Deep Sequencing of RNA from Three Different Extracellular Vesicle (EV) Subtypes Released from the Human LIM1863 Colon Cancer Cell Line Uncovers Distinct Mirna-Enrichment Signatures
journal contributionposted on 08.03.2021, 23:39 by H Ji, Maoshan Chen, David GreeningDavid Greening, W He, A Rai, W Zhang, Richard SimpsonRichard Simpson
© 2014 Ji et al. Secreted microRNAs (miRNAs) enclosed within extracellular vesicles (EVs) play a pivotal role in intercellular communication by regulating recipient cell gene expression and affecting target cell function. Here, we report the isolation of three distinct EV subtypes from the human colon carcinoma cell line LIM1863 - shed microvesicles (sMVs) and two exosome populations (immunoaffinity isolated A33-exosomes and EpCAM-exosomes). Deep sequencing of miRNA libraries prepared from parental LIM1863 cells/derived EV subtype RNA yielded 254 miRNA identifications, of which 63 are selectively enriched in the EVs - miR-19a/b-3p, miR-378a/c/d, and miR-577 and members of the let-7 and miR-8 families being the most prominent. Let-7a-3p∗, let-7f-1-3p∗, miR-451a, miR-574-5p∗, miR-4454 and miR-7641 are common to all EV subtypes, and 6 miRNAs (miR-320a/b/c/d, miR-221-3p, and miR-200c-3p) discern LIM1863 exosomes from sMVs; miR-98-5p was selectively represented only in sMVs. Notably, A33-Exos contained the largest number (32) of exclusively-enriched miRNAs; 14 of these miRNAs have not been reported in the context of CRC tissue/biofluid analyses and warrant further examination as potential diagnostic markers of CRC. Surprisingly, miRNA passenger strands (star miRNAs) for miR-3613-3p∗, -362-3p∗, -625-3p∗, -6842-3p∗ were the dominant strand in A33-Exos, the converse to that observed in parental cells. This finding suggests miRNA biogenesis may be interlinked with endosomal/exosomal processing.
This work was supported by National Health and Medical Research Council of Australia (NHMRC) program grant #487922. MC and AR are supported by a La Trobe University Research Scholarship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Pagination15p. (p. 1-15)
PublisherPublic Library of Science
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Science & TechnologyMultidisciplinary SciencesScience & Technology - Other TopicsCOLORECTAL-CANCEREXPRESSION PROFILESSIGNALING PATHWAYSEXOSOMAL PROTEINSMICRORNA CLUSTERTUMOR-SUPPRESSORUP-REGULATIONMETASTASISPROLIFERATIONBIOMARKERSCell Line, TumorTransport VesiclesHumansColorectal NeoplasmsMicroRNAsRNA, Small InterferingDNA PrimersGenetic MarkersMicroscopy, Electron, TransmissionBlotting, WesternSequence Analysis, RNAComputational BiologyCell CommunicationGene Expression Regulation, NeoplasticBase SequenceMolecular Sequence DataExosomesHigh-Throughput Nucleotide SequencingReal-Time Polymerase Chain ReactionGene OntologyGeneral Science & Technology