title: 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.
abstract: 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.
authors.affiliation: La Trobe Institute for Molecular Science (LIMS), La Trobe University, Melbourne, Victoria, Australia.
authors.forename: Hong
authors.initials: H
authors.lastname: Ji
authors.affiliation: La Trobe Institute for Molecular Science (LIMS), La Trobe University, Melbourne, Victoria, Australia.
authors.forename: Maoshan
authors.initials: M
authors.lastname: Chen
authors.affiliation: La Trobe Institute for Molecular Science (LIMS), La Trobe University, Melbourne, Victoria, Australia.
authors.forename: David W
authors.initials: DW
authors.lastname: Greening
authors.affiliation: Chongqing Key Laboratory for Disease proteomics; and State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Burn Research, Southwest Hospital, Third Military Medical University, Chongqing, China.
authors.forename: Weifeng
authors.initials: W
authors.lastname: He
authors.affiliation: La Trobe Institute for Molecular Science (LIMS), La Trobe University, Melbourne, Victoria, Australia.
authors.forename: Alin
authors.initials: A
authors.lastname: Rai
authors.affiliation: BGI-Shenzhen, Shenzhen, China.
authors.forename: Wenwei
authors.initials: W
authors.lastname: Zhang
authors.affiliation: La Trobe Institute for Molecular Science (LIMS), La Trobe University, Melbourne, Victoria, Australia.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Simpson
mesh_terms: D001483:Base Sequence; D015153:Blotting, Western; D002450:Cell Communication / Q000235:genetics*; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000235:genetics*; D019295:Computational Biology; D017931:DNA Primers / Q000235:genetics; D055354:Exosomes / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D063990:Gene Ontology; D005819:Genetic Markers / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D017423:Sequence Analysis, RNA; D022161:Transport Vesicles / Q000235:genetics* / Q000648:ultrastructure
chemical_list: D017931:DNA Primers; D005819:Genetic Markers; D035683:MicroRNAs; D034741:RNA, Small Interfering