Identification of pannexin 1-regulated genes, interactome, and pathways in rhabdomyosarcoma and its tumor inhibitory interaction with AHNAK
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA602752 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10954315_1.fastq.gz | Fastqsanger.gz | |||
| SRR10954315_2.fastq.gz | Fastqsanger.gz | |||
| SRR10954316_1.fastq.gz | Fastqsanger.gz | |||
| SRR10954316_2.fastq.gz | Fastqsanger.gz | |||
| SRR10954317_1.fastq.gz | Fastqsanger.gz |
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Project description:We have recently reported the down-regulation of pannexin 1 (PANX1) levels in rhabdomyosarcoma (RMS) and that restoration of its expression inhibits the progression of embryonal (eRMS) and alveolar (aRMS) rhabdomyosarcoma in vitro and in vivo. While we have shown that this PANX1-mediated inhibition of RMS progression involves reduction of cell proliferation and migration, as well as induction of apoptosis, the underlining molecular mechanisms at play remained to be elucidated. In the present study, using RNA sequencing (RNA-seq) combined with DAVID analysis, we identified the PANX1 interactome in RMS cells and showed that the Gene Ontology (GO) terms such as apoptosis and migration, and KEGG pathways such as MAPK and Rap1 Signaling pathways were amongst the most highly enriched processes in Rh30 (aRMS) cells expressing ectopic PANX1. We then used a proximity-dependent labeling approach (BioID) to screen for the PANX1 interactors in RMS cells, which was complemented by co-immunoprecipitation (co-IP) coupled to high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) performed using Myc-PANX1-enriched subcellular fractions. STRING protein network analyses of the overlapping hits from these two approaches revealed a PANX1 interactome with plasma membrane and cytoskeleton associated proteins from which ACTB and AHNAK were further validated by co-IP using whole cell lysates followed by Western blotting analysis. Using this new and comprehensive information, we have generated the first online PANX1 transcriptome and interactome databases that allow searching, in a user-friendly manner, for PANX1-regulated genes and PANX1 interactors. Moreover, AHNAK knockdown in PANX1-expressing Rh18 (eRMS) and Rh30 (aRMS) significantly abrogated the PANX1-mediated reduction in cell viability and migration, as well as the PANX1-mediated sensitivity to anoikis. Using a combination of genome-wide unbiased approaches, this study is thus the first to identify the PANX1 transcriptome and interactome, providing the research community with online searchable databases to access the PANX1-regulated genes and PANX1 interacting partners. We have shown that PANX1 interacts with AHNAK and that this interaction is critical to the PANX1-mediated inhibition of RMS cell viability and migration, and induction of apoptosis; thus demonstrating how this new information can be used to identify key members of the PANX1 signaling pathway and their relevance to PANX1 function.
2020-01-30 | MSV000084867 | MassIVE
Project description:We identified and interactome for seven diffent rhabdomyosarcoma oncofusions. The proliferative effects of the loss of these proteins was tested by a CRISPR loss of function library screen in four rhabdomyosarcoma cell lines engineered to be driven by 4 different oncofusions.
2026-03-26 | GSE314907 | GEO
Project description:Purpose: Our laboratory has recently reported novel tumor-suppressive roles of PANX1 in both embryonal (eRMS) and alveolar (aRMS) rhabdomyosarcoma, but the underlying molecular mechanism remains to be elucidated. Methods: PANX1-induced differential gene expression profile in alveolar rhabdomyosarcoma (RMS) cell line, Rh30, was generated by deep sequencing using Illumina HiSeq2000. The sequence reads that passed quality filters were analyzed by BOWTIE/TOPHAT pipeline with accessary programs SAMTOOLS (v1.0) and CUTADAPT (v1.7.1) followed by Cufflinks (v2.2.1). The protein expression of the differentially regulated genes was validated by Western blotting. Results: Using RNA-seq and DAVID analysis, we showed that the Gene Ontology (GO) terms such as apoptosis and migration, and KEGG pathways such as MAPK and Rap1 Signaling pathways were amongst the most highly enriched processes in Rh30 (aRMS) cells with ectopic PANX1 expression. We found that both GJA1 and its encoded protein connexin 43 (Cx43) were significantly increased in PANX1 expressing Rh30 (aRMS) cells. Conclusion: PANX1 transcriptome and proteome databases hold great potential for discovery of novel PANX1 tumor-suppressive mechanisms and therapeutic targets for RMS.
