IADR Abstract Archives
Oral Squamous Cell Carcinoma-Derived Extracellular Vesicles Horizontally Transfer miRNA-21 to Human THP1 Monocytes and Activate NF-kB Signaling Pathway
Objectives: Extracellular vesicles (EVs) are heterogeneous vesicles shedding from almost every cell type and carry different bio-macromolecules. Evidence regarding molecular and cellular mechanisms underlying cancer development indicates that immune changes occur during cancer development and progression. Alcohol is considered as one of the risk factors of oral squamous cell carcinoma(OSCC) and associated with poor prognosis. We postulated that EVs released from OSCC cells after ethanol exposure can modify immune function in the monocytes.
Methods: CAL27 cells were treated with different concentration of ethanol (25mM,50mM,100mM) and total number of EVs in the supernatant and their diameter were quantified by Nanoparticle Tracking Analysis. For functional co-culture studies, EVs were purified from CAL27 (50mM ethanol-treated and control) supernatant using an established filtration and centrifugation-based protocol. Isolated EVs were characterized by electron microscopy and Western blot. miRNA cargos of purified EVs were characterized by TaqMan MicroRNA Assay. Isolated CAL27-dervied EVs were co-cultured with human THP1 monocytes. NF-kB activity was measured by electrophoretic mobility shift assay (EMSA).
Results: Ethanol treatment of CAL27 at different concentrations significantly increased number of shedding EVs after 24h and 48h of ethanol administration (p<0.05). EVs had a mean diameter of 348nm. Isolated EVs expressed both CD63 and phosphatidylserine on their surface. Normalizing the amount of purified RNA from EVs per number of parental cells, we found increased levels of miRNA-21 in the EVs derived from ethanol-treated cells (p<0.05). Co-culture of EVs derived from ethanol-treated CAL27 with THP1 monocytes for 6h resulted in NF-kB pathway activation and increase in inflammatory cytokines like IL6 and MCP1 (p<0.05) and macrophage polarization. Introducing synthetics miRNA-21 mimic resulted in the same phenotype of NF-kB activation and cytokine production, indicating the ability of miRNA-21 in NF-kB activation.
Conclusions: Alcohol exposure increased the number of OSCC-derived EVs and those EVs modulated immune function through horizontal transfer of miRNA-21 and NF-kB activation.
Methods: CAL27 cells were treated with different concentration of ethanol (25mM,50mM,100mM) and total number of EVs in the supernatant and their diameter were quantified by Nanoparticle Tracking Analysis. For functional co-culture studies, EVs were purified from CAL27 (50mM ethanol-treated and control) supernatant using an established filtration and centrifugation-based protocol. Isolated EVs were characterized by electron microscopy and Western blot. miRNA cargos of purified EVs were characterized by TaqMan MicroRNA Assay. Isolated CAL27-dervied EVs were co-cultured with human THP1 monocytes. NF-kB activity was measured by electrophoretic mobility shift assay (EMSA).
Results: Ethanol treatment of CAL27 at different concentrations significantly increased number of shedding EVs after 24h and 48h of ethanol administration (p<0.05). EVs had a mean diameter of 348nm. Isolated EVs expressed both CD63 and phosphatidylserine on their surface. Normalizing the amount of purified RNA from EVs per number of parental cells, we found increased levels of miRNA-21 in the EVs derived from ethanol-treated cells (p<0.05). Co-culture of EVs derived from ethanol-treated CAL27 with THP1 monocytes for 6h resulted in NF-kB pathway activation and increase in inflammatory cytokines like IL6 and MCP1 (p<0.05) and macrophage polarization. Introducing synthetics miRNA-21 mimic resulted in the same phenotype of NF-kB activation and cytokine production, indicating the ability of miRNA-21 in NF-kB activation.
Conclusions: Alcohol exposure increased the number of OSCC-derived EVs and those EVs modulated immune function through horizontal transfer of miRNA-21 and NF-kB activation.