Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth most common form of cancer. It is commonly caused by smoking, alcohol, and Human Papilloma Virus (HPV). Due to its recent increase in incidence worldwide, it is important to investigate the molecular mechanisms responsible for HNSCC.
miRNAs typically perform post-transcriptional gene regulation by binding to the 3´untranslated region (UTR) of a messenger RNA (mRNA). Since their discovery, there has been a one miRNA to one target model for gene regulation. However, it is more the case that miRNAs in combination or co-operation are responsible, expanding the complexity of miRNA directed gene regulation.
Programmed Cell Death 4 (PDCD4) is an important tumour suppressor gene in HNSCC, containing three miR-499 binding sites and a single miR-21 binding site. Mutational analysis of these four individual binding sites was conducted to determine the contribution of each site to PDCD4 silencing. We determined that the first miR-499 site did not contribute to PDCD4 silencing, but that the presence of both the remaining miR-499 sites are required for PDCD4 suppression. This was demonstrated across several cell lines, which suggested that the requirement of two intact miR-499 binding sites is a ubiquitous mechanism for PDCD4 suppression.
The distance between the two functional miR-499 sites is 60nt, which is greater than the standard miRNA co-operation distance of 35nt. Bioinformatic analysis revealed that other targets share this distribution of miR-21 and miR-499 recognition sites, and may also be under the same form of regulation. Extending this analysis, we showed that thousands of genes have miRNA pairs 60nt apart.
In summary, the regulation of PDCD4 by the two distal miR-499 sites is unique. This suggests a higher degree of miRNA complexity in their role in gene regulation, and may impact a great number of targets. What is evident is that the one miRNA to one target model is not a true reflection of miRNA gene regulation.