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.
University of Helsinki researchers 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.
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.
Comparison of the differences in the mRNA levels associated with MVs and EXOs
a Non-supervised hierarchical clustering indicating normalized enrichment of the mRNA levels of the 46 most significant genes detected in the LNCaP and PC-3 MVs and EXOs in comparison with the PNT2 MVs or PNT2 EXOs. Volcano plots indicating changes in mRNA levels between: (b) LNCaP MVs vs PNT2 MVs; (c) LNCaP EXOs vs PNT2 EXOs; (d) PC-3 MVs vs PNT2 MVs; and (e) PC-3 EXOs vs PNT2 EXOs. Data is representative of three independent experiments per group. Data is reported as x-axis = log2 (fold change of sample/Ctrl), y-axis = negative log10 (P value). The horizontal blue line indicates a P value of 0.05. The vertical grey line indicates an absolute fold-change of 10. Red and green dots indicate increased or decreased mRNA levels of the genes of study in the MVs and EXOs in comparison to the PNT2 MVs and EXOs
This 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.