DEHP-elicited small extracellular vesicles miR-26a-5p promoted metastasis in nearby normal A549 cells.

Small extracellular vesicles (sEV) are small lipid bilayer particles released by cells. sEV have been shown to play critical roles in intercellular communication. Di (2-ethylhexyl) phthalate (DEHP), widely used as plasticizers, has been detected in the environment and human beings. DEHP was found to exist in the air particles and showed pulmonary toxicity. However, there's little knowledge about the role of sEV in mediating the toxicity of DEHP-induced lung toxicity. We hypothesized that sEV mediated the toxicity of DEHP through their cargo. To validate this, lung epithelial cells (A549) were exposed to various concentrations (0, 0.2, 2 and 20 μM) of DEHP for 48 h. sEV extracted from DEHP-exposed A549 cells were cultured with unexposed A549 cells. Results showed that DEHP induced the epithelial-mesenchymal transition (EMT) and promoted the migration and invasion ability of A549 cells. The number of released sEV significantly increased in the culture media in DEHP-exposed groups compared to unexposed groups. The sEV can enter the unexposed A549 cells and enhance its EMT and the ability of migration and invasion. Treatment with GW4869 in DEHP-exposed A549 cells almost blocked the effects of DEHP-elicited sEV in normal A549 cells. Sequencing and functional analysis showed that the enrichment of significantly differentially expressed sEV miRNAs were related to tumor etiology. MiR-26a-5p was significantly enriched in DEHP-elicited sEV. Inhibition of miR-26a-5p in DEHP-exposed cells led to the downregulation of miR-26a-5p in sEV, and thus abolished the effects of DEHP-elicited sEV in normal A549 cells, whereas overexpression of miR-26a-5p restored the effects. The transcription factors twist is one of the downstream targets in the effects of sEV-miR-26a-5p on EMT process. In all, our results showed that DEHP exposure promoted the secretion of miR-26a-5p in sEV, which subsequently enhanced the EMT, migration and invasion ability in neighboring normal cells via the twist.