2021-01-22 | GSE144102 | GEO
Project description:Rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children, is an aggressive cancer with a poor prognosis. Despite current management, the 5-year survival rate for patients with metastatic RMS is ∼30%; underscoring the need to develop better treatment strategies. We have recently reported that pannexin 1 (PANX1) levels are downregulated in RMS and that restoring its expression inhibits RMS progression. Here, we have surveyed and characterized the molecular changes induced by PANX1 re-expression in RMS. We cataloged transcriptomic changes in this context by RNA sequencing. At the protein level, we unveiled PANX1 interactors using BioID, complemented by co-immunoprecipitation coupled to high-performance liquid chromatography/electrospray ionization tandem mass spectrometry performed in PANX1-enriched fractions. Using these data, we generated searchable public databases for the PANX1 interactome and changes to the RMS transcriptome occurring when PANX1 expression is restored. STRING network analyses revealed a PANX1 interactome involving plasma membrane and cytoskeleton-associated proteins including the previously undescribed interactor AHNAK. Indeed, AHNAK knockdown abrogated the PANX1-mediated reduction in RMS cell viability and migration. Using these unbiased approaches, we bring insight to the mechanisms by which PANX1 inhibits RMS progression, identifying the cell migration protein AHNAK as a key modifier of PANX1-mediated changes in RMS malignant properties.
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Project description:Interactome CRISPR screen for rhabdomyosarcoma cells driven by four oncofusions
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Project description:The goal of this study was to explore in detail how the chromatin remodeler and NuRD subunit CHD4 controls the oncogenic signature of the tumor driver and fusion protein PAX3-FOXO1 in fusion-positive rhabdomyosarcoma. To this aim, we defined the interactome of CHD4 by LC-MS, identified its location in the genome by ChIP-seq, assessed its influence on DNA accessibility by DNase I hypersensitivity assays, and determined its target genes by RNA-seq.
2020-08-11 | PXD015231 | Pride
Project description:CoIP-MS/MS experiments to identify interacting proteins for human ZNF555 in muscle cancer rhabdomyosarcoma cells TE671.
2024-01-03 | PXD027542 | Pride
Project description:We identified the common protein interactome of rhabdomyosarcoma interacting proteins. Here we selected a subset of interactors, targeted them with sgRNA, and performed RNAseq is RH4-flag cells.
2026-03-26 | GSE314949 | GEO
Project description:Many biological processes are regulated by RNA-RNA interactions 1, nonetheless it remains formidable to analyze the entire RNA interactome. We developed a method, MARIO (MApping Rna-rna Interactions in vivO), to map protein-assisted RNA-RNA interactions in vivo. By circumventing the selection for a specific RNA-binding protein 2-5, our approach vastly expands the identifiable portion of the RNA interactome. Using this technology, we mapped the RNA interactome in mouse embryonic stem cells, which was composed of 46,780 RNA-RNA interactions. The RNA interactome was a scale-free network, with several lincRNAs and mRNAs emerging as hubs. We validated an interaction between two hubs, Malat1 and Slc2a3 using single molecule RNA fluorescence in situ hybridization. Base pairing was observed at the interaction sites of long RNAs, and was particularly strong in transposonRNA-mRNA and lincRNA-mRNA interactions. This reveals a new type of regulatory sequences acting in trans. Consistent with their hypothesized roles, the RNA interaction sites were more evolutionarily conserved than other regions of the transcripts. MARIO also provided new information on RNA structures, by simultaneously revealing the footprint of single stranded regions and the spatially proximal sites of each RNA. The unbiased mapping of the protein-assisted RNA interactome with minimum perturbation of cell physiology will greatly expand our capacity to investigate RNA functions. Three (3) ESC samples with different treatment (different digestion size and/or crosslinking method) and one (1) MEF sample were included to test our new approach for RNA-interactome mapping and the different samples were analyzed to show RNA interactome differences between them.
2016-06-23 | E-GEOD-61489 | biostudies-arrayexpress
Project description:Many biological processes are regulated by RNA-RNA interactions 1, nonetheless it remains formidable to analyze the entire RNA interactome. We developed a method, MARIO (MApping Rna-rna Interactions in vivO), to map protein-assisted RNA-RNA interactions in vivo. By circumventing the selection for a specific RNA-binding protein 2-5, our approach vastly expands the identifiable portion of the RNA interactome. Using this technology, we mapped the RNA interactome in mouse embryonic stem cells, which was composed of 46,780 RNA-RNA interactions. The RNA interactome was a scale-free network, with several lincRNAs and mRNAs emerging as hubs. We validated an interaction between two hubs, Malat1 and Slc2a3 using single molecule RNA fluorescence in situ hybridization. Base pairing was observed at the interaction sites of long RNAs, and was particularly strong in transposonRNA-mRNA and lincRNA-mRNA interactions. This reveals a new type of regulatory sequences acting in trans. Consistent with their hypothesized roles, the RNA interaction sites were more evolutionarily conserved than other regions of the transcripts. MARIO also provided new information on RNA structures, by simultaneously revealing the footprint of single stranded regions and the spatially proximal sites of each RNA. The unbiased mapping of the protein-assisted RNA interactome with minimum perturbation of cell physiology will greatly expand our capacity to investigate RNA functions. Three (3) ESC samples were treated with one (1) type of antisense oligonucleotides as is described in Kretz M. et al. (Nature. 2013 Jan 10;493(7431):231-5) to show the RNA interaction among specific RNAs and verify the results from MARIO
2016-06-23 | E-GEOD-65849 | biostudies-arrayexpress