Tumor-associated macrophages secret exosomal miR-155 and miR-196a-5p to promote metastasis of non-small-cell lung cancer

BACKGROUND: Understanding the molecular basis underlying metastasis of non-small cell lung cancer (NSCLC) may provide a new therapeutic modality for the treatment of NSCLC. However, the mechanisms by which tumor-associated macrophages (TAMs) affect NSCLC metastasis remain undefined. In this study, we aimed to discover a novel regulatory pathway involved in NSCLC metastasis. METHODS: Cell Counting Kit-8 (CCK-8), Transwell, western blot assays were used to assess cell viability, migration, invasion and epithelial-mesenchymal transition (EMT). Exosomes from macrophages medium were characterized, and in vitro cell coculture was further conducted to investigate M2 derived exosomes mediated crosstalk between TAMs and tumor cells. Besides, miRNA microarray was used to analyze miRNA expression profiles of M0 and M2 derived exosomes. Luciferase reporter assay was used to verify the potential binding between miRNA and mRNA. Moreover, 6-week-old male BALB/c nude mice were performed to establish transplantation tumor model using tail vein injection. Hematoxylin & eosin staining was used to detect the metastasis of tumor tissues. RESULTS: We found that M2 TAMs were the main TAMs in metastatic tissues of NSCLC patients and exosomes derived from M2 TAMs were able to promote cell viability, cell migration, cell invasion and EMT in NSCLC. We demonstrated that miR-155 and miR-196a-5p were abundant in M2 TAMs and exosomes secreted by M2 TAMs. Functional experiments demonstrated that the deletion of miR-155 and miR-196a-5p in M2 TAMs significantly prevented NSCLC metastasis in vitro and in vivo. To clarify the mechanism governing miR-155 and miR-196a-5p from M2 TAMs, we carried out bioinformatics analysis to predict potential target genes. Mechanistically, miR-155 and miR-196a-5p directly bound to the 3'-UTR of Ras association domain family member 4 (RASSF4), and negatively regulating RASSF4 expression. At last, rescue assays demonstrated that miR-155 and miR-196a-5p exerted its performance by RASSF4. CONCLUSIONS: Overall, we revealed a new regulatory pathway that was M2 TAMs secreted exosomal miR-155 and miR-196a-5p to promote NSCLC metastasis. This dynamic and reciprocal cross-talk between NSCLC and macrophages innovatively provided a potential opportunity for diagnosis and treatment of NSCLC.