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. ^
Biology, Molecular|Biology, Cell
"MicroRNAs Regulate Differentiation and Tumorigenicity of Neuroblastoma Cells"
(January 1, 2010).
ETD Collection for Fordham University.