| 1. |
- Lunavat, Taral R, et al.
(författare)
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BRAF(V600) inhibition alters the microRNA cargo in the vesicular secretome of malignant melanoma cells
- 2017
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424. ; 114:29
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Tidskriftsartikel (refereegranskat)abstract
- The BRAF inhibitors vemurafenib and dabrafenib can be used to treat patients with metastatic melanomas harboring BRAF(V600) mutations. Initial antitumoral responses are often seen, but drug-resistant clones with reactivation of the MEK-ERK pathway soon appear. Recently, the secretome of tumor-derived extracellular vesicles (EVs) has been ascribed important functions in cancers. To elucidate the possible functions of EVs in BRAF-mutant melanoma, we determined the RNA content of the EVs, including apoptotic bodies, microvesicles, and exosomes, released from such cancer cells after vemurafenib treatment. We found that vemurafenib significantly increased the total RNA and protein content of the released EVs and caused significant changes in the RNA profiles. RNA sequencing and quantitative PCR show that cells and EVs from vemurafenib-treated cell cultures and tumor tissues harvested from cell-derived and patient-derived xenografts harbor unique miRNAs, especially increased expression of miR-211-5p. Mechanistically, the expression of miR-211-5p as a result of BRAF inhibition was induced by increased expression of MITF that regulates the TRPM1 gene resulting in activation of the survival pathway. In addition, transfection of miR-211 in melanoma cells reduced the sensitivity to vemurafenib treatment, whereas miR-211-5p inhibition in a vemurafenib resistant cell line affected the proliferation negatively. Taken together, our results show that vemurafenib treatment induces miR-211-5p up-regulation in melanoma cells both in vitro and in vivo, as well as in subsets of EVs, suggesting that EVs may provide a tool to understand malignant melanoma progression.
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| 2. |
- Pathan, M., et al.
(författare)
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A novel community driven software for functional enrichment analysis of extracellular vesicles data
- 2017
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Ingår i: Journal of Extracellular Vesicles. - : Wiley. - 2001-3078. ; 6:1
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Tidskriftsartikel (refereegranskat)abstract
- Bioinformatics tools are imperative for the in depth analysis of heterogeneous high-throughput data. Most of the software tools are developed by specific laboratories or groups or companies wherein they are designed to perform the required analysis for the group. However, such software tools may fail to capture "what the community needs in a tool". Here, we describe a novel community-driven approach to build a comprehensive functional enrichment analysis tool. Using the existing FunRich tool as a template, we invited researchers to request additional features and/or changes. Remarkably, with the enthusiastic participation of the community, we were able to implement 90% of the requested features. FunRich enables plugin for extracellular vesicles wherein users can download and analyse data from Vesiclepedia database. By involving researchers early through community needs software development, we believe that comprehensive analysis tools can be developed in various scientific disciplines.
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| 3. |
- Hui, Xiao, et al.
(författare)
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Mast cell exosomes promote lung adenocarcinoma cell proliferation - role of KIT-stem cell factor signaling
- 2014
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Ingår i: Cell Communication and Signaling. - 1478-811X. ; 12:64
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Tidskriftsartikel (refereegranskat)abstract
- Background Human cells release nano-sized vesicles called exosomes, containing mRNA, miRNA and specific proteins. Exosomes from one cell can be taken up by another cell, which is a recently discovered cell-to-cell communication mechanism. Also, exosomes can be taken up by different types of cancer cells, but the potential functional effects of mast cell exosomes on tumor cells remain unknown. Methods and results Exosomes were isolated from the human mast cell line, HMC-1, and uptake of PKH67-labelled exosomes by the lung epithelial cell line, A549, was examined using flow cytometry and fluorescence microscopy. The RNA cargo of the exosomes was analyzed with a Bioanalyzer and absence or presence of the c-KIT mRNA was determined by RT-PCR. The cell proliferation was determined in a BrdU incorporation assay, and proteins in the KIT-SCF signaling pathway were detected by Western blot. Our result demonstrates that exosomes from mast cells can be taken up by lung cancer cells. Furthermore, HMC-1 exosomes contain and transfer KIT protein, but not the c-KIT mRNA to A549 cells and subsequently activate KIT-SCF signal transduction, which increase cyclin D1 expression and accelerate the proliferation in the human lung adenocarcinoma cells. Conclusions Our results indicate that exosomes can transfer KIT as a protein to tumor cells, which can affect recipient cell signaling events through receptor-ligand interactions.
