Detection of circulating miRNAs : comparative analysis of extracellular vesicle-incorporated miRNAs and cell-free miRNAs in whole plasma of prostate cancer patients

Funding Information: This study was supported by the Norwegian Financial Mechanism 2009–2014 under Project Contract No NFI/R/2014/045. The funding body had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. Publisher Copyright: © 2017 The Author(s).Background: Circulating cell-free miRNAs have emerged as promising minimally-invasive biomarkers for early detection, prognosis and monitoring of cancer. They can exist in the bloodstream incorporated into extracellular vesicles (EVs) and ribonucleoprotein complexes. However, it is still debated if EVs contain biologically meaningful amounts of miRNAs and may provide a better source of miRNA biomarkers than whole plasma. The aim of this study was to systematically compare the diagnostic potential of prostate cancer-associated miRNAs in whole plasma and in plasma EVs. Methods: RNA was isolated from whole plasma and plasma EV samples from a well characterised cohort of 50 patient with prostate cancer (PC) and 22 patients with benign prostatic hyperplasia (BPH). Nine miRNAs known to have a diagnostic potential for PC in cell-free blood were quantified by RT-qPCR and the relative quantities were compared between patients with PC and BPH and between PC patients with Gleason score ≥ 8 and ≤6. Results: Only a small fraction of the total cell-free miRNA was recovered from the plasma EVs, however the EV-incorporated and whole plasma cell-free miRNA profiles were clearly different. Four of the miRNAs analysed showed a diagnostic potential in our patient cohort. MiR-375 could differentiate between PC and BPH patients when analysed in the whole plasma, while miR-200c-3p and miR-21-5p performed better when analysed in plasma EVs. EV-incorporated but not whole plasma Let-7a-5p level could distinguish PC patients with Gleason score ≥ 8 vs ≤6. Conclusions: This study demonstrates that for some miRNA biomarkers EVs provide a more consistent source of RNA than whole plasma, while other miRNAs show better diagnostic performance when tested in the whole plasma.publishersversionPeer reviewe

Characterization of small RNA content in urinary and plasma extracellular vesicles from prostate cancer patients

Ārpusšūnas vezikulas (EVs) nodrošina starpšūnu komunikāciju fizioloģiskos procesos un dažādās saslimšanās, ieskaitot ļaundabīgos audzējus. EV RNS satura izpēte dotu iespēju atklāt jaunus prostatas vēža (PCa) biomarķierus. Šis darbs tiek fokusēts uz mazo RNS raksturošanu, kas iegūtas no PCa pacientu plazmas un urīna EVs, kā arī no PCa un normāliem prostatas audiem, izmantojot RNS sekvencēšanu. Rezultāti uzrādīja, ka gan plazmas, gan urīna EVs satur matrices RNS fragmentus, mikro RNS, garās-nekodējošās RNS, piwi-saistošās RNS un citas nekodējošās RNS, taču to proporcijas ievērojami variē starp indivīdiem. Daļa šo RNS ir paaugstināti ekspresētas vēža audos un varētu būt piemērotas kā PCa biomarķieri. Divas mikro RNS tika izvēlētas validācijai ar RT-qPCR, kas apstiprināja to piemērotību kalpot kā PCa biomarķieriem.Extracellular vesicles (EVs) mediate intercellular communication in physiological processes and various diseases, including cancer. Studying EV RNA content would allow finding new prostate cancer (PCa) biomarkers. This work focuses on characterization of small RNA content of EVs isolated from plasma and urine of PCa patients, and tumor and non-tumor prostate tissues using RNA sequencing. Results showed that both, plasma and urinary EVs contain messenger RNA fragments, micro RNAs, long non-coding RNAs, piwi-interacting RNAs and other non-coding RNAs, however their proportions vary immensely between individuals. A fraction of these RNAs are overexpressed in tumor tissue and may represent novel PCa biomarkers. Two of these micro RNAs were chosen for validation using RT-qPCR that confirmed their suitability as PCa biomarkers

Comparative analysis of transcriptome in matched tumor tissues and extracellular vesicles from biofluids of prostate cancer patients

Visdažādāko veidu šūnas izdala ārpusšūnas vezikulas (EVs) un ir pierādīts, ka tām ir nozīmīga loma prostatas vēža (PCa) attīstībā, jo tās spēj pārnest dažādas bioloģiskās molekulas, tostarp RNS, no vienas šūnas uz otru. Šis darbs ir vērsts uz plazmas un urīna EV, kā arī PCa audzēja un normālu prostatas audu transkriptoma izpēti, izmantojot RNS sekvencēšanas (RNA-seq) tehnoloģiju. Rezultāti uzrādīja, ka 1868 proteīnus kodējošie gēni un 1108 garās nekodējošās RNS (lncRNS) bija paaugstināti ekspresētas audzēja audos salīdzinājumā ar normāliem prostatas audiem, taču tikai 6% līdz 31% proteīnus kodējošie gēni un 1% līdz 5% lncRNS bija atrodamas EVs, kas liecina, ka EV ietvertā RNS saturs tikai daļēji atspoguļo audzēja saturu. Neskatoties uz to, PCa pacientu plazmas un urīna EVs saturēja RNS, kas nākušas no PCa audzēja.Extracellular vesicles (EVs) are released by various types of cells and are shown to play a role in prostate cancer (PCa) development by transferring a variety of biological molecules, including RNAs from one cell to another. This work focuses on analyzing the transcriptome of plasma and urinary EVs and matched tumor and normal prostate tissues by using RNA sequencing (RNA-seq) technology. Results showed that in total 1868 protein-coding genes and 1108 long non-coding RNAs (lncRNA) were overexpressed in tumor tissues versus normal tissues. However, only 6% to 31% of overexpressed protein-coding genes and 1% to 5% of overexpressed lncRNAs were present in EVs, suggesting that EV-enclosed RNA content only partially reflects the tumor content. Nevertheless, PCa patient plasma and urinary EVs did contain a fraction of PCa-derived RNAs