Immunotherapy for breast cancer using EpCAM aptamer tumor-targeted gene knockdown efficacy_UPF2 knockdown in EpCAMhi MDA-MB-231 breast cancer cell lines
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE156185 | GEO | 2021/02/05
REPOSITORIES: GEO
Similar Datasets
Project description:New strategies for cancer immunotherapy are needed since most solid tumors do not respond to current approaches. Here, we used EpCAM aptamer-linked small interfering RNAs (aptamer-siRNA chimeras (AsiCs)) to knockdown genes selectively in EpCAM+ tumors with the goal of making cancers more visible to the immune system to improve anti-tumor immunity. Gene targets were chosen whose knockdown was predicted to promote tumor neoantigen expression (Upf2 and Parp1), phagocytosis and antigen presentation (Cd47), or cause tumor cell death (Mcl1). Using scRNA-seq, we showed that knocking down the four targets simutaeoulsy potently improved the anti-tumor immunity mediated T cells and macrophage/monocytes, demonstrating the immunostimulatory capacity of EpCAM-AsiCs.
2021-02-05 | GSE156214 | GEO
Project description:Two core factors of the NMD machinery in D. melanogaster, Upf1 and Upf2, were knocked down using RNAi, as described in Rehwinkel et al. (2005) RNA, PMID: 16199763. Each of the two knockdowns were compared to mock RNAi knockdowns as described in Blanchette et al. (2005) Genes Dev, PMID: 15937219 in a dual channel experiment, using a custom splice-junction microarray design, see Blanchette et al. (2005), PMID: 15937219. The aim of the experiment was to identify which isoforms of alternatively spliced genes were affected by NMD knockdown and thereby gain insight into the NMD mechanism in Drosophila. The samples were hybridized in a dual channel setup, to custom designed Splice-Junction arrays manufactured by Agilent. A total of 6 independent knockdowns (3 Upf1 and 3 Upf2) were generated and hybridized to 6 different arrays. On each array a control sample was hybridized as well. The control sample is a pool of 3 independent mock RNAi knockdowns.
2010-05-18 | E-GEOD-13532 | biostudies-arrayexpress
Project description:Two core factors of the NMD machinery in D. melanogaster, Upf1 and Upf2, were knocked down using RNAi, as described in Rehwinkel et al. (2005) RNA, PMID: 16199763. Each of the two knockdowns were compared to mock RNAi knockdowns as described in Blanchette et al. (2005) Genes Dev, PMID: 15937219 in a dual channel experiment, using a custom splice-junction microarray design, see Blanchette et al. (2005), PMID: 15937219. The aim of the experiment was to identify which isoforms of alternatively spliced genes were affected by NMD knockdown and thereby gain insight into the NMD mechanism in Drosophila. The samples were hybridized in a dual channel setup, to custom designed Splice-Junction arrays manufactured by Agilent.
2009-06-03 | GSE13532 | GEO
Project description:A major task in analyzing alternative splicing (AS) by RNA-Seq is to explore and quantify the differential representation of various splice classes and the protein-coding potential of the mapped reads. To this end, we have generated a streamlined bioinformatic pipeline available to the scientific community, which maps RNA-Seq reads to a complex combinatorial database of exon-exon junctions for the unique junction discovery, PTC detection and identification of splice isoforms. Hence, we have incorporated a splice isoform inference and PTC detection algorithm, which facilitates a highly accurate mapping and prediction of splice isoform junctions and nonsense-mediated mRNA decay (NMD)-susceptibility. We used this pipeline to investigate the complexity of the transcriptome and global role of AS-NMD in vivo in mammalian cells, by taking advantage of our Upf2 conditional knock-out mouse. Using tissue-specific Cre deleter strains, we have previously demonstrated in vivo that NMD acts to degrade many transcripts that results from AS. Also, ablating UPF2 (a core NMD factor) in the mouse leads to rapid mortality and collapse of most organs tested (Thoren et al., 2010; Weischenfeldt et al., 2008), suggesting an essential function for NMD in AS. To explore the effect of NMD on global splicing and to generate and validate an attractive bioinformatic pipeline for the scientific community to study AS and NMD, we chose to analyze two different mammalian organ systems with distinct phenotypes upon UPF2 ablation to test the robustness and dynamic range of the analysis. In one end of the spectrum, we chose to analyze the liver, wherein removal of UPF2 results in failure in liver metabolism and a high mortality rate (Thoren et al., 2010). To profile a less affected tissue, we chose to analyze bone marrow-derived macrophages (BMMs). These are macrophages that differentiate in vitro and Upf2 deleted BMMs are completely devoid of NMD activity but nevertheless show no morphological or functional phenotype compared to wild-type controls (Weischenfeldt et al., 2008). Thus, we were able to make a direct comparison between these two tissues to demonstrate the potency of our Isoform and PTC detection pipeline described below, which will allow the scientific community to analyze transcriptome data for global AS and NMD-susceptibility. Examination of UPF2 WT vs. KO in two different murine tissues (liver, bone marrow-derived macrophages).
