Regulation of p14 ARFexpression by miR-24: A potential mechanism compromising the p53 response during retinoblastoma development

Abstract

Background: Most human cancers show inactivation of both pRB- and p53-pathways. While retinoblastomas are initiated by loss of the RB1 tumor suppressor gene, TP53 mutations have not been found. High expression of the p53-antagonist MDM2 in human retinoblastomas may compromise p53 tumor surveillance so that TP53 mutations are not selected for in retinoblastoma tumorigenesis. We previously showed that p14 ARFprotein, which activates p53 by inhibiting MDM2, is low in retinoblastomas despite high mRNA expression.Methods: In human fetal retinas, adult retinas, and retinoblastoma cells, we determined endogenous p14 ARFmRNA, ARF protein, and miR-24 expression, while integrity of p53 signalling in WERI-Rb1 cells was tested using an adenovirus vector expressing p14 ARF. To study p14 ARFbiogenesis, retinoblastoma cells were treated with the proteasome inhibitor, MG132, and siRNA against miR-24.Results: In human retinoblastoma cell lines, p14 ARFmRNA was disproportionally high relative to the level of p14 ARFprotein expression, suggesting a perturbation of p14 ARFregulation. When p14 ARFwas over-expressed by an adenovirus vector, expression of p53 and downstream targets increased and cell growth was inhibited indicating an intact p14 ARF-p53 axis. To investigate the discrepancy between p14 ARFmRNA and protein in retinoblastoma, we examined p14 ARFbiogenesis. The proteasome inhibitor, MG132, did not cause p14 ARFaccumulation, although p14 ARFnormally is degraded by proteasomes. miR-24, a microRNA that represses p14 ARFexpression, is expressed in retinoblastoma cell lines and correlates with lower protein expression when compared to other cell lines with high p14 ARFmRNA. Transient over-expression of siRNA against miR-24 led to elevated p14 ARFprotein in retinoblastoma cells.Conclusions: In retinoblastoma cells where high levels of p14 ARFmRNA are not accompanied by high p14 ARFprotein, we found a correlation between miR-24 expression and low p14 ARFprotein. p14 ARFprotein levels were restored without change in mRNA abundance upon miR-24 inhibition suggesting that miR-24 could functionally repress expression, effectively blocking p53 tumor surveillance. During retinal tumorigenesis, miR-24 may intrinsically compromise the p53 response to RB1 loss. © 2012 To et al; licensee BioMed Central Ltd.