specific sub-cellular distribution in the normal human central nervous system and in malignant brain tumors of glial origin. Altered sub-cellular localization and isoform distribution in malignant glioma indicate that tumor-specific changes in DRBP76-related gene products and their regulatory functions may contribute to the formation and/or maintenance of these tumors. To identify endogenous mRNA targets of DRBP76, we performed RNA-immunoprecipitation and genome-wide microarray analyses in HEK293 cells, and identified specific classes of transcripts encoding critical functions in cellular metabolism. Our data suggest that DRBP76 expression, isoform distribution and sub-cellular localization are profoundly disturbed upon malignant transformation in the CNS. Thus, DRBP76’s roles in co- or post-transcriptional gene regulation may contribute to the neoplastic phenotype. Keyword: RIP-chip 5 biological replicates of DRBP76 Immunoprecipitation samples, Mock Immunoprecipiation samples, Total mRNA samples were collected and analyzed using oligo microarrays. For each microarray, biological samples were labelled with Cy3 and human reference RNA was labelled with Cy5.

2010-05-04 | E-GEOD-20001 | biostudies-arrayexpress

Gene expression analysis of 4T1 tumors expressing mouse urokinase plasminogen activitor (uPA)

Project description:Analysis of uPA regulation of 4T1 tumor growth at gene expression level. The hypothesis tested in the present study was that overexpression of uPA in 4T1 tumor influence the tumor growth and metastasis related cell signaling pathway. 4T1 cells were stably transduced by lentiviral vector expressing mouse uPA or empty vector. After day 7 of inoculation of 4T1 cells into fatpad of Balb/c mice. Total RNA was extracted from tumor samples (triplicate) using Qiagen total RNA isolation kit. The Illumina MouseWG-6 v2.0 Expression BeadChip (Illumina, Wallingford, CT) was used for gene expression. The raw data from the fluorescence intensity measurements of each array experiment was processed using GeneSpringGX v.11.0 software (Agilent, Santa Clara, CA). Statistical analysis, fold change calculations, and hierarchical clustering of the data were also performed in GeneSpring software. Genes that expressed significantly differently with more than 1.5-fold change and a p-value of <0.05 with respect to controls were taken into consideration. Gene expression data were further validated by qRT-PCR analysis. Pathway analysis was performed by MetaCore software (GeneGo, Inc, St. Joseph, MI).

2012-05-30 | E-GEOD-38346 | biostudies-arrayexpress

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