Extracellular Vesicles Obtained From Lung Adenocarcinoma Cells Cultured Under Intermittent Hypoxia Induce M2 Macrophage Polarization via miR-20a-5p Delivery

Abstract

Introduction: Intermittent hypoxia (IH), an important feature of obstructive sleep apnea, enhances the function of lung cancer cell-derived extracellular vesicles (EVs) to exacerbate the immunosuppressive properties of macrophages. Herein, we investigated the effects of EVs obtained from lung adenocarcinoma cells cultured under IH on macrophage polarization. Methods: The M1-type and M2-type tumor-associated macrophages (TAMs) in tissues from lung adenocarcinoma cases with (n = 10) or without (n = 12) OSA were assessed by immunohistochemical studies. EVs obtained from A549 cells grown under normoxia (EV-NA) or IH (EV-IH) were isolated and cocultured with macrophages. MicroRNA sequencing was used to determine discrepant miRNAs in EVs, selecting miR-20a-5p for subsequent experiments. Next, reverse transcription-quantitative polymerase chain reaction, flow cytometry, luciferase reporter assay, western blotting assay, and gain- and loss-of-function assays were used to explore the mechanism by which miR-20a-5p promotes M2 macrophage polarization by targeting phosphatase and Tensin homolog gene (PTEN). Results: Stromal M2 TAMs were highly abundant in patients with lung adenocarcinoma and obstructive sleep apnea. Macrophages treated with EV-IH that highly expressed miR-20a-5p showed the M2 phenotype. Luciferase reporter assay confirmed PTEN as a target of miR-20a-5p. Transfection of miR-20a-5p mimics decreased PTEN expression, upregulated M2 polarization markers, and promoted Akt phosphorylation in macrophages, while transfection with a miR-20a-5p inhibitor had the opposite effects. Furthermore, miR-20a-5p inhibition in macrophages eliminated the PTEN downregulation, Akt phosphorylation, and upregulation of M2 polarization markers induced by EV-IH transfection. Conclusion: These findings indicate that EVs obtained from lung adenocarcinoma cells cultured under IH deliver miR-20a-5p to promote M2 macrophage polarization by targeting PTEN.