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| 4. |
- Lázaro-Ibáñez, Elisa, et al.
(författare)
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Distinct prostate cancer-related mRNA cargo in extracellular vesicle subsets from prostate cell lines
- 2017
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Ingår i: Bmc Cancer. - : Springer Science and Business Media LLC. - 1471-2407. ; 17:92
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Tidskriftsartikel (refereegranskat)abstract
- Background: Multiple types of extracellular vesicles (EVs), including microvesicles (MVs) and exosomes (EXOs), are released by all cells constituting part of the cellular EV secretome. The bioactive cargo of EVs can be shuffled between cells and consists of lipids, metabolites, proteins, and nucleic acids, including multiple RNA species from non-coding RNAs to messenger RNAs (mRNAs). In this study, we hypothesized that the mRNA cargo of EVs could differ based on the EV cellular origin and subpopulation analyzed. Methods: We isolated MVs and EXOs from PC-3 and LNCaP prostate cancer cells by differential centrifugation and compared them to EVs derived from the benign PNT2 prostate cells. The relative mRNA levels of 84 prostate cancer-related genes were investigated and validated using quantitative reverse transcription PCR arrays. Results: Based on the mRNA abundance, MVs rather than EXOs were enriched in the analyzed transcripts, providing a snapshot of the tumor transcriptome. LNCaP MVs specifically contained significantly increased mRNA levels of NK3 Homeobox 1 (NKX3-1), transmembrane protease serine 2 (TMPRSS2), and tumor protein 53 (TP53) genes, whereas PC-3 MVs carried increased mRNA levels of several genes including, caveolin-2 (CAV2), glutathione S-transferase pi 1 (GSTP1), pescadillo ribosomal biogenesis factor 1 (PES1), calmodulin regulated spectrin associated protein 1 (CAMSAP1), zinc-finger protein 185 (ZNF185), and others compared to PNT2 MVs. Additionally, ETS variant 1 (ETV1) and fatty acid synthase (FASN) mRNAs identified in LNCaP-and PC-3-derived MVs highly correlated with prostate cancer progression. Conclusions: Our study provides new understandings of the variability of the mRNA cargo of MVs and EXOs from different cell lines despite same cancer origin, which is essential to better understand the the proportion of the cell transcriptome that can be detected within EVs and to evaluate their role in disease diagnosis.
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| 5. |
- Lunavat, Taral R
(författare)
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Extracellular Vesicles and RNA interference in tumors
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Extracellular vesicles (EVs) including apoptotic bodies (ABs), microvesicles (MVs), exosomes (EXOs) and cell derived artificial nanovesicles (NVs) are important mediators of cell-to-cell communication, in part by transferring bioactive molecules such as DNA, mRNA, miRNA, siRNA, proteins, and lipids. These EVs are released by many cell types, including melanoma cells, and are found in many body fluids. EVs derived from various cell types differ in their molecular composition making them as important diagnostic and prognostic markers. The overall aim of this thesis was to use small-RNA sequencing techniques to define the molecular RNA cargo in the EV subsets described above as well as to examine the functional relevance of the EV-associated miRNA and siRNA on recipient cells. Characterization of EVs showed distinct RNA profiles in ABs, MVs, and EXOs, and there were significantly greater amounts of total RNA in EXOs compared to ABs and MVs. Small RNA sequencing revealed distinct repertoires of noncoding RNAs in the EV subsets. EXOs contained unique sets of miRNAs, which were shown to be differentially expressed in melanoma tumors compared with benign naevi in previously published studies, thus making them potentially useful as carriers of therapeutic agents. This study demonstrates that distinct sets of RNA molecules are present in subsets of EVs, and this provides unique insights into the contribution of extracellular RNA in cancer development and progression. The BRAFV600E inhibitor vemurafenib inhibited the growth of in vitro melanoma cell cultures, and EVs isolated from the treated cells had significantly higher RNA and protein contents compared to EVs from non-treated cells. Small RNA sequencing revealed distinct non-coding RNA species with significant alterations in miRNA between treated and non-treated cell-derived EVs. Moreover, treated cells and the EVs derived from them showed significant upregulation of miR-211 in vitro and in vivo. Furthermore, when vemurafenib-treated cell-derived EXOs were transferred to BRAFWT cells, KCNMA1 and IGF2R, genes that are known to play roles in tumor progression, were