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Cellular and extracellular vesicle miRNA profiles in GFP-PC3 and cavin-1-PC3 cell lines.

ABSTRACT: This study assessed exosomal and cellular miRNA profiles in GFP-PC3 cells and ectopically expressed cavin-1 PC3 cell lines. To understand the consequences of cavin-1 on extracellular vesicle (EV)-contained miRNAs, a small RNA-seq experiment was performed on cells and secreted EVs for GFP-PC3 and cavin-1-PC3 cells. Small RNA were selected for sequencing using the NEBNext Small RNA library Prep Kit. Libraries were size selected using a Perkin Elmer Labchip XT. Whole cell and EV libraries were multiplexed from three biological replicates and sequenced on an Illumina NextSeq500 mid-output run at a read depth of 1million reads per sample. While the raw data was mapped to the human miRNAome using RNA-STAR and miRBase (v21) as reference, 317 mature miRNAs were identified in EVs at varying abundances depending on cell of origin. The 317 miRNAs identified in both cell and EVs were further processed. After RUVseq normalization, DESeq2 analysis was used to identify differences between cellular miRNA profiles and EV miRNA profiles from the two different cell lines. This analysis ultimately defined the repertoire of selectively and passively exported exosomal miRNAs mediated by non-caveolar caveolin-1 and cavin-1 in a prostate cancer cell line. Aim: To assess exosomal miRNA compositional changes evoked by ectopic expression of cavin-1 in the PC3 advanced prostate cancer cell line. Objectives: 1. Determine differential expression of miRNAs in cells and 2. determine differential abundance of miRNAs in extracellular vesicles secreted from cavin-1 expressing or GFP expressing cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE109356 | GEO | 2021/01/14

REPOSITORIES: GEO

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Project description:Purpose: The goal of this study was to determine the microRNA (miRNA) content of extracellular vesicles (EVs) derived from murine mesenchymal stem cells (mMSC), and evaluate reproducibility among distinct EV productions. We also aimed at assessing the effect of freeze-drying on EV miRNA content, by performing sequencing on freeze-dried EVs and calculating statistical difference between unmodified and freeze-dried EVs. Methods: mMSC-derived EVs were obtained from mMSC in culture in reduced serum medium Opti-MEM by differential centrifugation, with a final step at 100,000 g for 110 min at 4°C. EV pellets (freeze-dried (n=3) or not (n=2)) were resuspended in Qiazol lysis buffer and RNA was extracted following RNeasy Micro kit. cDNA libraries for sequencing were prepared using the TruSeq Small RNA Sample Preparation Kit. Amplified cDNA constructs were purified on 6 % PAGE gel and DNA molecules corresponding to 15–50 nucleotide transcripts were excised, eluted from gel, and concentrated. Image analyses and base calling were performed using the HiSeq Control Software and Real-Time Analysis component (Illumina). Before statistical analysis, genes with less than 15 reads (cumulating all the analysed samples) were filtered out. Differentially expressed miRNA were identified using three Bioconductor packages: edgeR, DESeq and DESeq2. Results: Considering miRNAs detected with at least 5 counts (in terms of normalised counts), 339 miRNAs were identified and miRNA content was highly conserved among the two batches tested, with 237 miRNAs out of the 339 present in both batches (70%). Statistical analysis did not evidence statistical difference between unmodified EVs (n=2) and freeze-dried EVs (n=3) (DESeq2, p<0.05). No statistical difference was found using other Bioconductor packages DESeq and edgeR. These results indicated conservation of miRNA content following freeze-drying. Conclusion: mMSC-EV miRNA content was comparable between the two EV productions analysed, indicating reproducibility. Some of the miRNAs identified were consistent with previously published results on MSC-derived EVs. Freeze-drying conserved miRNA content.

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Cellular and extracellular vesicle miRNA profiles in GFP-PC3 and cavin-1-PC3 cell lines.

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