microRNA-449a (miR-449a) has been identified to function as a tumor suppressor in several types of cancers. However, the role of miR-449a in neuroblastoma has not been intensively investigated. We recently found that the overexpression of miR-449a significantly induces neuroblastoma cell differentiation, suggesting its potential tumor suppressor function in neuroblastoma. In this study, we further investigated the mechanisms underlying the tumor suppressive function of miR-449a in neuroblastoma. We observed that miR-449a inhibits neuroblastoma cell survival and growth through 2 mechanisms--inducing cell differentiation and cell cycle arrest. Our comprehensive investigations on the dissection of the target genes of miR-449a revealed that 3 novel targets- MFAP4, PKP4 and TSEN15 -play important roles in mediating its differentiation-inducing function. In addition, we further found that its function in inducing cell cycle arrest involves down-regulating its direct targets CDK6 and LEF1. To determine the clinical significance of the miR-449a-mediated tumor suppressive mechanism, we examined the correlation between the expression of these 5 target genes in neuroblastoma tumor specimens and the survival of neuroblastoma patients. Remarkably, we noted that high tumor expression levels of all the 3 miR-449a target genes involved in regulating cell differentiation, but not the target genes involved in regulating cell cycle, are significantly correlated with poor survival of neuroblastoma patients. These results suggest the critical role of the differentiation-inducing function of miR-449a in determining neuroblastoma progression. Overall, our study provides the first comprehensive characterization of the tumor-suppressive function of miR-449a in neuroblastoma, and reveals the potential clinical significance of the miR-449a-mediated tumor suppressive pathway in neuroblastoma prognosis.
Keywords: 13-cis retinoic acid; Bio-control; Bio-miR-449a; CASP2; CCNE2; CDC25A; CDK6; Caspase 2; Cyclin E2; FDR; FLOT2; Flotillin 2; GAP43; GAPDH; HDAC1; LEF1; MFAP4; Oligo; PKP4; RA; STMN1; TSEN15; biotinylated control oligonucleotides; biotinylated miR-449a mimic; cell cycle arrest; cell division cycle 25A; cyclin-dependent kinase 6; differentiation; differentiation therapy; false discovery rate; glyceraldehyde-3-phosphate dehydrogenase; growth associated protein 43; histone deacetylase 1; lymphoid enhancer-binding factor 1; miR-449a; miRNA; microRNA; microRNA-449a; microfibril-associated protein 4; neuroblastoma; oligonucleotides; plakophilin 4; stathmin 1; tRNA splicing endonuclease 15 homolog.