MicroRNA-1285 Inhibits The Expression Of P53 By Directly Targeting Its 3' Untransalted Region

p53 plays critical roles in the modulation of multiple cellular processes. The regulation of p53 is complicated and remains elusive. microRNAs (miRNAs) are a class of endogenously expressed small non-coding RNAs that regulate gene expression post-transcriptionally. In this study, we investigated whether p53 could be regulated by miRNAs.Firstly, we used TargetScan software to identify miRNAs that might target p53. Then a high-throughput luciferase assay was used to identify miRNAs that target the 3'UTR of p53.We found that miR-1285 can downregulate the expression of p53 by directly binding its 3'UTR. Notably, miR-612, which has the same seed region sequence as miR-1285,did not significantly alter expression of the p53 3'UTR reporter.According to the TargetScan prediction, the p53 mRNA 3'UTR contains two sequence motifs, which are nearly perfectly complementary to the miR-1285 sequence. We constructed p53 3'UTR luciferase reporters with one or two target sites mutated. The luciferase activities with p53 3'UTR mutant constructs decreased less than with the wild-type construct. This suggests that miR-1285 can regulate p53 expression by directly binding two target sites in the 3'UTR.Subsequently, we introduced miR-1285 mimics or control RNA into several p53 wild-type tumor cell lines, including MCF-7, SH-SY5Y and HepG2.Both p53 mRNA and protein levels were reduced when miR-1285 was overexpressed in these cells. Furthermore, miR-1285 antisense oligonucleotides were transfected into SH-SY5Y cells, which display relatively high expression of miR-1285.The results showed that miR-1285 silencing led to increased expression levels of p53 mRNA and protein.Furthermore, we determined the effect of miR-1285 on the expression of p21,a master gene downstream of p53.The luciferase activity of WWP-Luc, a p21 promoter based luciferease reporter, was significantly decreased when co-transfected with miR-1285 in SH-SY5Y cells. Consistent with this, both p21 mRNA and protein levels were suppressed by miR-1285 in SH-SY5Y cells.In conclusion, our study provides the first evidence that miR-1285 directly regulates the expression of p53 by binding its 3'UTR.