Role of microRNA-335-5p in gastric cancer derived extracellular vesicles

Background: MicroRNA-335-5p (miR-335) has been reported to be dysregulated in various types of cancer, including gastric cancer (GC). Recently, we have shown the downregulation of miR-335 in GC tissues relative to their paired adjacent non-tumour tissues. We have also demonstrated that miR-335 is downregulated in plasma samples from GC patients. Here, we aimed to investigate the expression of miR-335 in GC derived extracellular vesicles (EVs) in plasma. Moreover, we focused on exosomes loaded with miR-335, their effect after incorporation in GC cells, and their biodistribution in vivo. Methods: EVs were isolated from a cohort of 12 plasma patients' samples, from supernatants from two GC cell lines, a primary tumour derived cell line AGS and metastatic derived cell line HS746T, and from cells transfected with miR-335 mimics and characterized by western blot and nanosight. MiR-335 expression levels in plasma EVs, cell lines, transfected cells and their EVs, as well as expression of target genes of miR-335, were analysed by qPCR. Incorporation of EVs into cells was quantified by flow cytometry. In biodistribution studies, EVs were labeled with fluorescent dye DiR, injected intravenously in the tail of mice (3 per condition) and their distribution in time was evaluated using in vivo visualizing machine. Brain, heart, lung, spleen, kidney, liver, stomach, colon, intestine and bone marrow were evaluated. Plasma samples were obtained with written informed consent from patients. Animal studies were approved by ethical committee. Results: Our cohort of patients show a tendency that plasma EVs isolated from GC patients contain less miR-335 when compared to healthy donors. In vitro data demonstrate that upon uptake of miR335-enriched EVs by GC cells, the expression of CDH11 and PLAUR is altered in a similar manner as these genes are regulated in GC cells transfected with miR-335. In vivo studies in mice shows, that after intravenous injection of these EVs labeled with DiR, EVs enriched in miR-335 show different distribution in time in several organs, including stomach, in comparison to control EVs. Summary/Conclusion: MiR-335 is present in EVs isolated from both plasma and GC cell culture supernatants. EVs enriched in miR-335 show functional properties after cell uptake and different biodistribution in mice.