MicroRNAs Regulate Differentiation and Tumorigenicity of Neuroblastoma Cells
Neuroblastoma is the most common extracranial solid tumor in children. Both cellular heterogeneity and N-myc amplification influence the survival of NB patients. miRNAs are naturally occurring small (19-23 nucleotides) non-coding RNAs which down-regulate expression of protein-coding genes by binding to 3'-UTRs and directing degradation and/or inhibiting translation of the cognate mRNAs. Deregulated miRNAs have been implicated in many cancers including neuroblastoma. These studies sought to identify miRNAs that are involved in neuroblastoma tumorigenicity. miRNAs differentially expressed with respect to cell phenotype and level of N-myc overexpression were identified from a microarray and verified by qRT-PCR. From these analyses I showed five miRNAs that differ with phenotype (miR-124, miR-375, miR-21, miR-221, and miR-335) and three miRNAs that differ with N-myc overexpression level (miR-17-92 cluster members, miR-17-5p, miR-18a and miR-20a). Of the phenotype-specific miRNAs, miR-21, -221 and -335 characterize the non-neuronal, non-tumorigenic substrate adherent S cell phenotype as shown by qRT-PCR of steady state levels and induced differentiation studies. In contrast, miR-124 is a neuronal miRNA; it is highest in N-type neuroblasts and increases with RA-induced neuronal differentiation. Moreover, increased expression of exogenous miR-124 induced differentiation along a catercholaminergic neuronal pathway as shown by smaller cell bodies, elongated neurites and a 3.7-fold increase in 3H-norephinephrine uptake. Importantly, miR-124-induced differentiation resulted in a 5–7-fold reduction in malignant potential. Lastly, the miR-375 appears to be associated with a neuroendocrine phenotype—present in N- and I-type cells (cancer stem cells), barely detectable in S cells and markedly decreased when cells were induced to differentiate to S. Moreover, experimental down-regulation of miR-375 increased the neuronal-specific HuD RNA-binding protein, showing it is a target of miR-375. The expression levels of miR-17-92 cluster members are 3.1–4.8-fold higher in N-myc amplified cell lines compared to non-amplified cell lines. Experimental down-regulation of N-myc decreased the mean expression of these members by 70%; thus N-myc regulates expression of the cluster. N-myc is also a target for regulation by miR-17-5p as shown by: (1) N-myc mRNA and miR-17-5p levels are negatively correlated in N- myc amplified cell lines; (2) N-myc 3'-UTR contains functional miR-17-5p binding sites; (3) down-regulation of miR-17-5p significantly increased N-myc mRNA and protein and (4) increased expression of miR-17-92 cluster decreased N-myc. Most importantly, increased expression of miR-17-92 cluster in an N-myc non-amplified cell line increased the colony-forming efficiency 3.3-fold, suggesting that overexpression of the miR-17-92 cluster with N-myc amplification may contribute to N-myc-related tumorigenicity. Finally, gel mobility shift assays showed that overlapping miR-17-5p and HuD protein binding sites in the N-myc 3'-UTR could influence the expression of N-myc. Altogether my studies have shown that miRNAs play vital roles in regulating cell malignancy and differentiation in human neuroblastoma cells.
Molecular biology|Cellular biology
Samaraweera, Leleesha, "MicroRNAs Regulate Differentiation and Tumorigenicity of Neuroblastoma Cells" (2010). ETD Collection for Fordham University. AAI3452811.