PB0932 - miR-5683 from gastric cancer cells inhibits endothelial TFPI contributing to thrombotic risk in patients with gastric cancer

Background: Advanced gastric cancer (AGC) has a high incidence of venous thromboembolism (VTE). We previously described genes whose differential expression correlated with VTE in primary tumor from 100 AGC patients from Agamenon registry (PMID: 35052827). One gene overexpressed among VTE patients corresponded to a microRNA, miR-5683, whose association with thrombosis is unknown.

Aims: To investigate the link between miR-5683 and VTE in AGC.

Methods: In silico approach by using TargetScanHuman, revealed TFPI as a target of miR-5683. EA.hy926 endothelial cells were transfected with miR-5683 mimic and a scrambled control (SCR). Transfections were verified by RT-qPCR. Expression of secreted TFPI was analyzed by western-blot. Extracellular vesicles (EVs) were isolated by ultracentrifugation of secretomes from AGS and Kato-III gastric cancer cells transfected with miR-5683 mimic and SCR. The presence of miR-5683 in these EVs was checked by RT-qPCR. EVs from transfected AGS were incubated with EA.hy926 and the intracellular incorporation of miR-5683 was checked by RT-qPCR, and the intracellular and secreted TFPI by western-blot.

Results: Transfecting different concentrations of miR-5683 mimic significantly increased its expression in EA.hy926 (Figure-1A). miR-5683 reduced TFPI secreted by EA.hy926 in a dose-dependent manner, being the differences significant between 50 nM miR-5683 and SCR (Figure-1B). mir-5683 increased in EVs from AGS and Kato-III cells transfected with miR-5683 mimic, compared to EVs from cells transfected with SCR (Figure-2). Finally, EA.hy926 exposed to EVs from AGS cells transfected with miR-5683 mimic increased the intracellular expression of this miRNA (Figure-3A), which reduced intracellular and secreted TFPI (Figure-3B).

Conclusion(s): TFPI is a target of miR-5683, which is secreted from gastric cancer cells through EVs that are able to transfer their content to endothelial cells, reducing TFPI expression. Thus, we hypothesize increased levels of miR-5683 in gastric cancer increase the thrombotic risk in AGC patients through the reduction of TFPI by intercellular exchange between tumor cells and endothelium.