down-regulated and this resulted in growth attenuation. Overall, miR-211 could be used as a biomarker of response in patients diagnosed with BRAF-mutant melanoma. This study also provides the framework for further investigations into the function of miR-211 in melanoma cells and EVs as well as in cells that might receive miRNA from EVs. Artificial EXO-mimetic NVs were developed by serial extrusion, and they showed similar characteristics as EXOs. Exogenous loading of GFP-siRNA in NVs led to down-regulation of GFP in endothelial cells. Cell-derived NVs carrying endogenously expressed Myc-siRNA showed significant down-regulation of human cMyc both transcriptionally as well as translationally in lymphoma cells. These NVs were efficiently loaded with siRNA and were taken up by recipient cells resulting in the reduction of target gene expression. In conclusion, this study suggests that EXO-mimetic NVs can be a platform for delivering siRNA to cells. Taken together, EVs have significant therapeutic potential. EVs have emerged as a novel and functionally important vehicle of cell-cell communication that can mediate multiple biological effects. In addition, these vesicles might provide unique signatures that can be used as biomarkers of response to drug treatment.
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| 6. |
- Lunavat, Taral R, et al.
(författare)
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RNAi delivery by exosome-mimetic nanovesicles - Implications for targeting c-Myc in cancer
- 2016
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Ingår i: Biomaterials. - : Elsevier BV. - 0142-9612. ; 102, s. 231-238
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Tidskriftsartikel (refereegranskat)abstract
- To develop RNA-based therapeutics, it is crucial to create delivery vectors that transport the RNA molecule into the cell cytoplasm. Naturally released exosomes vesicles (also called "Extracellular Vesicles") have been proposed as possible RNAi carriers, but their yield is relatively small in any cell culture system. We have previously generated exosome-mimetic nanovesicles (NV) by serial extrusions of cells through nano-sized filters, which results in 100-times higher yield of extracellular vesicles. We here test 1) whether NV can be loaded with siRNA exogenously and endogenously, 2) whether the siRNA-loaded NV are taken up by recipient cells, and 3) whether the siRNA can induce functional knock-down responses in recipient cells. A siRNA against GFP was first loaded into NV by electroporation, or a c-Myc shRNA was expressed inside of the cells. The NV were efficiently loaded with siRNA with both techniques, were taken up by recipient cells, which resulted in attenuation of target gene expression. In conclusion, our study suggests that exosome-mimetic nanovesicles can be a platform for RNAi delivery to cell cytoplasm.
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| 7. |
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| 8. |
- Tüzesi, Ágota, 1982, et al.
(författare)
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Pediatric brain tumor cells release exosomes with a miRNA repertoire that differs from exosomes secreted by normal cells
- 2017
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Ingår i: Oncotarget. - : Impact Journals, LLC. - 1949-2553. ; 8:52, s. 90164-90175
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Tidskriftsartikel (refereegranskat)abstract
- High-grade gliomas (HGGs) are very aggressive brain tumors with a cancer stem cell component. Cells, including cancer stem cells, release vesicles called exosomes which contain small non-coding RNAs such as microRNAs (miRNAs). These are thought to play an important role in cell-cell communication. However, we have limited knowledge of the types of exosomal miRNAs released by pediatric HGG stem cells; a prerequisite for exploring their potential roles in HGG biology. Here we isolated exosomes released by pediatric glioma stem cells (GSCs) and compared their repertoire of miRNAs to genetically normal neural stem cells (NSCs) exosomes, as well as their respective cellular miRNA content. Whereas cellular miRNAs are similar, we find that the exosomal miRNA profiles differ between normal and tumor cells, and identify several differentially expressed miRNAs. Of particular interest is miR-1290 and miR-1246, which have previously been linked to 'stemness' and invasion in other cancers. We demonstrate that GSC-secreted exosomes influence the gene expression of receiving NSCs, particularly targeting genes with a role in cell fate and tumorigenesis. Thus, our study shows that GSCs and NSCs have similar cellular miRNA profiles, yet differ significantly in the repertoire of exosomal miRNAs and these could influence malignant features of HGG. © Tuzesi et al.
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