Identification of new p53 target microRNAs by bioinformatics and functional analysis

Background: The tumor suppressor p53 is a sequence-specific transcription factor that regulates an extensive network of coding genes, long non-coding RNAs and microRNAs, that establish intricate gene regulatory circuits influencing many cellular responses beyond the prototypical control of cell cycle, apoptosis and DNA repair. Methods: Using bioinformatic approaches, we identified an additional group of candidate microRNAs (miRs) under direct p53 transcriptional control. To validate p53 family-mediated responsiveness of the newly predicted target miRs we first evaluated the potential for wild type p53, p63 beta and p73 beta to transactivate from p53 response elements (REs) mapped in the miR promoters, using an established yeast-based assay. Results: The REs found in miR-10b, -23b, -106a, -151a, -191, -198, -202, -221, -320, -1204, -1206 promoters were responsive to p53 and 8 of them were also responsive to p63 beta or p73 beta. The potential for germline p53 mutations to drive transactivation at selected miR-associated REs was also examined. Chromatin Immuno-Precipitation (ChIP) assays conducted in doxorubicin-treated MCF7 cells and HCT116 p53(+/+) revealed moderate induction of p53 occupancy at the miR-202, -1204, -1206, -10b RE-containing sites, while weak occupancy was observed for the miR-23b-associated RE only in MCF7 cells. RT-qPCR analyses cells showed modest doxorubicin-and/or Nutlin-dependent induction of the levels of mature miR-10b, -23b, -151a in HCT116 p53(+/+) and MCF7 cells. The long noncoding RNA PVT1 comprising miR-1204 and -1206 was weakly induced only in HCT116 p53(+/+) cells, but the mature miRs were not detected. miR-202 expression was not influenced by p53-activating stimuli in our cell systems. Conclusions: Our study reveals additional miRs, particularly miR-10b and miR-151a, that could be directly regulated by the p53-family of transcription factors and contribute to the tuning of p53-induced responses.