Abstract
Neuroblastoma, a childhood tumor derived from neural crest progenitor cells, represents the most common extracranial solid tumor in children. Among these, high-risk neuroblastoma (HRNB) accounts for most cases, with a survival rate of less than 50%. The poor prognosis associated with HRNB is largely attributed to tumor heterogeneity, disrupted molecular pathways, and therapeutic failure, reflecting the shortcomings of traditional multimodal therapy. Key oncogenes, including MYCN and ALK, are central drivers of neuroblastoma pathogenesis, and targeting these molecular markers could lead to more effective and less toxic therapies. MicroRNAs (miRNAs), which are small non-coding RNAs that regulate gene expression post-transcriptionally, have shown therapeutic promise in various malignancies, including HRNB. Specifically, miR-34a and miR-124 have been identified as downregulated in HRNB cell lines. miR-34a targets the MYCN oncogene, while miR-124 has been shown to induce differentiation in neuroblastoma cells and enhance sensitivity to chemotherapeutic agents such as cisplatin. This project investigated the therapeutic potential of miR-34a and miR-124 in neuroblastoma by exploring their replenishment as targeted therapies and assessing their synergistic effects with the platinum-based chemotherapeutic cisplatin.
One of the key challenges in utilizing miRNA therapies is the efficient delivery of miRNAs to target cells. Traditional delivery methods, including polyethylenimine (PEI) and lipofectamine, often suffer from high cytotoxicity or suboptimal efficiency. To overcome these issues, we explored the use of a novel bioreducible polyethylenimine (PEI-SS) as a transfection agent. PEI-SS is a high molecular weight PEI mimic that was designed to reduce the cytotoxicity seen with conventional PEI, while maintaining efficient miRNA delivery. We hypothesized that PEI-SS would demonstrate comparable miRNA loading efficiency to gold standard PEI25k, but with significantly reduced cytotoxicity.
To test this hypothesis, we synthesized PEI-SS by crosslinking low molecular weight PEI with disulfide bonds, which can be cleaved in reducing intracellular environments. We used PEI-SS to deliver miR-34a and miR-124 to HRNB cell lines and observed effects on cytotoxicity, transfection efficacy, and synergy with cisplatin. Our results indicated that PEI-SS efficiently loaded miR-34a and miR-124, achieving transfection efficacy comparable to PEI25k but with significantly lower cytotoxicity. Treatment with miR-34a and miR-124 reduced cell viability. However, a clear synergistic effect between miRNA treatment and cisplatin was not observed in the HRNB cell line SK-N-SH, underscoring the complexity of their interaction and the necessity for further investigation across different HRNB cell lines.
Additionally, molecular biology techniques such as gene expression analysis will be employed to more deeply characterize the effects of miR-34a and miR-124 on HRNB cells. These findings suggest that PEI-SS is a promising transfection agent for miRNA delivery and could be bioengineered for combination therapy with chemotherapeutic agents like cisplatin.
In future applications, polyplexes formed by PEI-SS and miRNAs could be loaded into mesenchymal stem cells (MSCs), which possess tumor-homing abilities, particularly for neuroblastoma. This approach could enable targeted delivery of miRNA and chemotherapy agents directly to tumor sites, offering a more precise and effective treatment for HRNB patients. Overall, PEI-SS provides a novel, non-toxic platform for miRNA-based therapies and has the potential to be integrated into more effective, personalized treatment regimens for HRNB.