PO-482 Analysis of small RNA cargo in urinary and plasma EVs and matching prostate cancer and normal prostate tissues

Abstract

IntroductionExtracellular vesicles (EVs) are membrane-enclosed structures released to the extracellular environment by different cell types, including cancer cells. Cancer derived-EVs have been shown to contain specific RNA cargo. Though, to what extent the EV cargo isolated from biofluids represents the tumour content is still under debate. The aim of this study was to characterise the small RNA content in plasma and urinary EVs collected from prostate cancer (PCa) patients before and after prostatectomy and to compare it with the matching tumour and normal tissue, in order to analyse their correlation and to identify RNA biomarkers that are released in plasma or urine from the tumour tissue.Material and methodsEVs were isolated from plasma and urine samples of 4 patients using size exclusion chromatography. EVs were quantified using Nanosight and their purity and size were assessed by electron microscopy and Western blot analysis. EVs were treated with Proteinase K and RNase A prior RNA isolation. Small RNA libraries were built and sequenced using Ion Proton and Illumina platforms. Specific miRNAs candidates were selected and validated by qPCR.Results and discussionsRNA deep sequencing analysis revealed that urinary EV RNA cargo is mainly comprised by miRNAs (51.68%), fragments of mRNAs (35.7%) and long non-coding RNAs (linc) (4.36%); while plasma EVs contained higher proportion of mRNA fragments (63.53%), miRNAs (12.31%) and processed pseudogenes (8.29%). Correlation analysis revealed low to moderate yet statistically significant correlation (≤0.4; p<0.001) between EV small RNA content and tumour tissue, while the correlation between EVs and normal prostate tissues was significantly lower. The highest correlation was observed between pre- and post-operation EV samples from the same biofluid (0.7–0.9; p<0.001). Among the different RNA species, miRNAs presented a moderate correlation with the tumour (≤0.3; p<0.001). A total of 822 different mature miRNAs were identified. A number of miRNAs overexpressed in tumour vs. adjacent normal tissue and present in urinary and plasma EVs were identified in each patient. From those, urinary derived miR-375 was validated and could serve as a biomarker for monitoring of PCa.ConclusionThis study provided a catalogue of small RNA species found in plasma and urinary EVs of PCa patients and revealed a number of miRNAs that are overexpressed in PCa and released in EVs.