Urinary extracellular vesicles contain mature transcriptome enriched in circular and long noncoding RNAs with functional significance in prostate cancer

Abstract

Long noncoding (lnc)RNAs modulate gene expression alongside presenting unexpected source of neoantigens. Despite their immense interest, their ability to be transferred and control adjacent cells is unknown. Extracellular Vesicles (EVs) offer a protective environment for nucleic acids, with pro and antitumourigenic functions by controlling the immune response. In contrast to extracellular nonvesicular RNA, few studies have addressed the full RNA content within human fluids' EVs and have compared them with their tissue of origin. Here, we performed Total RNA-Sequencing on six Formalin-Fixed-Paraffin-Embedded (FFPE) prostate cancer (PCa) tumour tissues and their paired urinary (u)EVs to provide the first whole transcriptome comparison from the same patients. UEVs contain simplified transcriptome with intron-free cytoplasmic transcripts and enriched lnc/circular (circ)RNAs, strikingly common to an independent 20 patients' urinary cohort. Our full cellular and EVs transcriptome comparison within three PCa cell lines identified a set of overlapping 14 uEV-circRNAs characterized as essential for prostate cell proliferation in vitro and 28 uEV-lncRNAs belonging to the cancer-related lncRNA census (CLC2). In addition, we found 15 uEV-lncRNAs, predicted to encode 768 high-affinity neoantigens, and for which three of the encoded-ORF produced detectable unmodified peptides by mass spectrometry. Our dual analysis of EVs-lnc/circRNAs both in urines' and in vitro's EVs provides a fundamental resource for future uEV-lnc/circRNAs phenotypic characterization involved in PCa.

Keywords: CircRNA; EV; TSA; lncRNA; neoantigen.

FIGURE 1

uEVs are enriched in circRNAs and some lncRNAs. (a). Full transcriptome of paired liquid and solid biopsies of prostate cancer patients. Experimental procedure from prostate tumour biopsies and urines collections to RNA sequencing, through FFPE biopsies and uEVs isolation. (b). Mean gene expression of paired Tumour against uEVs (n = 83,980 RNAs if at least one counts, n = 21,397 if at least five counts for cirRNAs and 20 counts for lncRNAs and other RNAs). DEseq2 normalized counts, for each type of RNA are plotted; circRNAs (orange), lncRNAs (green), all other types of RNAs (grey). Each dot represents all transcripts for each gene. R² = 0.35 and R² = 0.16 for 2029 circRNAs and 3228 lncRNAs, respectively (> = 5 counts for cirRNAs, > = 20 counts for lncRNAs and other RNAs). (c). Density plot showing the distribution of log2 fold change uEVs/Tumour ratio per gene types. The right side of dotted line correspond to enriched genes in uEVs compared to Tumour tissues. The left side of dotted line correspond to the enriched genes in tumours compared to uEVs. Each colour represents an RNA type. Number of total differentially enriched genes in Tumour and uEVs are indicated on the right with the number of enriched RNA in EVs in bracket. (d). Interaction between the normalized number of counts of the 311 upregulated circRNAs (top) and 274 upregulated lncRNAs (bottom) in paired Tumour and uEVs for each patient (P1 to P6). Each dot corresponds to a circRNA or lncRNA upregulated in uEVs compared to Tumour with Log2 FC = 0,5–1 (blue), Log2 FC < 0,5 (grey), Log2 FC > 1 (red). The number of counts for the same RNA, in Tumour and in uEV are linked together with a line as shown for four circRNAs in the zoom window of patient 6. For each patient are indicated the numbers of circRNA and lncRNA with a log2(FC) > 1. (e). Heatmap display unsupervised hierarchical clustering with euclidean distance (CED) of 311 circRNAs (left) and 274 lncRNAs (right) up regulated in uEVs (FC > = 1.5, at least 20 counts) in each of the individual samples from six Tumour biopsies (green), paired six uEVs (black) and 20 independent uEVs (pink) from prostate cancer patients. Colour scales represent z‐scores of log10(TPM+1)

FIGURE 2

Intronic RNAs are depleted in uEVs compared to Tumour. (a). Genomic read counts distribution by percentage across exon, intron, 3′UTR, 5′UTR, intergenic and promoter in frozen, FFPE and uEVs biopsies. (b). Distribution of log10(exonic read counts/intronic read counts) normalized by length from Tumour (blue) and uEVs (red) samples for lncRNA (top, 2151 in tumours, 1393 in uEVs) and mRNA (bottom, 13,084 in tumours, 11,483 in uEVs) annotations. (c). Metagene of mean coverage for two first exons, last exon, first intron and last intron of 25,166 mRNAs and lncRNAs from Tumour (blue) and uEVs (red) samples. (d). GGbio‐generated RNA read profiling along minus (−; pink) and plus (+; bleu) strands of chr8:74198516–74398516 in Tumour and uEVs specimens. Arrow lines represent introns and rectangles represent exons of GENCODE‐annotated protein‐coding gene JPH1 (pink) and part of GDAP1. The maximum value of coverage, read count is shown in the left panel of read mapping. Some intronic reads are indicated for the two genes

