Background
Non-small-cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer cases and urgently requires the identification of novel biomarkers and mechanistic targets to control metastasis. In this study, we investigated whether exosome-mediated delivery of miR-338-3p inhibits NSCLC progression by suppressing Close Homolog of L1 (CHL1) expression and downstream MAPK signaling pathways.
 
Methods
The expression levels of CHL1 and miR-338-3p were quantified in NSCLC tissues, cell lines (A549 and SK-MES-1), and patient serum samples using qRT-PCR and Western blot analysis. Functional roles of CHL1 and miR-338-3p were evaluated through gain-of-function and loss-of-function experiments, including MTS proliferation assays and flow cytometry-based apoptosis detection. Dual-luciferase reporter assays were employed to confirm direct binding of miR-338-3p to the 3′ untranslated region (3′UTR) of CHL1. Exosomes isolated from serum samples were characterized via transmission electron microscopy (TEM) and immunoblotting for exosomal markers CD63 and TSG101. The intercellular transfer of exosomes derived from BEAS-2B epithelial cells to NSCLC cells was experimentally modeled, with pharmacological inhibition of exosome secretion achieved using GW4869. Activation status of the MAPK pathway was assessed by measuring protein levels of ERK5, JNK, MEK4, and p38.
 
Results
CHL1 was significantly upregulated in non-small cell lung cancer (NSCLC) tissues and cell lines, and its expression was positively associated with advanced disease stage and metastasis. Elevated CHL1 levels were correlated with poorer overall survival in The Cancer Genome Atlas (TCGA) cohorts. Functional studies demonstrated that CHL1 overexpression promoted cellular proliferation and inhibited apoptosis, whereas CHL1 knockdown exerted opposing effects and attenuated MAPK signaling activity. miR-338-3p was identified as a direct regulator of CHL1, with luciferase reporter assays confirming its binding to the CHL1 3' untranslated region. Inhibition of miR-338-3p enhanced NSCLC cell proliferation and reduced apoptosis, while restoration of miR-338-3p expression suppressed tumor growth and induced apoptotic responses. In a cohort of 66 patient samples, circulating exosomal miR-338-3p levels were significantly lower in NSCLC patients compared to healthy controls and were inversely correlated with pathological subtype, M classification, and lymph node metastasis. Exosomes derived from BEAS-2B cells effectively delivered miR-338-3p to A549 and SK-MES-1 cells, resulting in downregulation of CHL1 and suppression of MAPK signaling, which collectively led to reduced proliferation and increased apoptosis. Notably, treatment with GW4869, an exosome secretion inhibitor, abrogated this intercellular transfer and its functional consequences.
Conclusion
Exosomal miR-338-3p acts as a mobile tumor-suppressive molecule that targets CHL1 and suppresses MAPK signaling, thereby inhibiting NSCLC progression and metastasis. The miR-338-3p/CHL1/MAPK axis represents a promising mechanistic biomarker and a potential therapeutic target for exosome-based strategies in lung cancer management.