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 ~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.
背景/目的：外泌体是由所有细胞释放的纳米级细胞外囊泡，在细胞间通讯中发挥关键作用，并携带影响周围或远处细胞的致瘤特性。外泌体分子相互作用组的复杂性及其对受体细胞的影响尚不清楚。本研究旨在通过多组学和生物信息学方法，解析肺腺癌A549细胞来源外泌体的分子谱和相互作用组。方法：我们利用动态光散射、冷冻透射电镜、蛋白质印迹和流式细胞术，对体外培养的A549细胞上清液中分离的外泌体进行了全面的形态学和理化表征。通过蛋白质组学和miRNA高通量分析，结合生物信息学网络分析，阐明外泌体分子载物。同时，与正常肺成纤维细胞MRC-5来源的外泌体进行了miRNA比较分析。结果：外泌体平均尺寸约为40纳米，呈盘状脂质双层结构，四跨膜蛋白CD9和CD63被验证为外泌体标志物。蛋白质组学分析鉴定出68种蛋白质，主要与细胞外基质组织和代谢过程相关。miRNA测序揭示了72种miRNA，其中hsa-miR-619-5p、hsa-miR-122-5p、hsa-miR-9901、hsa-miR-7704和hsa-miR-151a-3p尤为突出，参与调控代谢过程、基因表达和致瘤通路。通过蛋白质基因组学方法整合蛋白质组和miRNA数据，鉴定出受外泌体miRNA靶向和蛋白质相互作用双重影响的基因，包括ERBB2、CD44和APOE，这些影响通过协同或拮抗相互作用实现。差异分析显示，与MRC-5外泌体相比，A549外泌体具有独特的miRNA谱，与癌症相关生物过程相关；值得注意的是，hsa-miR-619-5p成为未来临床生物标志物研究的有前景候选分子。网络分析还揭示了潜在外泌体受体细胞中受多个上调肿瘤相关miRNA靶向的基因。结论：这项整合性研究深入揭示了肺腺癌A549细胞来源外泌体的分子相互作用组，为未来研究外泌体载物及其在肿瘤细胞通讯、生长和进展中的作用奠定了基础。
肿瘤(癌症)患者之家