MicroRNAs of Bone Marrow Mesenchymal Stem Cell-Derived Exosomes Regulate Acute Myeloid Leukemia Cell Proliferation and Apoptosis
Acute myeloid leukemia (AML) is a malignant hematologic disorder characterized by impaired differentiation and uncontrolled proliferation of myeloid precursor cells, leading to bone marrow failure. Despite advancements in chemotherapy and hematopoietic stem cell transplantation, therapeutic outcomes remain suboptimal due to drug resistance and high relapse rates. Emerging evidence suggests that exosomes, nano-sized extracellular vesicles secreted by cells, play pivotal roles in intercellular communication and disease progression. Bone marrow mesenchymal stem cells (BMSCs) are a rich source of exosomes, which carry functional biomolecules such as microRNAs (miRNAs) that may modulate cancer cell behavior. This study investigates the therapeutic potential of BMSC-derived exosomes in AML and elucidates the role of specific miRNAs in regulating leukemic cell proliferation and apoptosis.
Isolation and Characterization of BMSC-Derived Exosomes
Exosomes were isolated from BMSCs and AML-derived KG-1a cells using polyethylene glycol-based precipitation. Validation via Western blot confirmed the presence of exosomal markers TSG101, HSPA8, and Alix in both BMSC- and KG-1a-derived exosomes (Figure 1A). Nanoparticle tracking analysis (NTA) revealed exosome diameters ranging from 50–100 nm, consistent with typical exosome size (Figure 1B). These results confirmed the successful isolation of exosomes with characteristics suitable for downstream functional analyses.
BMSC-Derived Exosomes Suppress AML Cell Proliferation and Cell Cycle Progression
To evaluate the impact of BMSC-derived exosomes on AML cells, KG-1a cells were treated with varying exosome concentrations (1–20 µg/mL) for 48 hours. The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay demonstrated a dose-dependent suppression of KG-1a proliferation. At 10 µg/mL and 20 µg/mL, exosomes reduced proliferation by 50% and 60%, respectively (P < 0.0001 vs. untreated controls; Figure 2A). Flow cytometry revealed significant G0/G1 phase arrest (70% vs. 55% in controls; P < 0.01) and reduced S-phase entry (15% vs. 25%; Figure 2B), indicating that exosomes halted cell cycle progression. Furthermore, apoptosis rates in KG-1a cells increased from 5% in controls to 25% after exosome treatment (P < 0.001; Figure 2C). These findings established 10 µg/mL as the optimal therapeutic concentration for subsequent experiments.
Differential miRNA Profiles in BMSC-Derived Exosomes
High-throughput RNA sequencing identified 1,167 differentially expressed miRNAs between BMSC- and KG-1a-derived exosomes, with 153 upregulated and 1,014 downregulated miRNAs (Figure 3A–B, Supplemental Table 1). Hierarchical clustering and scatter plots highlighted distinct miRNA profiles, underscoring the unique molecular cargo of BMSC-derived exosomes. Gene ontology (GO) analysis linked target genes of differentially expressed miRNAs to critical processes such as cell proliferation, apoptosis, and cell cycle regulation (Figure 3C). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis further implicated these miRNAs in cancer-associated pathways, including PI3K-Akt, MAPK, and TNF signaling (P < 0.05; Figure 3D).
Validation of Candidate miRNAs and Functional Studies
Quantitative PCR (qPCR) validated the upregulation of hsa-miR-124-5p (P < 0.01) and hsa-miR-143-3p (P < 0.0001) in BMSC-derived exosomes compared to KG-1a exosomes, while hsa-miR-100-5p was downregulated (P < 0.01; Figure 4A). A miRNA-mRNA interaction network predicted SMC4—a gene implicated in chromosomal stability and cancer progression—as a key target of hsa-miR-124-5p (Figure 4B).
To assess functional relevance, hsa-miR-124-5p was knocked down in BMSCs using specific inhibitors. Exosomes from knockdown BMSCs failed to suppress KG-1a proliferation (P < 0.0001 vs. BMSC exosomes at 48–72 hours; Figure 5A) or induce apoptosis (8% vs. 25% in BMSC exosome-treated cells; P < 0.001; Figure 5C). Similarly, G0/G1 arrest was abolished (55% vs. 70%; P < 0.05; Figure 5B). In contrast, hsa-miR-143-3p knockdown enhanced apoptosis (P < 0.001; Supplemental Figure 1), suggesting divergent roles for these miRNAs.
Mechanistic Role of hsa-miR-124-5p in SMC4 Regulation
BMSC-derived exosomes significantly reduced SMC4 mRNA (P < 0.001) and protein (P < 0.0001) levels in KG-1a cells (Figure 6A–B). This suppression was reversed upon hsa-miR-124-5p knockdown, restoring SMC4 expression to baseline (P < 0.0001). SMC4, essential for mitotic chromosome condensation, is overexpressed in AML stem cells, and its downregulation aligns with reduced leukemic cell viability. These results establish hsa-miR-124-5p as the primary mediator of BMSC exosome effects via SMC4 targeting.
Discussion and Clinical Implications
This study demonstrates that BMSC-derived exosomes inhibit AML cell proliferation, induce cell cycle arrest, and promote apoptosis through miRNA-dependent mechanisms. The hsa-miR-124-5p/SMC4 axis represents a novel therapeutic target, as SMC4 knockdown mimics exosome-mediated anti-leukemic effects. These findings align with prior studies showing BMSC exosomes suppress oral and colorectal cancers via miRNA transfer. However, this study is the first to link hsa-miR-124-5p to AML pathogenesis.
Notably, hsa-miR-124-5p is underexpressed in AML cells, and its restoration via BMSC exosomes may counteract leukemogenic pathways. The PI3K-Akt and MAPK pathways, enriched in KEGG analysis, are critical for AML survival, suggesting that exosomal miRNAs may concurrently target multiple oncogenic networks.
Limitations and Future Directions
While this study provides mechanistic insights, key limitations include the lack of in vivo validation and primary patient-derived AML cells. Future work should assess exosome efficacy in animal models and clinical samples. Additionally, the contribution of other miRNAs (e.g., hsa-miR-143-3p) and non-miRNA cargo (proteins, lipids) warrants exploration.
Conclusion
BMSC-derived exosomes exert anti-leukemic effects by delivering hsa-miR-124-5p, which suppresses SMC4 expression and disrupts AML cell proliferation and survival. This study highlights the potential of BMSC exosomes as a novel biological therapy for AML, offering a dual advantage of targeted miRNA delivery and reduced off-target toxicity.
doi.org/10.1097/CM9.0000000000001138
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