Mechanism of exosomal microRNA-1234-3p of lung cancer cells in promoting immune escape by regulating functions of tumor-associated macrophages and T lymphocytes

Objective To investigate the effects of exosomal microRNA-1234-3p (miR-1234-3p) of lung cancer cells on the malignant behaviors of lung cancer cells, the polarization of tumor-associated macrophages (TAMs), and the functions of T cells, as well as its potential molecular mechanisms. Methods Exosomes were extracted from lung cancer A549 cells, and their morphology was observed using transmission electron microscopy. The expression of exosomal marker proteinstumor susceptibility gene 101 (TSG101), CD63 and CD9was detected by western blot. The relative expression level of miR-1234-3p was measured using real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). The expression of exosomal miR-1234-3p was inhibited by transfecting, the proliferation, migration, and invasion abilities of A549 cells were detected using the CCK-8 method, wound healing assay, and Transwell invasion assay, respectively. The exosomes were co-cultured with macrophages and T lymphocytes separately. The expression of TAM polarization markers CD206 and CD163 was detected by western blot. The secretion levels of cytokinesinterleukin (IL)-6, IL-10, and transforming growth factor-β (TGF-β)were measured using an enzyme-linked immunosorbent assay (ELISA). The proliferation and apoptosis abilities of T lymphocytes, as well as the expression of functional moleculesγ-interferon (IFN-γ) and programmed death receptor 1 (PD-1) were detected through cell experiments. The expression levels of proteins related to the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway were detected by western blot. Results The isolated exosomes were round double-layer vesicles with a diameter of 50 to 200 nm and expressed TSG101, CD63 and CD9 proteins. The expression level of miR-1234-3p in A549 cells was higher than that in human normal lung epithelial cells BEAS-2B, and the expression level of miR-1234-3p in A549 cell exosomes was higher than that in parental A549 cells, with statistically significant differences (P < 0.001). After inhibiting exosomal miR-1234-3p, the proliferation, migration, and invasion abilities of A549 cells decreased, with statistically significantdifferences(P < 0.001). After inhibiting exosomal miR-1234-3p, the levels of M2-type polarization marker proteins CD206 and CD163 in macrophages decreased, the levels of cytokines IL-10 and TGF-β decreased, and the level of IL-6 increased, with statistically significant differences (P < 0.001), indicating that the polarization of macrophages toward the M2 type was inhibited. In T lymphocytes, after inhibiting exosomal miR-1234-3p, cell viability increased, the apoptosis rate decreased, the expression level of the functional molecule IFN-γ increased, and the expression level of PD-1 decreased, with statistically significant differences (P < 0.01 or P < 0.001). After inhibiting exosomal miR-1234-3p, the protein levels of phosphorylated (p)-PI3K and p-AKT in A549 cells decreased, with statistically significant differences (P < 0.001). Conclusion Exosomal miR-1234-3p of lung cancer cells may promote the malignant behaviors of lung cancer cells, the polarization of TAMs toward the M2 type, and the dysfunction of T lymphocytes by activating the PI3K/AKT signaling pathway, thereby regulating the immunosuppressive microenvironment and promoting the progression of lung cancer.