N.OT04.Distinct mechanisms of microRNA sorting into cancer cell-derived extracellular vesicle subtypes Morayma Temoche-Diaza, Matthew Shurtleffa, Ryan Nottinghamb, Jun Yaob, Alan Lambowitzb, Randy SchekmanaaOT04.Identification of EV secretion-associated gene involved in melanoma progression by microRNA-based screening Nobuyoshi Kosakaa, Fumihiko Urabeb, Tomofumi Yamamotoc, Yurika Sawad and Takahiro Ochiyaa Department of Molecular and Cellular Medicine, Institute of Healthcare Science, Tokyo Healthcare University, Shinjyuku-ku, Japan; bDivision of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tokyo, Japan; cDepartment of Molecular and Cellular Medicine, Institute of Medical Science, Tokyo Medical University, Tokyo, Japan; d Department of Molecular and Cellular Medicine, Institute of Medical Science, Tokyo Health-related University, Tokyo, JapanaUniversity of California, Berkeley, Berkeley, USA; bUniversity of Texas, Austin, Austin, USAIntroduction:It has been shown that extracellular vesicles (EVs) derived from cancer cells dictate their surrounding microenvironmental cells or distant cells inside the future metastatic organs for the benefit of cancer cells. Hence, revealing the molecular mechanisms underlying the production of EVs would prove to become a precious contribution for establishing EV-targeted therapy against cancer. However, the precise mechanism of EV production, particularly in cancer cells, remains unclear. Here, we established a microRNA-based screening system to determine the molecules involved in EV production from melanoma cells. Solutions: Melanoma cell lines, A375 cells, have been employed in this study. Combined with the ultra-sensitive EV detection strategy (Yoshioka), ExoScreen, we have screened nearly 2000 GP-Ib alpha/CD42b Proteins web miRNAs in melanoma cells. To confirm the results of ExoScreen, we employed the nanoparticle tracking analysis. Target genes of miRNAs were identified by the combination of gene expression evaluation and target prediction bioinformatics.Introduction: Extracellular vesicles (EVs) encompass a range of vesicles secreted to the extracellular space. EVs happen to be implicated in advertising tumour metastasis but the molecular compositions of tumourderived EV sub-types along with the mechanisms by which molecules are sorted into EVs remain largely unknown. As such dissecting different EV sub-populations and analysing the molecular mechanisms behind active cargo sorting is required. Methods: The highly metastatic breast cancer cell line, MDA-MB-231, was used as the model cell line for this study. Iodixanol linear gradient allowed for the separation of EV sub-populations. miRNA profiling and TGIRTsequencing was CD252/OX40 Ligand Proteins custom synthesis utilised to study the miRNA content in the distinct EV sub-populations. Cell fractionation and cell-free miRNA packaging reconstitutions, coupled with in vivo confirmation, in cultured cells, had been utilised to study the molecular mechanisms of miRNA sorting. Benefits: We found that a minimum of two distinct EV subpopulations are released by MDA-MB-231 cells. Their differential biochemical properties recommend diverse subcellular origins (endosomes vs. direct budding in the plasma membrane). Moreover, they are governed by distinct mechanisms of miRNA sorting (active vs. passive). By utilizing biochemical and genetic tools, we identified that the Lupus La protein is responsible for mir122 sorting into EVs in vitro and in vivo. Furthermore, in vitro studiesJOURNAL OF EXTRACELLULAR VESICLESshowed that the Lupus La protein interacts with mir122 with incredibly higher af.
