Abstract 2286: p53-miR-dependent post-transcriptional circuits: mechanisms, targets and inter-individual variation

Abstract

Abstract Using bioinformatics approaches, we identified a group of candidate microRNAs (miRs) for direct p53 transcriptional control. To validate p53 family-mediated control of the newly predicted target miRs we evaluated the potential for wild type p53, p63α and p73α to transactivate from p53 response elements (REs) mapped at the miR promoters. Results obtained with an established yeast-based assay indicated that the REs found at miR-10b, -23b, -106a, -151, -191, -198, -202, -221, -320, -1204, -1026 genes were responsive to p53 of which 8 were also responsive to p63α or p73α. ChIP assays in doxorubicin-treated HCT116 cells -p53+/+ vs p53-/– revealed moderate p53 occupancy at the miR-202, -1206 sites, and weak occupancy at miR-10b, -191. RT-qPCR analyses in HCT116 and MCF7 cells showed modest doxorubicin- and/or p53-dependent regulation for miR-23b, -151, -191. We then focused on miR-191 and its potential impact on MDM4, an important modulator of p53. The A>C SNP (rs4245739) in the proximal portion of the MDM4 3′UTR occurs in a predicted binding site for miR-191, with the A allele resulting in a mismatch at the predicted miR-191 seed. This SNP is in strong association with the tag-SNP (rs2369244) within the MDM4 haplotype that was reported to impact on cancer risk. Using MCF7 and HCT116 cells that are heterozygous for rs4245739, we examined at the endogenous MDM4 gene level the impact of varying the expression of miR-191. A slight allelic imbalance was observed by allele-specific qPCR when miR-191 was over-expressed, and the co-transfection of miR-34a, for which seed sequences are predicted in the distal portion of the MDM4-3′UTR, had an additive effect, leading to ∼20% reduction of the C-allele. Western blot analyses indicated that steady-state MDM4 protein levels were reduced by miR-191 and/or miR-34a overexpression. Our study reveals additional miRs that could be directly regulated by the p53-family of transcription factors and could contribute to the tuning of p53-induced responses with the possibility of inter-individual variations due to functional SNPs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2286. doi:1538-7445.AM2012-2286