miR-34a is a potential tumor suppressor gene on 1p36 in neuroblastoma Free

LB-71

A 10 mega base region of 1p36 is commonly lost in MYCN amplified neuroblastoma, and is a predictive factor for the most aggressive phenotype. Despite over a decade of investigational research, no definitive tumor suppressor gene has been identified in this region . Micro RNAs are non-coding small RNAs involved in normal cell development and cancer biology. We have identified 6 miRNAs located on 1p36 (miR-200a, a^*, b; 429; 551a; and 34a) of which three are predicted to target MYCN gene by computational approach (miR-200b, 429 and 34a). We have found that the expression of mir-34a correlates with 1p36 loss. By knock in experiments, we have found that miR-34a consistently suppresses cell growth. Using FACS analysis, we discovered that reduced cell growth by miR-34a is due to a decreased BrdU in-corporation, and increased apoptosis in IMR32 cells. Western blotting showed miR-34a significantly suppressed MYCN protein expression in IMR32 cells. When using a luciferase reporter containing MYCN 3’-UTR transfected into both Hela and IMR32 cells, miR-34a could also repress luciferase activity demonstrating that MYCN is a direct target of miR-34a. In order to identify other targets of miR-34a, we performed microarray experiments on IMR32 cells transfected with miR-34a. Of the genes altered, 44 were induced and 187 suppressed by mir-34a at 72 hrs. Of the induced genes, ~50% were highly expressed in neural tissues including brain. However, of the suppressed genes, only 7 (4%) were predicted targets of miR-34a, indicating the presence of other potential targets of this microRNA. We conclude that miR-34a causes global suppression of gene expression and cell growth, and induction of neural differentiation in a NB cell line. Furthermore we demonstrate that MYCN is a direct target of mir-34a, which maps to a region commonly lost in MYCN amplified neuroblastoma. Our studies showed that mir-34a is a potential tumor suppressor gene in NB and loss of its expression may result in an aggressive phenotype in the presence of MYCN amplification.