Identification of a CSC-Associated miRNA Signature in NSCLC and Functional Characterization of hsa-let-7a-3p

Background: Metastasis is the leading cause of mortality in lung cancer and is regulated by multiple molecular mechanisms, including microRNAs (miRNAs). Although cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT) contribute to metastatic progression, the miRNA networks underlying these phenotypes remain poorly characterized in non-small cell lung cancer (NSCLC). Aim: To identify miRNA signatures associated with CSCs and EMT in NSCLC and functionally characterize hsa-let-7a-3p. Methods: EMT was induced in A549 and NCI-H1975 cells by dCas9-mediated activation of TWIST, whereas CSC-enriched populations were generated by CD133-based sorting and stem cell culture conditions. Small RNA sequencing, bioinformatic analyses, qPCR validation, and functional assays were performed to identify and characterize phenotype-associated miRNAs. Results: Small RNA sequencing identified distinct miRNA expression profiles associated with EMT and CSC enrichment. Comparative analysis identified 13 commonly downregulated and 13 commonly upregulated miRNAs shared by CSCs from A549 and H1975 cells, suggesting conserved post-transcriptional regulatory mechanisms. Functional enrichment and miRNA-target interaction network analyses linked the miRNA signatures to pathways involved in epithelial plasticity, stemness, and tumor progression, including Wnt, TGF-β, mTOR, focal adhesion, adherens junction, and regulation of the actin cytoskeleton. Among the dysregulated miRNAs, hsa-let-7a-3p was consistently upregulated in CD133⁺ CSC-enriched cells from both NSCLC cell lines. Functional assays showed that hsa-let-7a-3p overexpression significantly reduced clonogenic capacity and showed a trend toward decreased invasion without affecting proliferation. Conclusion: This study identifies miRNA signatures associated with CSC-enriched and EMT-associated phenotypes in NSCLC and demonstrates that these signatures represent coordinated post-transcriptional regulatory programs involved in epithelial plasticity, stemness, and metastatic progression. Functional validation of hsa-let-7a-3p further supports its role as a context-dependent regulator of CSC biology and highlights the potential of miRNA signatures as diagnostic biomarkers in NSCLC.