2012-05-01 | E-GEOD-26561 | biostudies-arrayexpress
Project description:Nonsense-mediated decay (NMD) is an evolutionarily conserved surveillance mechanism that targets mRNAs undergoing premature translation termination for rapid degradation as well as normal physiological transcripts. From yeast to humans, NMD requires the function of three conserved Up-frameshift (Upf) factors (Upf1, Upf2, Upf3). Interestingly, in humans, mutations in UPF2 and UPF3B are associated with intellectual disability and autism spectrum disorder (ASD). However, the neurobiological mechanism by which deficient NMD leads to neurodevelopmental disorders remains unknown. Consequently, no treatment is currently available. Here we report that mice lacking Upf2 in the murine forebrain (Upf2 fb-KO mice) show endophenotypes associated with ASD and deficits in learning and memory. In addition, Upf2-mediated deficient NMD leads to abnormal long-term potentiation (LTP) in the hippocampus. Like patients with mutations in UPF2, Upf2 fb-KO mice showed neuroanatomical abnormalities including a smaller corpus callosum. Surprisingly, transcriptomic analysis revealed elevated mRNA expression of immune-related genes in the hippocampus of Upf2-fb-KO mice, which was accompanied by increased inflammation and progressive infiltration of peripheral immune cells. More importantly, treatment with an FDA-approved immunosuppressant, cyclophosphamide (CyP), significantly reduces brain inflammation, improves the neuroanatomical defects and the deficits in LTP and memory deficits, and reverses some of the ASD-like behaviors, notably the social deficits. Collectively, our results reveal the biological basis underlying neurodevelopmental disorders associated with dysfunctional NMD. Moreover, these findings suggest that immunosuppressant drugs, like CyP, may provide a new therapeutic approach for the treatment of patients with mutations in core NMD components.
2020-06-29 | GSE81591 | GEO
Project description:A major task in analyzing alternative splicing (AS) by RNA-Seq is to explore and quantify the differential representation of various splice classes and the protein-coding potential of the mapped reads. To this end, we have generated a streamlined bioinformatic pipeline available to the scientific community, which maps RNA-Seq reads to a complex combinatorial database of exon-exon junctions for the unique junction discovery, PTC detection and identification of splice isoforms. Hence, we have incorporated a splice isoform inference and PTC detection algorithm, which facilitates a highly accurate mapping and prediction of splice isoform junctions and nonsense-mediated mRNA decay (NMD)-susceptibility. We used this pipeline to investigate the complexity of the transcriptome and global role of AS-NMD in vivo in mammalian cells, by taking advantage of our Upf2 conditional knock-out mouse. Using tissue-specific Cre deleter strains, we have previously demonstrated in vivo that NMD acts to degrade many transcripts that results from AS. Also, ablating UPF2 (a core NMD factor) in the mouse leads to rapid mortality and collapse of most organs tested (Thoren et al., 2010; Weischenfeldt et al., 2008), suggesting an essential function for NMD in AS. To explore the effect of NMD on global splicing and to generate and validate an attractive bioinformatic pipeline for the scientific community to study AS and NMD, we chose to analyze two different mammalian organ systems with distinct phenotypes upon UPF2 ablation to test the robustness and dynamic range of the analysis. In one end of the spectrum, we chose to analyze the liver, wherein removal of UPF2 results in failure in liver metabolism and a high mortality rate (Thoren et al., 2010). To profile a less affected tissue, we chose to analyze bone marrow-derived macrophages (BMMs). These are macrophages that differentiate in vitro and Upf2 deleted BMMs are completely devoid of NMD activity but nevertheless show no morphological or functional phenotype compared to wild-type controls (Weischenfeldt et al., 2008). Thus, we were able to make a direct comparison between these two tissues to demonstrate the potency of our Isoform and PTC detection pipeline described below, which will allow the scientific community to analyze transcriptome data for global AS and NMD-susceptibility.
