Background

The tumour suppressor gene Programmed Cell Death 4 (PDCD4) has been shown to be dysregulated in various cancers including head and neck cancer. However its regulation is not well known. MicroRNAs (miRNAs) are small non-coding RNAs that bind to the 3’ untranslated region (UTR) of their mRNA target to mediate gene silencing. Conventionally only a single miRNA is required and there are few reports on the simultaneous regulation of a gene by multiple miRNA. Here we provide new insights for the regulation of PDCD4 by two miRNAs, miR-21 and miR-499.

Methods

To understand the direct interaction of these miRNAs to PDCD4, mutants were created to represent the four key binding sites and tested using a luciferase reporter. The exact contributions of miR-21 and miR-499 binding sites were determined using titration assays. Furthermore, siRNA KO of Ago2 was performed to evaluate the role of Ago2 in its interaction with miR-21 and miR-499.

Results

miR-21 and miR-499 bind independently to the PDCD4 3’UTR. The first miR-499 site appears to be redundant as assessed by the endpoint luciferase assay and titration. Interestingly, the last two miR-499 binding sites appear to be critical as mutations at either site abolish silencing activity suggesting a co-dependence. Furthermore, it appears that the miR-21 site has a contribution to miR-499 binding to the 3’UTR. Knockdown of Ago2 reduced but did not completely prevent binding by either miRNA.  

Conclusion

We show that the miR-21 and miR-499 binding sites contribute differently to PDCD4 regulation. Our data sheds new light on the complex regulation of tumour suppressor genes by multiple miRNAs. This regulation may be widespread as other genes also contain the miR-21 and miR-499 combination.