Background and Aims
p53, a transcription factor that participates in multiple cellular functions, is considered the most important tumour suppressor in the cell. Previous evidence suggests that post-transcriptional deregulation of p53 is related to tumorigenesis, tumour progression and therapy resistance. Several p53 regulatory proteins such as MDM2 and MDM4 have been found to modulate the stability and activity of p53. MicroRNAs have also been shown to participate in the post-transcriptional regulation of p53 and modify both protein levels and p53 activity. To further understand the role of miRNAs in p53 modulation, this study has investigated the role of miR-766 in p53 regulation and its impact on cellular fate.
Methodology
Wild-type (MCF10A, MCF7, SBC-3, U2OS and STA-ETI) and mutant TP53 (MDA-MB-468 and MDA-MB-231) breast and sarcoma cell lines were used to elucidate the role of mir-766 in post-transcriptional regulation of p53. Following transfection with mir-766 overexpression and control constructs, cell cycle distribution and proliferation were assessed through Propidium Iodide staining, and Cell-TitreGlo/colony formation assays respectively. Protein and mRNA expression levels of p53 and its known target genes was assessed through real-time PCR and western blot analysis, 24 and 48 hours post transfection (Lipofectamine).
Results
Following mir-766 overexpression, wild-type p53 protein levels significantly increased in immortalized normal breast (MCF10A), breast cancer (MCF7), lung cancer (SBC-3) and sarcoma (U2OS, STA-ETI, TC252) cell lines, accompanied with decreased MDM4 protein levels. CDKN1A, a down-stream target of p53, was also up-regulated after exogenous expression of miR-766. However, over-expression of miR-766 in either MB231 or MB468 cells had no effect on p53 protein levels, suggesting that miR-766 would not affect p53 level if p53 is mutant. Functionally, over-expression of miR-766 repressed cell proliferation in MCF10A, U2OS and SBC-3 cell lines, and induced significant cell cycle arrest in U2OS and SBC-3 cell lines.
Interestingly, five miR-766 binding sites located in the 3’UTR of MDM4 were identified through in Silico prediction analysis, suggesting that miR-766 participates in p53 regulation by targeting MDM4. Further studies are to be carried out in future.
Conclusion
MiR-766 functions as a positive regulator of p53 by reducing MDM4. The data support that over-expression of miR-766 can increase the levels of wild type p53 protein, decrease the levels of its principle regulator MDM4, and induce cell proliferation repression in cells.