FIGURE 3

Depleted lncRNAs in uEVs are nuclear. (a). Experimental procedure, starting from 22Rv1 cell line fractionation polyA RNA‐seq, to propose cytoplasmic or nuclear localization of up‐regulated genes in uEVs and up regulated genes in Tumour biopsies. (b). Stacked barplot distribution, by percentage, of cytoplasmic (blue), nuclear (red), both (yellow) or nonpolyA RNAs (grey) of up‐regulated genes in Tumour (8336) and up regulated genes in uEVs (4925). 15.4% and 57.8% upregulated RNAs, respectively, in tumours and uEVs are cytoplasmic; 23.6% and 2.9% are nuclear; 57.2% and 32% are both. (c). Density distribution of log2 (fold change cytoplasmic/nuclear ratio) per RNA types (7732 RNAs from Tumour and 4556 RNAs from uEVs), mRNA (purple), pseudogene (yellow), lncRNA (green) in Tumour (top) and uEVs (bottom). The left side of dotted line in both graphs corresponds to the nuclear RNAs, the right side corresponds to cytoplasmic RNAs

FIGURE 4

uEVs‐enriched circRNAs contain essential circRNAs and are common to PCa cell lines EVs. (a). Venn diagram showing number of over‐represented circRNAs in prostate cancer uEVs (n = 311), 171 essential circRNAs defined by Chen et al., circRNAs expressed (at least five DEseq normalized counts) in PC3, LNCaP and DU145 PCa cell lines (cell circRNAs, n = 704) and in cell‐EVs (cEVs circRNAs, n = 11,418). (b). List of the 14 essential circRNAs up‐regulated in uEVs c. Sequencing read coverage from back splicing of GOLPH3 circRNA, from chromosome 5:32124716–32174319, is shown using GGplot2 in Tumour, uEVs, PC3, LNCaP and DU145 cEVs and in cells. The maximum value of coverage read count is shown in the left panel of read mapping. Parental transcript ENST000000265070.7 is schematized by blue rectangles representing exons and black arrow lines representing introns (shrunk to 100 nt). Junction of back splicing is indicated in light blue.

FIGURE 5

uEVs‐enriched lncRNAs contain cancer lncRNAs and are common to PCa cell lines EVs. (a). Venn diagram showing number of over‐represented lncRNAs in prostate cancer uEVs (n = 274), 485 functional cancer lncRNAs defined in CLC2(Vancura et al., 2021), lncRNAs expressed (at least 20 DEseq normalized counts) in PC3, LNCaP and DU145 PCa cell lines (cell lncRNAs, n = 2312) and in cell‐EVs (cEVs lncRNAs, n = 3078). (b). List of the 28 CLC2 upregulated uEVs lncRNAs including 25 common with cEVs (black) and three exclusive to uEVs (grey). (c). Sequencing read coverage of PCAT6 lncRNA (strand +) is shown using GGplot2 in Tumour, uEVs, DU145‐, LNCaP‐ and PC3‐cEVs and in cells. The maximum value of coverage read count is shown in the left panel of read mapping. Metagene transcript is schematized by grey rectangles representing exons and arrow line representing introns

FIGURE 6

uEVs‐enriched RNAs show lncRNA‐encoding neoantigens shared by prostate cell lines. (a). Workflow of ORFs, peptides and neoantigens prediction from lncRNAs enriched in Tumour (n = 1993) and uEVs (n = 274). (b). Correlation between hydrophobicity and observed retention time for Neoantigen peptides (red dots), PC3 lncRNA peptides (blue dots and regression line with 95% confidence interval, R² = 0.482), peptides from lncRNAs upregulated in uEVs (cyan) and peptides derived from Uniprot‐annotated human proteins (grey dots and orange regression line, R² = 0.889) from three cell lines (PC3, LNCAP and HCT116 in N = 5 biological replicate). (c). Heatmap representing the relative expression by log10(TPM+1), of POLR2A mRNA and 15 uEVs‐neoLncRNAs, encoding the 351 strong unique neoantigens within uEVs, Tumour, PC3‐, LNCaP‐ and DU145‐cells and their respective EVs and RPF from PC3 ribosome profiling dataset. Highlighting of peptides by mass spectrometry is indicated by a cross and writing in red. (d). Example of EV‐neoLncRNA. IGV‐generated public PC3 prostate cancer cell line ribosome occupancy (Hsieh et al., 2012) and uEVs RNA‐seq profiling along plus (+, blue) and minus (−, pink) strands of POLR2A mRNA and (e). of AL354920.1 EV‐neoLncRNA. Blue arrow‐lines and rectangles represent introns and exons of metatranscripts, respectively. RPM, reads per million mapped reads. Open reading frames (ORFs, red rectangles), starting from AUG codon, of the most abundant transcripts for POLR2A and AL354920.1, are indicated. The sequence of one AL354920.1 ORF from the frame 1, generated with GGplot2, is presented and the regions of the 20 translated neopeptides are in blue and red. Red sequence is the strongest 9‐mer neoantigen