miR-99b-5p was significantly higher expressed in malignant compared to benign EVs. 92 miRNAs were enriched and 48 miRNAs were depleted in PC3 EVs compared to PC3 cells, which could be confirmed by qRT-PCR. miRNAs differentially expressed in PC3 EVs were validated with qRT-PCR in EVs derived from additional cell lines and patient plasma and from matched tissue samples. Non-coding RNA expression profiling of PC3 metastatic PCa cells and their EVs was performed by next generation sequencing (NGS). Nanoparticle tracking analysis (NTA), Western blot, electron microscopy, and flow cytometry analysis were used for the characterization of EVs. EVs were isolated from different benign and malignant prostate cell lines and blood plasma from patients with PCa (n = 18) and controls with benign prostatic hyperplasia (BPH) (n = 7). The role of EV RNA in prostate cancer (PCa) is still largely unknown. Their nucleic acids content offers new opportunities for biomarker research in different solid tumors. This workflow, however, detected tumor-specific mutations in blood less often in EV-RNA than in cfDNA.Įxtracellular vesicles (EVs) are shed by many different cell types. Expression profiling showed that the EVs from patients resemble more healthy donors than tumor cell lines supporting that EVs are mostly derived from healthy tissue.Ĭonclusions: We provide a workflow for ctEV detection by spin column-based generic isolation of EVs and PCR-based measurement of gene expression and mutant transcripts in EV-RNA derived from cancer patients’ blood plasma. The workflow detected only ctEVs with mutant transcripts in plasma of patients with high amounts (>20%) of circulating tumor DNA (ctDNA). Plasma CD9-positive EV and GAPDH EV-RNA levels were significantly different between the preservation tubes. Results: Our workflow applied on cell lines revealed a high concordance between cellular mRNA and EV-RNA in expression levels as well as variant allele frequencies for PIK3CA, KRAS and BRAF. RNA from EVs was isolated with the ExoRNeasy protocol and evaluated for transcript expression levels of 96 genes by RT-qPCR and genotyped by digital PCR. EVs were isolated with the ExoEasy protocol from this plasma and from conditioned medium of 6 cancer cell lines and characterized according to MISEV2018-guidelines. Methods: For this purpose, blood from 20 patients and 15 healthy blood donors (HBDs) was collected in different preservation tubes (EDTA, BCT, CellSave) and processed into plasma within 24 hours from venipuncture. The goal of this study was to detect circulating tumor-derived EVs (ctEVs) by mutant mRNA transcripts (EV-RNA) in plasma of patients with solid cancers and compare the occurrence of ctEVs with circulating tumor DNA (ctDNA) in cell-free DNA (cfDNA).
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