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TRACE-seq: “TRanslatomic” Analysis Captured in Extracellular vesicles using sequencing

ABSTRACT: Purpose: We develop a new methodology to monitor live cells in vitro as well as in vivo. Method: Each population of HEK 293T cells was transfected with the TRACE-seq construct (GFP-C-YTHDF1/GBP1-CD9) and a negative control construct (GFP-C-YTHDF1/GBP1-CD9) which contains no mRNA catcher. The analysis was made in duplicate with microvesicles (MVs) mRNA content isolated from cell culture media and their corresponding cell lysate. Samples were generated with the SMART-seq2 protocol and the Nextera kit (Illumina). Data were collected using 50 × 8 × 50 reads on a HiSeq. Reads were aligned to hg19 using STAR and counting reads associated genes were detected with the FeatureCounts module. Also, a QC qPCR was made on these samples and the cDNA library quality was analyzed by a High sensitivity DNA chip assay. Results: From the 3604 genes selected for this analysis, RNA-seq data showed a non negligeable increase of the correslation between MVs mRNA content and cell lysate from the HEK293T transfected with the TRACE construct. Conclusion: Our methodology bring a representative part of the transcriptome from the cell into MVs which is a usefull method for in vivo analysis. Moreover, our study represents the first detailed analysis of a new undestructive, fully compatible for in vivo, monitoring process of the cells transcriptome.

ORGANISM(S): Homo sapiens

PROVIDER: GSE162425 | GEO | 2020/12/02

REPOSITORIES: GEO

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Project description:Background: In previous studies we showed that IL-27 shares several effects with IFN-γ in human cancer cells. To identify novel extracellular mediators, potentially involved in epithelial ovarian cancer (EOC) biology, we analyzed the effect of IL-27 or IFN-γ on the secretome of EOC cells in culture. Methods: The secretome of cytokine-treated or untreated SKOV3 cells was analyzed by nano-UHPLC-MS/MS and eluting peptides by an Orbitrap Fusion Tribrid mass spectrometer. Extracellular GBP1 was studied by ELISA, immunoprecipitation, GTP-agarose pull-down, and Western blotting. GBP and STAT proteins were analysed on cell lysates by Western blot. GBP1 was transfected using lipofectamine reagent and cell viability measured by MTT assay. Results: IL-27 and IFN-γ modulate the extracellular release of a limited fraction of proteins that were also induced in the whole cell. Among these proteins we focused our attention on GBP1, a guanylate-binding protein and GTPase, which is a mediator of several biological activities of IFNs. Interestingly GBP1, 2, and 5 were induced by cytokine treatment in EOC cells, but only GBP1 was secreted. ELISA and immuno-blotting analyses showed that cytokine-stimulated EOC cells release full-length GBP1 molecule in vitro, through non-classical pathways, not involving microvesicles. Moreover, soluble, full-length GBP1 accumulates in the ascites of most EOC patients, suggesting a potential role of extracellular GBP1 in the tumor environment. EOC cells enriched from ascites showed constitutive tyrosine-phosphorylated STAT1 and STAT3 proteins and GBP1 expression, supporting a role of STAT-activating cytokines in GBP1 induction in vivo. Data from the TCGA dataset of EOC indicate that high GBP1 gene expression correlates with a better overall survival. Accordingly GBP1 transfection reduced SKOV3 cell proliferation, suggesting a potential anti-tumor activity of GBP1. Conclusions: Our data show for the first time that soluble GBP1 is released by cytokine-stimulated EOC cells in vitro and accumulates in EOC patients’ ascites. GBP1 expression may have anti-tumor effects, in EOC, as suggested by TCGA dataset analysis and in vitro transfection experiments. Further studies to identify the biological role of soluble GBP1 in the tumor environment of EOC are warranted.

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TRACE-seq: “TRanslatomic” Analysis Captured in Extracellular vesicles using sequencing

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