Abstract Background: The SRC-family of kinases (SFKs) has been broadly implicated in the pathogenesis of prostate cancer. Fyn, a member of the SFKs, mediates mitogenic signals and regulates proliferation, adhesion and motility. Cumulative data indicate that Fyn serves as a proto-oncogene and is known to be over-expressed in several types of malignancies. MicroRNAs are noncoding RNAs that are aberrantly expressed in cancer and seem to influence tumor behavior and progression. In a previous study performed in human embryonic kidney (HEK 293T) cells, we have validated the binding specificity of miR-125a-3p and Fyn 3’UTR using Luciferase assay and established the regulation of Fyn expression and signaling by miR-125a-3p. In the current study we aimed to characterize the interplay between miRNA-125a-3p and Fyn in prostate cancer. Methods: Prostate cancer (PC3) cell line, were transiently transfected with empty miR-vec (control) or with miR-125a-3p and their cell cycle and apoptotic rate were measured by fluorescence activated cell sorting (FACS). The migration ability was measured by transwell assay and scratch assay. Proliferation was assessed by MTT assay. Quantitative PCR and western blot analysis were implicated to determine the effect of miR-125a-3p on Fyn expression as well as on the activation state of its downstream effectors/proteins FAK, Akt and paxillin. Extraction of miRNA from formalin fixed paraffin-embedded (FFPE) cancerous and matched adjacent prostate tissues was performed with miRNeasy FFPE Kit on a cohort of 20 human prostate cancer specimens. Results: Overexpression of miR-125a-3p in PC3 cells reduced significantly the expression of Fyn mRNA and protein and led to impaired cell viability to an arrest at the G2/M phase of the cell cycle. Moreover, overexpressing of miR-125a-3p reduced the activity of FAK, Akt and paxillin and the secretion of VEGF to the culture media. In miRNA125a-3p-overexpressing PC3 cells the migration was compromised by 50% and cell viability decreased by 50%. Moreover, we observed an inverse correlation between the expression of miR-125a-3p and the Gleason score of tissue samples obtained from patients diagnosed with prostate cancer, whereas the expression of miR-125a-3p was extremely reduced in Gleason score higher than 8. Conclusions: Our study indicates miR-125a-3p signature in cellular pathways accounted for cell viability and migration of prostate cancer cells. The interplay between miR-125a-3p and Fyn may represent a new plausible therapeutic target in prostate cancer. Citation Format: Lihi Ninio Many, Irit Ben-Aharon, Sofia Zilber, Mattan Levy, Salomon M. Stemmer, Ruth Shalgi. The role of miRNA-125a-Fyn interplay in prostate cancer tumorigenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4168. doi:10.1158/1538-7445.AM2013-4168