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Establishment and characterization of CRISPR/Cas9-mediated Merlin/NF2 knockout human mesothelial cell line Met-5A

ABSTRACT: Establishment and characterization of CRISPR/Cas9-mediated Merlin/NF2 knockout human mesothelial cell line Met-5A

PROVIDER: PRJNA476769 | ENA |

REPOSITORIES: ENA

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Establishment and characterization of CRISPR/Cas9-mediated Merlin/NF2 knockout human mesothelial cell line Met-5A

Project description:To investigate the effect of NF2 on gene expression, we have employed whole genome microarray expression profiling using MeT-5A cells.

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lncRNA expression alterations in mesothelial cells treated with exosomes

Project description:To investigate whether exosomes from ovarian cancer cell lines could change lncRNA expression in mesothelial cells, exosomes from ovarian cancer cells (SKOV3 cells, A2780 cells) were added to MeT-5A cells, PBS treatment was set as blank control. Total RNAs of MeT-5A cells treated with exosomes were extracted, and then lncRNA sequencing was performed.

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Establishment and characterization of NF2/CDKN2A-double knockout gene in human mesothelial cell line generated by using CRISPR/Cas9 technology

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Analysis of the transcriptomic profile of WT mouse embryonic fibroblast (MEF WT) and Nf2-knockout mouse embryonic fibroblast (MEF Nf2 KO) cell lines

Project description:Merlin is the tumor suppressor protein encoded by the NF2 gene. The expression of Merlin is remarkably decreased in metastatic breast cancer tissues irrespective of the breast cancer subtype. In order to ascribe clinical relevance, we re-capitulated the loss of Merlin in breast cancer cells. Merlin deficiency elicited a markedly invasive phenotype. In order to overcome the challenge of embryonic lethality of a total Nf2-knockout, we generated a unique mammary-specific Nf2-knockout mouse mammary tumor model. Both, the Nf2-knockout mouse embryonic fibroblasts (MEF) and Merlin-deficient breast tumor cells displayed a robust invasive phenotype. Transcriptomic assessment of Nf2-knockout MEFs revealed notable alterations in glutathione transferase and antioxidant networks indicating a role for Merlin in redox biology. This programmatic alteration resonated with the pathways that emerged from breast tumor cells engineered for Merlin deficiency.

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Proteomic analysis of the NF2/Merlin Interactome by Proximity Biotinylation

Project description:Neurofibromatosis type 2 is an inherited neoplastic disease consisting of schwannomas, meningiomas, and ependymomas that is caused by inactivation of the tumor suppressor gene NF2. The NF2 gene product, merlin, has no intrinsic catalytic activity; its tumor suppressor function is mediated through the proteins with which it interacts. However, there is no consensus about which merlin interactions are necessary for tumor suppression. We used proximity biotinylation followed by mass spectrometry and direct binding assays to characterize the proteins that associate with merlin and merlin mutants in immortalized Schwann cells. We identified 52 proteins that associate with merlin, including a previously unreported merlin binding protein, ASPP2. Our results identify merlin as a component of mechanosensing signal transduction pathways in cell junctions, in the context of a specific set of structures and molecules through which it acts, in a cell type relevant to schwannoma formation.

2019-04-25 | PXD012017 | Pride

Treatment of extracellular vesicles change gene expression in mesothelial cells

Project description:To investigate whether the EVs from cancer cell lines could change gene expression in mesothelial cells, the EVs from five types of cells (ES-2 cells, A2780 cells, SKOV3 cells, HOSE1 cells and HOSE2 cells) were added to MeT-5A cells (Fig. 2a). All three selected cancer cell lines expressed metastatic phenotypes in mouse model (Fig. 1c), and EVs from HOSE2 cell line, established together with HOSE1 cell line, was also set as control. Total mRNAs were extracted by MeT-5A cells treated with EVs, and then microarray analysis was performed.

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RNA expression profiling in primary and immortalized human mesothelial cells, and in human vein and microvascular endothelial cells

Project description:Mesothelial cells, which interact with endothelial cells, are widely used in research including cancer and drug development, have not been comprehensively profiled. We therefore performed RNA sequencing of polarized, primary peritoneal (HPMC) and immortalized pleural mesothelial cells (MeT-5A), and compared it to endothelial cells from umbilical vein (HUVEC) and cardiac capillaries (HCMEC).

2023-05-24 | E-MTAB-12021 | biostudies-arrayexpress

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Project description:To determine the transcriptional program that imparts adhesion capacity to healthy mesothelial cells, we analyzed subtle gene expression changes by performing highly parallel single-cell RNA-seq genome-wide expression profiling of individual mesothelial cells exposed to stress (Extended Figure 6A) using the Drop-seq workflow (Pubmed ID 26000488). We individually sequenced >16,000 cells from Met-5A mesothelial cells at various time-points after exposure to a 15 min desiccation shock, as well as under control unstressed conditions.

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Merlin/NF2 Suppresses Tumorigenesis by Inhibiting the E3 Ubiquitin Ligase CRL4DCAF1 in the Nucleus

Project description:Current models imply that the FERM domain protein Merlin, encoded by the tumor suppressor NF2, inhibits mitogenic signaling at or near the plasma membrane. Here, we show that the closed, growth inhibitory form of Merlin accumulates in the nucleus, binds to the E3 ubiquitin ligase CRL4DCAF1, and suppresses its activity. Depletion of DCAF1 blocks the promitogenic effect of inactivation of Merlin. Conversely, enforced expression of a Merlin-insensitive mutant of DCAF1 counteracts the antimitogenic effect of Merlin. Re-expression of Merlin and silencing of DCAF1 induce a similar, tumor-suppressive program of gene expression. Tumor-derived mutations invariably disrupt Merlinâs ability to interact with or inhibit CRL4DCAF1. Finally, depletion of DCAF1 inhibits the hyperproliferation of Schwannoma cells from NF2 patients and suppresses the oncogenic potential of Merlin-deficient tumor cell lines. We propose that Merlin suppresses tumorigenesis by translocating to the nucleus to inhibit CRL4DCAF1. To examine if Merlin controls gene expression through inhibition of CRL4DCAF1, and define the general function of this ligase, we compared the gene expression program activated by expression of Merlin or by depletion of DCAF1 in Merlin-null FC-1801 mouse Schwannoma cells

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RNA sequencing of endothelial cells from mouse brain of WT and EC-specific Nf2-deleted mice

Project description:Understanding of transciptome changes of endothelial cells (ECs) in mouse brain by Nf2/Merlin is unknown. Here, we performed bulk RNA sequencing to investigate the changes of expression patterns by Nf2/Merlin deletion in mouse brain ECs

2022-03-24 | GSE186189 | GEO

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