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Although aberrant microRNA (miRNA) is expressed in different types of human cancer tissues, its pathophysiologic role and the relevance of tumorigenesis and metastasis are still largely unknown.Here, we defined miRNAs involved in cancer metastasis (metastamirs) using an established mouse model for peritoneal dissemination of human scirrhous gastric carcinoma cells.Highly metastatic derivatives (44As3 cells) were derived from the parental cells originally isolated from patients (HSC-44PE cells).Using microarray analysis to identify differentially expressed miRNAs in 44As3 and HSC-44PE cells, we focused on miR-516a-3p as a candidate antimetastatic miRNA (antimetastamir) whose functions in cancer had not been studied.We confirmed attenuated expression of miR-516a-3p in 44As3 cells compared with HSC-44PE cells by Northern blot analysis and quantitative reverse transcriptase PCR.Stable ectopic overexpression in 44As3-miR-516a-3p cells permitted identification of sulfatase 1 as a direct target of the miRNA, through use of the isobaric tagging reagent iTRAQ and the QSTAR Elite Hybrid LC-MS/MS system.Sulfatase 1 is known to remove 6-O-sulfates from heparan sulfate proteoglycans on the cell surface, causing release of membrane-bound Wnt ligands from cells.Consistent with this function, Western blot analyses revealed high levels ofWnt3a, Wnt5a, and nuclear b-catenin accumulation in 44As3 cells but relatively reduced levels in 44As3-miR-516a-3p cells.Notably, orthotopic inoculation of nude mice with 44As3-miR-516a-3p cells yielded significantly longer survival periods compared with mice inoculated with control 44As3 cells.Through atelocollagen-mediated delivery of an miR-516a-3p expression vector into orthotopic 44As3 tumors, we documented its feasibility as a treatment agent.Our findings define the miRNA miR-516-3p as an antimetastamir with potential therapeutic applications in blocking metastatic dissemination of gastric cancers.