Endocrine Abstracts

MicroRNAs (miRNAs) are small non-coding RNAs of ~22 nucleotides that negatively regulate gene expression through imperfect base pairing to the 3′ untranslated regions (UTRs) of target mRNAs. We have investigated the role of the miR-15a–miR-16-1 cluster in pituitary tumourigenesis, as it functions in other cancers as a tumour suppressor via regulation of the cell-cycle regulator cyclin D1. We have used two approaches: 1) in vitro studies examining for altered expression of miR-15a, miR-16-1 and cyclin D1 in pituitary tumours, mainly somato-lactotrophinomas, that develop in mice deleted for a multiple endocrine neoplasia type-1 allele (Men1^+/−); and 2) in vivo studies examining the effects of antagomirs, which are modified oligonucleotide miRNA inhibitors, on mouse pituitaries. Mice were kept in accordance with UK Home Office welfare guidelines and project licence restrictions. Use of total pituitary RNA from wild-type (Men1^+/+) and Men1^+/− mice for quantitative reverse transcriptase-PCR (qRT-PCR) revealed significant downregulation of miR-15a (−4.2-fold change, P<0.05) and miR-16-1 (−3.8-fold change, P<0.005) expression in pituitary tumours compared to Men1^+/+ pituitary tissue. Furthermore, cyclin D1 mRNA (+2.9-fold change, P<0.005) and cyclin D1 protein (>10-fold change) were increased in Men1^+/− pituitary tumours, and the greater cyclin D1 protein expression is consistent with miRNA-mediated post-transcriptional regulation of cyclin D1 mRNA. In addition, miR-15a and miR-16-1 expression were strongly correlated, consistent with transcription from the same genomic locus (r²=0.89), and there was an inverse correlation between miR-15a and miR-16-1 with cyclin D1 mRNA expression (r²=0.86 and r²=0.67 respectively). In vivo, direct transauricular injection of antagomirs specific for miR-15a and miR-16-1, into the pituitaries of Men1^+/+ mice resulted in a twofold increase in cyclin D1 mRNA. Thus, these findings, which support a role for the miR-15a–miR-16-1 cluster in pituitary tumourigenesis via regulation of cyclin D1, may facilitate the development of novel therapeutic strategies.