Abstract
Melanoma is the deadliest of all skin cancers, accounting for over 80% of all skin-cancer related death globally. The population of New Zealand is furthermore at significantly increased risk of melanoma compared to the majority of other countries, with a melanoma incidence rate and melanoma-related mortality rate almost tenfold greater than the global average. Thus, investigation into the pathophysiology and treatment of melanoma is of vital importance to the population of New Zealand and the world at large.
The tumour suppressor p53 is almost ubiquitously lost in cancer, frequently through mutation. In melanoma however p53 is wildtype in ~95% of cases, yet is still functionally inactivated through non-mutational means. For this reason, investigation of non-mutational p53 inactivation mechanisms is of high importance to understanding melanoma pathophysiology and potential treatment targets. One mechanism which has been suggested to interfere with p53 activity in cancer is the aberrant expression of p53 isoforms. The TP53 gene is capable of producing 12 distinct protein isoforms through the use of alternative promoter sites and alternative splicing, some of which have been observed to directly regulate the activity of canonical p53. Thus, dysregulation of these isoforms has the potential to dysregulate p53 activity significantly, contributing to loss of tumour suppression and oncogenesis.
miRNAs are small non-coding RNAs which act as sequence-specific regulators of gene expression primarily through translational repression, and are also observed to be aberrantly expressed in cancer. The majority of miRNA target sequences are located in the 3’UTR of mRNA transcripts, where miRNAs bind and induce mRNA degradation to downregulate the associated protein product. As the alpha, beta, and gamma isoforms of p53 have unique 3’UTR sequences, miRNAs have the potential to differentially regulated the expression of these isoforms, possibly contributing to their aberrant expression in cancer and melanoma specifically. Thus, we sought to assess whether a selection of miRNAs predicted to bind the p53 3’UTR would differentially regulate the expression of p53 isoforms based on their unique 3’UTR sequences.
To this end we transfected A375 melanoma cells with mimics of miR-122, miR-223, miR-376, miR-1305 and miR-26a alongside dual luciferase plasmid constructs in which the expression of luciferase was regulated by the alpha, beta, or gamma p53 3’UTR sequence, seeking to see if these miRNAs would differentially regulate the expression of luciferase which could be quantified via measurement of luminescence.
We found that overexpression of miR-122, miR-223, miR-376, miR-1305, and miR-26a did not induce significant downregulation of luciferase protein expression regulated by any of the p53 alpha, beta, or gamma 3’UTRs relative to negative controls, suggesting that these miRNAs do not differentially regulate p53 isoforms in cancer.