Examination of PTTG-Binding Factor (PBF) mRNA reveals functional miRNA target sites and exon-skipping splice variants in thyroid cells

Dysregulation of the processing and stability of mRNA encoding oncogenes or tumour suppressor proteins is a critical event in the pathogenesis of cancer. Previously, we demonstrated that the proto-oncogene PTTG-binding factor (PBF) is overexpressed in thyroid, pituitary and breast tumours. Critically, high PBF expression is associated with reduced disease-specific survival in thyroid cancer. However, the mechanisms responsible for regulating PBF expression are unknown. In this study, we examined PBF mRNA in human thyroid cancer FFPE specimens by touchdown 1-step RT-PCR analysis. Four different PBF mRNA splice variants (SV) were identified through direct sequencing. Altered expression of an SV lacking exons 4 and 5 was evident in 27/30 thyroid tumours, in two main groups with either up- (1.5±0.2; n=9) or down-regulation (0.42±0.06; n=18), and both with a significant twofold difference in relative expression compared to full-length PBF mRNA (P<0.05). We next investigated whether PBF mRNA contained any target sites for regulatory miRNAs that might induce mRNA degradation. Bioinformatic analysis revealed a number of putative miRNA interaction sites in the 3’ UTR region of the PBF gene. Five miRNAs (i.e. miR-122, -124, -365, -506 and -647) were selected for further study based on evolutionary conservation of sequence. Importantly, a significant reduction in endogenous PBF mRNA levels was observed following transfection of thyroid cancer TPC-1 cells using synthetic mimics for miR-506-3p (56.9±4.0%; P<0.001), miR-122-5p (52.5±10.6%; P<0.01) and miR-124-3p (40.6±5.6%; P<0.01) after 48 hr. In contrast, there was no change with miR-647 (P=NS) or miR-365a-3p (P=NS). In summary, these results implicate exon-skipping as a possible mechanism in dysregulating PBF activity in thyroid cells. Furthermore, our findings suggest that the stability of PBF mRNA is dependent on miRNAs such as miR-506 and miR-124, the expression of which are frequently down-regulated in tumours and associated with increased cell motility and invasion capability of malignant cells.