2012-05-01 | GSE26561 | GEO
Project description:Eukaryotic cells have evolved a mechanism called nonsense mediated mRNA decay (NMD) that detects and degrades aberrant mRNAs that contain premature termination codons (PTCs). NMD has recently acquired broader importance as it has been found to regulate not only aberrant mRNAs but also a great diversity of transcripts, including wild type genes in mammals, Drosophila and yeast. Seven proteins are the core of the NMD complex: SMG1, SMG2 (UPF1), SMG3 (UPF2), SMG4 (UPF3), SMG5, SMG6 and SMG7. Plants have orthologues of most of these proteins. We have identified and characterized a number of alleles of UPF1, UPF3 and SMG5 and made 35S:UPF2 RNAi (upf2i) transgenic plants, all of which share some phenotypic characteristics. Transcriptome analysis of upf1 and upf3 mutants has identified a subset of genes coregulated by UPF1 and UPF3 and, therefore, by NMD (unpublished data). By doing further transcriptome analysis on smg5 mutants and upf2i plants we aim to build a more robust subset of NMD targets in Arabidopsis. RNA will be extracted from 17 day-old mutant, transgenic and wild type seedlings grown at 22-24 C under constant light. 6 samples were used in this experiment
2011-10-06 | E-GEOD-32671 | biostudies-arrayexpress
Project description:The RNA helicase UPF1 interacts with mRNAs, mRNA decay machinery, and the terminating ribosome to promote nonsense-mediated mRNA decay (NMD). Structural and biochemical data have revealed that UPF1 exists in an enzymatically autoinhibited “closed” state. Upon binding the NMD protein UPF2, UPF1 undergoes an extensive conformational change into a more enzymatically active “open” state, which exhibits enhanced ATPase and helicase activity. However, mechanically deficient UPF1 mutants can support efficient NMD, bringing into question the roles of UPF1 enzymatic autoinhibition and activation in NMD. Here, we identify two additional important features of the activated open state: slower nucleic acid binding kinetics and enhanced ATP-stimulated nucleic acid dissociation kinetics. Computational modeling based on empirical measurements of UPF1, UPF2, and RNA interaction kinetics predicts that the majority of UPF1-RNA binding and dissociation events in cells occur independently of UPF2 binding. We find that UPF1 mutants with either reduced or accelerated dissociation from RNA have NMD defects, whereas UPF1 mutants that are more dependent on UPF2 for catalytic activity remain active on well-established NMD targets. These findings support a model in which the kinetics of UPF1-mRNA interactions are important determinants of cellular NMD efficiency.
2024-05-29 | GSE246484 | GEO
Project description:The RNA helicase UPF1 interacts with mRNAs, mRNA decay machinery, and the terminating ribosome to promote nonsense-mediated mRNA decay (NMD). Structural and biochemical data have revealed that UPF1 exists in an enzymatically autoinhibited “closed” state. Upon binding the NMD protein UPF2, UPF1 undergoes an extensive conformational change into a more enzymatically active “open” state, which exhibits enhanced ATPase and helicase activity. However, mechanically deficient UPF1 mutants can support efficient NMD, bringing into question the roles of UPF1 enzymatic autoinhibition and activation in NMD. Here, we identify two additional important features of the activated open state: slower nucleic acid binding kinetics and enhanced ATP-stimulated nucleic acid dissociation kinetics. Computational modeling based on empirical measurements of UPF1, UPF2, and RNA interaction kinetics predicts that the majority of UPF1-RNA binding and dissociation events in cells occur independently of UPF2 binding. We find that UPF1 mutants with either reduced or accelerated dissociation from RNA have NMD defects, whereas UPF1 mutants that are more dependent on UPF2 for catalytic activity remain active on well-established NMD targets. These findings support a model in which the kinetics of UPF1-mRNA interactions are important determinants of cellular NMD efficiency.
2024-05-29 | GSE246483 | GEO