Abstract B76: MicroRNAs regulate cell phenotype and N-myc expression in human neuroblastoma cells

Abstract

Abstract Identification of miRNAs that regulate neuroblast growth and differentiation could be important in understanding the onset and progression of the childhood cancer neuroblastoma, and in developing new therapeutics to fight this often fatal disease. Neuroblastoma tumors and derived cell lines are heterogeneous, with three basic phenotypes - neuroblastic (N), substrate-adherent (S), and cancer stem cells (I). These types differ significantly in their morphology, biochemistry and tumorigenic potential. Five miRNAs differentially expressed with respect to cell phenotype were identified from a miRNA microarray, validated by RT-PCR, and quantified by real-time RT-PCR taqman assays in 14 different cell lines (6 N-, 5 I- and 3 S-types). Out of these miRNAs, three are highly expressed in non-tumorigenic S-type cell lines compared to I- and N-type cell lines: miR-21 (10- fold, p<0.05), miR-221 (18-fold, p<0.05) and miR-335 (136-fold, p<0.05). Moreover, miRNA expression increased significantly with BrdU-induced I- to S-type differentiation of BE(2)-C. By contrast, miR-124a levels are 10-fold higher in N-type cells compared to both I- and S-type cells and are elevated 2-fold (p<0.05) following retinoic acid-induced neuronal differentiation of I-type BE(2)-C. Transient transfection of miR-124a into I-cells induced a neuronal-like phenotype with increased neurite growth, supporting its active role in neuronal differentiation. Another neuronal-specific miRNA, miR-375, expressed in N- and I- but not in S-type cells, significantly decreased with induced differentiation from an I- to S-type phenotype. A second factor affecting survival of neuroblastoma patients is overexpression/amplification of the protooncogene N-myc. Thus, the microarray was sorted for miRNAs whose levels correlated directly or inversely with N-myc gene copy number. Candidate miRNAs were quantified by real-time RT-PCR taqman assays in the same 14 cell lines (8 N-myc amplified and 6 non-amplified). Three members of the miR-17-92 cluster (miR-17-5p, miR-18a and miR-20a) are highly expressed in N-myc-amplified cell lines compared to non-amplified cells (p<0.05). Moreover, siRNA-induced reduction of N-myc protein (2.5-fold) down-regulated expression levels of all three miRNAs by 70% (p<0.05), suggesting N-myc up-regulates the expression of the miR-17-92 cluster. A screen of potential targets of the miR-17-92 cluster members revealed, interestingly, that N-myc mRNA itself is a predicted target of miR-17-5p. Down-regulation of this miRNA (95%) by transient transfection with specific inhibitors increased N-myc mRNA 2-fold (p<0.05) and protein 1.5-fold (p<0.05), confirming and extending results of Cloonan et al (2008). In N-myc amplified cell lines, N-myc mRNA levels negatively correlate with levels of miR-17-5p. Together these data reveal a possible negative feedback loop between N-myc and miR-17-5p. In conclusion, miRNAs that are differentially expressed with respect to both phenotype and N-myc amplification status may significantly contribute to the progression and aggressiveness of neuroblastoma. Citation Information: Cancer Res 2009;69(23 Suppl):B76.