MiRNA and proteomic profiling of small extracellular vesicles secreted by Toxoplasma gondii cultured with two different human cervical epithelial feeder layers.

Toxoplasma gondii has been reported to exert antitumor effects by inducing apoptosis and cell cycle arrest; however, the biological roles of small extracellular vesicles released by the parasite remain poorly understood. Small extracellular vesicles derived from parasite-host coculture systems may encapsulate molecular signals that reflect interactions between T. gondii and distinct epithelial microenvironments. In this study, tachyzoites of the T. gondii RH strain were cocultured with human cervical adenocarcinoma HeLa cells or normal cervical epithelial H8 cells, and small extracellular vesicles were isolated from coculture supernatants. High-throughput small RNA sequencing and liquid chromatography-tandem mass spectrometry were used to profile microRNA and protein cargo, followed by differential expression and functional enrichment analyses. Vesicles from both coculture systems contained components of both parasite and host origin, indicating bidirectional molecular exchange. Compared with vesicles derived from coculture with normal epithelial cells, those obtained from tumor cell coculture showed distinct enrichment of microRNAs and proteins associated with cell proliferation, DNA damage responses, and apoptotic regulation. Pathway analyses highlighted the involvement of cell cycle checkpoints, p53-related signaling, and programmed cell death. Overall, the molecular composition of parasite-derived vesicles varied according to the epithelial feeder layer, with tumor cell-associated vesicles exhibiting a stronger association with tumor-related regulatory pathways. These findings provide insight into parasite-host communication mediated by extracellular vesicles and suggest a potential role for T. gondii-derived vesicles in modulating tumor-associated biological processes.