Development of Shortened miR-506-3p Mimics Exhibiting Strong Differentiation-Inducing Activity in Neuroblastoma Cells.

microRNA mimics are synthetic RNA molecules that imitate the mature miRNA duplexes and their functions. These mimics have shown promise in treating cancers. Nucleotide chemical modifications of microRNA mimics have been investigated and have improved the stability of miRNA mimics. However, the potential therapeutic benefit of mimic analogs based on sequence modifications has not been explored. miR-506-3p was identified as a differentiation-inducing microRNA in neuroblastoma cells, suggesting the potential of applying the miR-506-3p mimic in neuroblastoma differentiation therapy. In this study, we explored the possibility of developing shortened miR-506-3p analogs that can maintain differentiation-inducing activities comparable to the wild-type miR-506-3p mimic. We found that deleting up to two nucleotides at either the 3' end or within the middle region of the miR-506-3p sequence fully maintained the differentiation-inducing activity when compared to the wild-type mimic. Deleting up to four nucleotides from the 3' end or deleting three nucleotides in the middle positions diminished the differentiation-inducing activity, but the analogs still maintained differentiation-inducing activities that were significantly higher than the negative control oligo. The shortened analog designs potentially benefit patients from two perspectives: (1) the reduced cost of manufacturing shortened analogs, and (2) the reduced non-specific toxicity due to their smaller molecular sizes.

Systematic Analysis of miR-506-3p Target Genes Identified Key Mediators of Its Differentiation-Inducing Function.

Background/Objectives: miR-506-3p has been demonstrated to be a strong inducer of neuroblastoma cell differentiation, highlighting the potential of applying miR-506-3p mimics to neuroblastoma differentiation therapy. However, the target genes of miR-506-3p that mediate its differentiation-inducing function have not been fully defined. This study aims to comprehensively investigate the targetome of miR-506-3p regarding its role in regulating neuroblastoma cell differentiation. Methods: We combined gene expression profiling and functional high-content screening (HCS) to identify miR-506-3p target genes that have differentiation-modulating functions. For evaluating the potential clinical relevance of the identified genes, we analyzed the correlations of gene expressions with neuroblastoma patient survival. Results: We identified a group of 19 target genes with their knockdown significantly inducing cell differentiation, suggesting that these genes play a key role in mediating the differentiation-inducing activity of miR-506-3p. We observed significant correlations of higher mRNA levels with lower patient survival with 13 of the 19 genes, suggesting that overexpression of these 13 genes plays important roles in promoting neuroblastoma development by disrupting the cell differentiation pathways. Conclusions: Through this study, we identified novel target genes of miR-506-3p that function as strong modulators of neuroblastoma cell differentiation. Our findings represent a significant advancement in understanding the mechanisms by which miR-506-3p induces neuroblastoma cell differentiation. Future investigations of the identified 13 genes are needed to fully define their functions and mechanisms in controlling neuroblastoma cell differentiation, the understanding of which may reveal additional targets for developing novel differentiation therapeutic agents.

Identification of CDKN3 as a Key Gene that Regulates Neuroblastoma Cell Differentiation.

We conducted a high-content screening (HCS) in neuroblastoma BE(2)-C cells to identify cell cycle regulators that control cell differentiation using a library of siRNAs against cell cycle-regulatory genes. We discovered that knocking down expression of cyclin dependent kinase inhibitor 3 (CDKN3) showed the most potent effect in inducing neurite outgrowth, the morphological cell differentiation marker of neuroblastoma cells. We then demonstrated that CDKN3 knockdown increased expression of neuroblastoma molecular differentiation markers, neuron specific enolase (NSE), ßIII-tubulin and growth associated protein 43 (GAP43). We further showed that CDKN3 knockdown reduced expression of cell proliferation markers Ki67 and proliferating cell nuclear antigen (PCNA), and reduced colony formation of neuroblastoma cells. More importantly, we observed a correlation of high tumor CDKN3 mRNA levels with poor patient survival in the investigation of public neuroblastoma patient datasets. In exploring the mechanisms that regulate CDKN3 expression, we found that multiple strong differentiation-inducing molecules, including miR-506-3p and retinoic acid, down-regulated CDKN3 expression. In addition, we found that N-Myc promoted CDKN3 expression at the transcriptional level by directly binding to the CDKN3 promoter. Furthermore, we found that CDKN3 and two additional differentiation-regulating cell cycle proteins identified in our HCS, CDC6 and CDK4, form an interactive network to promote expression of each other. In summary, we for the first time discovered the function of CDKN3 in regulating neuroblastoma cell differentiation and characterized the transcriptional regulation of CDKN3 expression by N-Myc in neuroblastoma cells. Our findings support that CDKN3 plays a role in modulating neuroblastoma cell differentiation and that overexpression of CDKN3 may contribute to neuroblastoma progression.