Molecular Profiling of A549 Cell-Derived Exosomes: Proteomic, miRNA, and Interactome Analysis for Identifying Potential Key Regulators in Lung Cancer

Simple Summary: Exosomes are small extracellular vesicles released by all cells that play a crucial role in cell-to-cell communication. Exosomes derived from cancer cells carry tumorigenic properties, transmitting these signals to surrounding cells within the tumor microenvironment or to distant sites. However, the complexity of exosome cargo remains poorly understood. In this study, we isolated exosomes from human lung adenocarcinoma A549 cell cultures, characterized their physicochemical and morphological properties, and analyzed their proteomic and miRNA cargo through high-throughput profiling and bioinformatics. Key molecules within the exosomal cargo were identified, and network analysis revealed their potential effects on the physiology of potential recipient cells. Furthermore, we compared the miRNA profiles of malignant A549 exosomes with those from normal lung fibroblast MRC-5 cells, identifying tumor-associated miRNAs for potential biomarker exploration in clinical samples. This study advances our understanding of exosomal molecular components and their interactions, opening avenues for further research into their roles in cancer. Background/Objectives: Exosomes, nano-sized extracellular vesicles released by all cells, play a key role in intercellular communication and carry tumorigenic properties that impact surrounding or distant cells. The complexity of the exosomal molecular interactome and its effects on recipient cells still remain unclear. This study aims to decipher the molecular profile and interactome of lung adenocarcinoma A549 cell-derived exosomes using multi-omics and bioinformatics approaches. Methods: We performed comprehensive morphological and physicochemical characterization of exosomes isolated from cell culture supernatant of A549 cells in vitro, using DLS, cryo-TEM, Western blot, and flow cytometry. Proteomic and miRNA high-throughput profiling, coupled with bioinformatics network analysis, were applied to elucidate the exosome molecular cargo. A comparative miRNA analysis was also conducted with exosomes derived from normal lung fibroblast MRC-5 cells. Results: Exosomes exhibited an average size of similar to 40 nm and disk-shaped lipid bilayer structures, with tetraspanins CD9 and CD63 validated as exosomal markers. Proteomic analysis identified 68 proteins, primarily linked to the extracellular matrix organization and metabolic processes. miRNA sequencing revealed 72 miRNAs, notably hsa-miR-619-5p, hsa-miR-122-5p, hsa-miR-9901, hsa-miR-7704, and hsa-miR-151a-3p, which are involved in regulating metabolic processes, gene expression, and tumorigenic pathways. Th integration of proteomic and miRNA data through a proteogenomics approach identified dually affected genes including ERBB2, CD44, and APOE, impacted by both exosomal miRNA targeting and protein interactions through synergistic or antagonistic interactions. Differential analysis revealed a distinct miRNA profile in A549 exosomes, associated with cancer-related biological processes, compared to MRC-5 exosomes; notably, hsa-miR-619-5p emerged as a promising candidate for future clinical biomarker studies. The network analysis also revealed genes targeted by multiple upregulated tumor-associated miRNAs in potential exosome-recipient cells. Conclusions: This integrative study provides insights into the molecular interactome of lung adenocarcinoma A549 cell-derived exosomes, providing a foundation for future research on exosomal cargo and its role in tumor cell communication, growth, and progression.