Characterization of RNA in exosomes secreted by human breast cancer cell lines using next-generation sequencing

Abstract

Exosomes are nanosized (30-100 nm) membrane vesicles secreted by most cell types. Exosomes have been found to contain various RNA species including miRNA, mRNA and long non-protein coding RNAs. A number of cancer cells produce elevated levels of exosomes. Because exosomes have been isolated from most body fluids they may provide a source for non-invasive cancer diagnostics. Transcriptome profiling that uses deep-sequencing technologies (RNA-Seq) offers enormous amount of data that can be used for biomarkers discovery, however, in case of exosomes this approach was applied only for the analysis of small RNAs. In this study, we utilized RNA-Seq technology to analyze RNAs present in microvesicles secreted by human breast cancer cell lines.Exosomes were isolated from the media conditioned by two human breast cancer cell lines, MDA-MB-231 and MDA-MB-436. Exosomal RNA was profiled using the Ion Torrent semiconductor chip-based technology. Exosomes were found to contain various classes of RNA with the major class represented by fragmented ribosomal RNA (rRNA), in particular 28S and 18S rRNA subunits. Analysis of exosomal RNA content revealed that it reflects RNA content of the donor cells. Although exosomes produced by the two cancer cell lines shared most of the RNA species, there was a number of non-coding transcripts unique to MDA-MB-231 and MDA-MB-436 cells. This suggests that RNA analysis might distinguish exosomes produced by low metastatic breast cancer cell line (MDA-MB-436) from that produced by highly metastatic breast cancer cell line (MDA-MB-231). The analysis of gene ontologies (GOs) associated with the most abundant transcripts present in exosomes revealed significant enrichment in genes encoding proteins involved in translation and rRNA and ncRNA processing. These GO terms indicate most expressed genes for both, cellular and exosomal RNA.For the first time, using RNA-seq, we examined the transcriptomes of exosomes secreted by human breast cancer cells. We found that most abundant exosomal RNA species are the fragments of 28S and 18S rRNA subunits. This limits the number of reads from other RNAs. To increase the number of detectable transcripts and improve the accuracy of their expression level the protocols allowing depletion of fragmented rRNA should be utilized in the future RNA-seq analyses on exosomes. Present data revealed that exosomal transcripts are representative of their cells of origin and thus could form basis for detection of tumor specific markers.

Extracellular Vesicle MicroRNAs: Biomarker Discovery in Various Diseases Based on RT-QPCR

“…Meanwhile, these observations were confirmed by similar research on lung cancer, in which the mean overall exosome concentration and exosome miRNA concentration was significantly different from controls, while the expression profile future science group Extracellular vesicle microRNAs Review of the previously identified 12 miRNAs in exosomes was comparable to tumor tissue-derived miRNAs [45]. By using RNA-seq, it was revealed that exosome miRNA content reflected miRNA content of the donor cells [46]. In addition, Let-7 miRNA family was shown to be enriched in both cellular and extracellular fractions (exosomes and culture media) of a metastatic gastric cancer cell line, AZ-P7a, but decreased in a low metastatic cell line, AZ-521.…”

Differential fates of biomolecules delivered to target cells via extracellular vesicles

“…Because tumor-derived exosomes contain fragmented ribosomal RNA (24) and genomic DNA (25-27), we anticipated fragmentation of the reporter mRNA in exosomes. We therefore examined the integrity of Luc-RFP mRNA in MVs via RT-PCR using four sets of primers along the Luc coding region, shown in Fig.…”

Exosomes derived from B16F0 melanoma cells alter the transcriptome of cytotoxic T cells that impacts mitochondrial respiration

“…These nanoscaled extracellular vesicles are actively released by cells to transfer proteins, mRNA, and noncoding RNAs between cells. Interestingly, tumor cells release exosomes at a greater rate than normal cells, which suggests that they may play an important role in cancer [15]. Some of the earliest exosome studies suggest that exosomes mediate B-cell transformations [16,17].…”